Banana Breeding, Progress and Challenges (Michael Pillay) (9781439800171) (2011)

Banana Breeding
Edited by
Michael Pillay
Abdou Tenkouano
Boca Raton London New York
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This book is dedicated to our families, who enrich our lives, and to
our late colleagues Dirk Vuylsteke, Paul Speijer, John Hartman (IITA),
and Phil Rowe (FHIA) for their contributions to banana breeding.
Contents
Foreword............................................................................................................................................ix
Manjit S. Kang
Introduction........................................................................................................................................xi
Ivan W. Buddenhagen
Preface............................................................................................................................................ xiii
Editors.............................................................................................................................................xvii
Contributors.....................................................................................................................................xix
Chapter 1 General Plant Morphology of Musa..............................................................................1

Deborah Karamura, Eldad Karamura, and Guy Blomme
Chapter 2 Evolution and Genetic Relationships in Banana and Plantains................................... 21

Uma Subbaraya, Marimuthu S. Saraswathi, and Michael Pillay
Chapter 3 Genetic Resources for Banana Improvement.............................................................. 41

Markku Häkkinen and Richard Wallace
Chapter 4 Genomes, Cytogenetics, and Flow Cytometry of Musa............................................. 53

Michael Pillay and Abdou Tenkouano
Chapter 5 Genetics of Important Traits in Musa......................................................................... 71

Eli Khayat and Rodomiro Ortiz
Chapter 6 Major Diseases of Banana........................................................................................... 85

Guy Blomme, Simon Eden-Green, Mohammed Mustaffa,
Bartholemew Nwauzoma, and Raman Thangavelu
Chapter 7 Integrated Pest Management of Banana.................................................................... 121

Thomas Dubois and Daniel L. Coyne
Chapter 8 Reproductive Biology................................................................................................ 145

Jeanie Anne Fortescue and David William Turner
Chapter 9 Breeding Techniques................................................................................................. 181

Abdou Tenkouano, Michael Pillay, and Rodomiro Ortiz
vii
viii Contents
Chapter 10 Mutations and Cultivar Development of Banana...................................................... 203

Shri Mohan Jain, Bradley Till, Prasnna Suprasanna, and Nicolas Roux
Chapter 11 Biotechnology in Musa Improvement....................................................................... 219

Leena Tripathi
Chapter 12 Genotype by Environment Interaction and Musa Improvement............................... 237

Rodomiro Ortiz and Abdou Tenkouano
Chapter 13 Quality Improvement of Cultivated Musa................................................................ 251

Edson Perito Amorim, Sebastião de Oliveira e Silva,
Vanusia Batista de Oliveira Amorim, and Michael Pillay
Chapter 14 Postharvest Processed Products from Banana.......................................................... 269

Cherukatu Kalathil Narayana and Michael Pillay
Chapter 15 Propagation Methods in Musa.................................................................................. 285

Michael Pillay, Christopher A. Cullis, David Talengera, and Leena Tripathi
Chapter 16 Hybrid Distribution to Farmers: Adoption and Challenges...................................... 305

Abdou Tenkouano, Michael Pillay, and Ousmane Coulibaly
Chapter 17 Molecular Breeding of Other Vegetatively Propagated Crops: Lessons for

Banana....................................................................................................................... 321
Michael Pillay, Abdou Tenkouano, and Rodomiro Ortiz
Chapter 18 Future Prospects........................................................................................................ 351

Rodomiro Ortiz, Michael Pillay, and Abdou Tenkouano
Index............................................................................................................................................... 355
Foreword
Food production must be increased year after year to keep pace with population growth. At the cur-
rent population growth rate of 1.2%, world population is expected to reach 9 to 10 billion by 2050.
On the basis of this estimate, food production would need to be doubled in the next 30 years or so
and tripled in the next 50 years. We will need to do this without causing ecological damage to our
natural resources and the environment.
Although cereals are expected to continue to be the most important calorie providers in the world,
crops like bananas will also remain important calorie-providing staples throughout the world, espe-
cially in developing tropical countries. Because of the importance of banana in some parts of the
world, especially Africa, attention must be focused on it as a staple food.
Several previously published books have included chapters on bananas, but there is no recent
book that provides in-depth coverage of all aspects of banana breeding and genetics, including
biotechnology. In some recently published books, individual chapters can be found on banana
improvement, but all aspects of banana breeding, genetics, biotechnology, genetic resources, and
morphology have not received treatment in sufficient detail, especially in light of the fact that major
advances have occurred in modern methods of banana breeding and related aspects during the past
couple of decades. Thus, there was a need to bring together these advances in a single title. The
current book, Banana Breeding: Progress and Challenges, edited by Michael Pillay and Abdou
Tenkouano, fills this need.
The book is a wide-ranging compilation of chapters by various experts. The book begins with
a chapter on the general plant morphology of Musa. Subsequently, chapters such as Evolution and
Genetic Relationships in Bananas and Plantains, Genetic Resources for Banana Improvement,
Genomes, Cytogenetics, and Flow Cytometry of Musa, and Genetics of Important Traits in Musa are
included. Two chapters cover the major diseases and pests of banana. Five chapters cover the central
focus of the book, including Reproductive Biology, Breeding Techniques, Mutations and Cultivar
Development of Banana, Biotechnology in Musa Improvement, and Genotype by Environment
Interaction and Musa Improvement. The latter chapter should help provide tools for selecting both
narrowly adapted and broadly adapted cultivars. The chapters on quality improvement of cultivated
Musa and postharvest processed products provide researchers and teachers with information to
improve quality aspects of banana and how to reduce postharvest losses, respectively.
Because of its vegetative reproduction, the chapter on propagation methods in Musa should prove
valuable to small-scale farmers in providing enough planting material. In the chapter Molecular
Breeding of Other Vegetatively Propagated Crops: Lessons for Banana, the book draws on the
strengths and weaknesses of other vegetatively propagated crops to avoid mistakes and to make
achievement of success more certain.
The editors and other authors have vast experience in banana breeding, genetics, biotechnology,
molecular-marker technology, tissue culture, and other areas. Thus, all the chapters are authorita-
tive contributions. I must congratulate all of them for generating a first-class publication that should
be useful to researchers, teachers, and extension personnel around the world. This book is expected
to have a major impact on banana research and teaching. This comprehensive book should serve as
a ready reference for all researchers and teachers interested in banana breeding and production.
Manjit S. Kang
ix
Introduction
Finally we have a book centering on banana breeding. It is even entitled Banana Breeding. This is
a most welcome addition to the classical book by N. W. Simmonds, The Evolution of the Bananas,
now some 50 years old.
This book is a wide-ranging compilation of chapters by various authors covering plant morphology,
origin, genetic resources, reproductive biology, diseases, pests, quality improvement, propagation, and
distribution to farmers, as well as the central focus chapters covering breeding, genetics, and biotech-
nology. There is even a chapter comparing breeding with three other major clonal tropical crops.
When one considers that banana breeding based on modern science is now some 90 years
old and yet farmers are still mainly growing the diverse clones selected by villagers thousands
of years ago from their natural environment, one must ask why. How could so many diverse and
excellent clones have arisen by purely natural events of pollination, seed set, seed germination,
and then selection?
The answers are complex, but things must have been very different then. Indeed, in an excel-
lent chapter by Fortescue and Turner on reproductive biology, it is made very clear that low seed
set and germination today are major detriments to breeding progress and that little research has
been applied to understand the physiological/biochemical reasons for ovule abortion/low seed set.
When early man first found and brought into cultivation parthenocarpic plants, they were probably
quite fertile. As plants were moved about, encountering genetically diverse bananas, seed set was
probably abundant. With time, seed set was negatively selected and, hence, has led to present-day
sterility. Some parthenocarpic clones are still quite fertile, but exploration and evaluation for this
character has been neglected. Even our understanding of parthenocarpy itself and its inheritance go
back to work now 50 years old.
But the breeding programs themselves have waxed and waned as support varied and objectives
changed. There are six existing breeding programs outside the center of origin of Musa, and only
two minor efforts in India and none at all in the Southeast Asia center of diversity, where wild
bananas still exist and clones are still being domesticated. Research on coevolved pathosystems is
neglected, and no feedback from the natural systems into breeding exists.
Many chapters in this book reveal the enormity of molecular research applied to bananas and
the attempts to apply molecular techniques to banana breeding itself. Molecular-assisted selection
is still in its infancy and much dynamism continues in the molecular field.
Breeding techniques and breeding philosophies are expertly detailed in a chapter by Tenkouano
et al. It is clear that much has been learned to direct the future of breeding. Excellent bibliographies
in many chapters provide a valuable documentation of the diverse and enormous scattered research
activity on bananas of the last 50 years.
Breeding bananas started with the simple objective of a Fusarium wilt-resistant ‘Gros Michel.’
Breeding objectives changed and proliferated as new programs started and local farmers’ needs
were addressed. Objectives are now very diverse and complex, and they differ in different regions.
Much has been learned of banana evolution through molecular science. Yet breeders still use only a
very limited pool of parents compared with the great natural diversity existing. It is clear that much
research is still needed to assess and to reduce reproductive barriers.
A perusal of these chapters with the literature and an examination of the experience on which
they are based reveal a wealth of knowledge and views not readily available elsewhere. It is an excel-
lent new resource on bananas and banana breeding.
Ivan W. Buddenhagen
xi
Preface
This book comprises a collection of chapters written by experts in banana research. Banana is one
of the most important agricultural crops, providing food, income, and employment for millions of
people, especially in the tropics. The crop is threatened by various diseases and pests that are being
compounded by environmental change. Breeding resistant cultivars appears to be the only sustain-
able solution. The crop has been largely neglected and there are a few isolated breeding programs
in the world. The last major book covering a diverse range of topics in bananas and plantains was
written over 10 years ago. Since then, a large body of new information on banana has emerged, and
this is reflected in the number of publications in various research areas. We believe that there is a
need for updated information in banana breeding and new responses to old challenges facing the
crop. The purpose of this book is to portray our personal perspectives on the challenges facing the
crop. To enable us to cover a wider range of topics, we have enlisted the ideas of leading experts,
including agronomists, biologists, biotechnologists, breeders, crop improvement and integrated pest
management (IPM) specialists, plant pathologists, and taxonomists. In this book our aim is to con-
centrate the current information and provide an accessible source of information to those interested
in banana research, especially the development of new disease- and pest-resistant cultivars. This
book provides basic as well as advanced information for those interested in learning more about
banana as well for those pursuing further research in the crop.
Chapter 1, written by Deborah Karamura, Eldad Karamura, and Guy Blomme, individuals with
vast experience in bananas and plantains, provides a detailed botanical description of the plant. In
addition to describing the aerial shoot corm and root systems, they also address the role of morphol-
ogy in classifying banana and the confounding effects of mutations and genotype x environment
(GxE) interactions. They emphasize the importance of distinguishing cultivars, even those pro-
duced by breeding programs, with regards to breeders’ rights and the fact that traders and buyers
can select the cultivar of their choice.
In Chapter 2, Uma Subbaraya, Marimuthu S. Saraswathi, and Michael Pillay draw on their
personal experiences to present a comprehensive treatment of the evolution, diversification, and
molecular genetic relationships in bananas and plantains. The early recordings of banana in India
and indigenous knowledge of the uses of banana is a unique aspect of this chapter. The authors also
indicate the various research needs in banana with regards to molecular taxonomy, especially of
unique germplasm available in India.
Markku Häkkinen, a highly respected field botanist, and Richard Wallace provide information
in Chapter 3 on some of the new banana germplasm that have been recently identified. They report
on members of the five sections and their potential usefulness for conventional breeding. They con-
clude by stating that “The incorporation of genetic traits (disease and pest resistance, drought and
cold tolerance, and so forth) from these sections will play an important role in the development of
new, improved hybrid bananas for use by future generations.”
In Chapter 4, Michael Pillay and Abdou Tenkouano provide a comprehensive treatise of the
genomes in Musa, the part played by molecular cytogenetics in identifying the genomes, and the
role of genomes in Musa classification. They formulate from their personal and practical expe-
riences the opinion that conventional and molecular cytogenetics have played and will continue
to play a vital role in breeding of banana. Various biochemical markers to identify the different
genomes are discussed. The importance of flow cytometry, especially in ploidy identification, is
highlighted. Recent information on cytogenetical aspects of fertility is addressed briefly.
The genetics of important traits are discussed in Chapter 5 by Eli Khayat and Rodomiro Ortiz,
two individuals with vast experience and knowledge in banana genetics. The chapter presents new
xiii
xiv Preface
perspectives on the genetics of plant architecture, fruit parthenocarpy, fruit ripening and senes-
cence, nematode resistance, and resistance to black leaf streak disease.
In Chapter 6, Guy Blomme, Simon Eden-Green, Mohammed Mustaffa, Bartholemew Nwauzoma,
and Raman Thangavelu use their firsthand field experiences to provide an extensive review of the
Sigatoka diseases, Fusarium wilt, Xanthomonas wilt, and viral diseases of banana. This is perhaps
the first comprehensive treatise of Xanthomonas wilt of banana.
Thomas Dubois and Daniel Coyne present an excellent overview of integrated pest management
of banana in Chapter 7. In addition to the major pests—nematodes and the banana weevil—that
generally receive the most attention, they outline a wide range of pests associated with banana culti-
vation. They define integrated pest management (IPM) and present an important assessment on how
IPM principles can be applied to various banana cultivation systems.
Chapter 8 is a comprehensive treatment of the reproductive biology of banana by Jeanie Fortescue
and David Turner. The authors’ probing writing style and personal experiences make this chapter
one of the best treatments written on this topic. The floral biology, breeding systems, pollen and seed
production, and reproductive systems are fully discussed. This is excellent reading for researchers
wishing to start banana breeding programs.
Chapter 9 by Abdou Tenkouano, Michael Pillay, and Rodomiro Ortiz highlights the experience
of these banana breeders with their work in Africa and presents some of their own research find-
ings. A short history of banana breeding is followed by the main reasons for breeding in the crop.
The chapter outlines the objectives and difficulties of banana breeding and the progress made in this
field. Future breeding goals are elaborated.
Shri Mohan Jain, Bradley Till, Prasnna Suprasanna, and Nicolas Roux, specialists in mutation
research, discuss the value of mutations in producing new banana cultivars in Chapter 10. The
chapter is ideal for anyone interested in using mutations in banana since it addresses the best
methods for inducing mutations, the types of materials to use, and the steps to follow after muta-
tion induction. The value of targeting-induced local lesions in genomes (TILLING) in banana is
also introduced.
The value of the many facets of biotechnology in Musa improvement is addressed by Leena
Tripathi in Chapter 11. The application of tissue culture in Musa research is discussed. The role of
genomics and transgenic technology for Musa genetic improvement is highlighted, with examples
of genes that will be useful for developing transgenic banana. The challenges facing researchers in
the development of transgenics, especially in less-developed countries, are elaborated.
In Chapter 12, Rodomiro Ortiz and Abdou Tenkouano give an overview of the progress made
in research related to GxE interactions in banana. The components of phenotypic stability for some
traits are discussed. The chapter reviews how to manage GxE to have efficient selection schemes
and multilocation testing before release of improved cultivars. Broad-sense heritability (H2) and
repeatability (R) estimates for growth, bunch, and fruit traits in triploid Musa germplasm are pro-
vided. The authors discuss new ways of using GxE information, inclusive of market-related infor-
mation, to develop end-use approaches to breeding and to target new cultivars to areas where they
are most likely to add value.
In Chapter 13, Edson P. Amorim, Sebastião de Oliveira e Silva, Vanusia B. de Oliveira Amorim,
and Michael Pillay address the nutritional value of banana. Breeding objectives for quality improve-
ment are discussed. The role of biofortification and breeding strategies for developing biofortified
cultivars with improved nutritional quality is highlighted.
Cherukatu K. Narayana and Michael Pillay list some of the various postharvest products obtained
from banana in Chapter 14. The need for new products from banana to reduce large postharvest
losses is discussed. The authors conclude by stating that bananas represent a great potential raw
material for food and nonfood processing industries.
Chapter 15 by Michael Pillay, Christopher A. Cullis, David Talengera, and Leena Tripathi reviews
various ways of propagating banana. Embryo culture of hybrid seeds from breeding programs is dis-
cussed. The role of micropropagation is reviewed. Low-cost techniques to rapidly multiply banana
Preface xv
seedlings are reviewed. Since micropropagation is usually associated with somaclonal variation,
molecular methods to detect somaclonal variants are outlined.
The success of plant breeding programs is measured by the extent to which the breeding products
are adopted and used by the growers, profitably and durably. Beyond the genetic products, perhaps
the more challenging task for the breeders is to understand and help put in place the complex bat-
tery of institutional and transactional measures that create a conducive delivery framework. These
issues are discussed by Abdou Tenkouano, Michael Pillay, and Ousmane Coulibaly in Chapter 16.
The authors also elaborate on the asymmetric nature of cultivar-based transactions in an evolving
market within domestic, regional, and international contexts.
Molecular breeding in banana has not been as progressive as that of other crops. In Chapter 17
Michael Pillay, Abdou Tenkouano, and Rodomiro Ortiz review some aspects of molecular breeding
in potato, cassava, and sugarcane. The breeding challenges, production constraints, and breeding
objectives of these crops are similar to those of banana. Greater progress has been made in the
search for molecular markers, mapping, and developing transgenics for a number of traits in potato,
cassava, and sugarcane. These are useful lessons for banana scientists.
Chapter 18, by Rodomiro Ortiz, Michael Pillay, and Abdou Tenkouano, is a brief chapter on
future prospects in Musa research.
We trust that those interested in banana and plantain will find the information in this book use-
ful and stimulating. This book is intended for students, teachers, and banana breeders. We hope
that academics throughout the world interested in developing tropical crops will also find use for
this book.
We thank all the authors for their valuable contributions and for sharing their knowledge to make
this book a success.
Editors
Michael Pillay (BSc, UHDE, BEd, BA, BSc [Hons] MS,
PhD) is a professor in the Department of Biosciences at
Vaal University of Technology, Vanderbijlpark, South
Africa. He completed a BSc degree in botany and zool-
ogy and a university higher diploma in education at the
University of Durban-Westville (now the University of
KwaZulu–Natal), a BEd and BA from the University of
South Africa (UNISA), a BSc (Hons) at the University of
Durban-Westville, an MS (agronomy) at Louisiana State
University, and a PhD at Virginia Polytechnic Institute and
State University.
Dr. Pillay is the author and coauthor of many articles and
book chapters. He served as an editor for the American Journal
of Agronomy and is on the editorial board of the Journal of Crop Improvement. Dr. Pillay’s research
interests are biotechnology, breeding, molecular genetics, genetics, plant sciences, plant systemat-
ics, cytogenetics, tissue culture, and germplasm conservation of crop plants. Dr. Pillay started his
career as an educator (1974–1984) before obtaining a scholarship to Louisiana State University, in
the United States. After completing three postdoctoral fellowships in the United States, he joined
the International Institute of Tropical Agriculture (IITA) in Nigeria as an associate scientist to work
on cytogenetics and molecular biology of bananas and plantains. He was then transferred to Uganda
as a scientist and banana breeder/molecular biologist.
He joined Vaal University of Technology in 2007 as an associate professor, was employed at
UNISA as professor in 2009, and rejoined Vaal University of Technology in 2010, where his main
interests are research and teaching.
Abdou Tenkouano (BSc, MSc, PhD) is the director of the

Regional Center for Africa, AVRDC—The World Vegetable
Center in Arusha, Tanzania, and formerly a banana and
plantain breeder at the International Institute of Tropical
Agriculture. Dr. Tenkouano obtained his MSc in plant breed-
ing and his PhD in genetics from the Department of Soil and
Crop Sciences, Texas A&M University, College Station, Texas,
and his BSc in agronomy and ingénieur agronome in rural
development (agricultural planning and resource management)
from the University of Ouagadougou, Burkina Faso.
Dr. Tenkouano is the author of numerous articles and book
chapters in a wide range of topics on banana and plantain.
His research interests lie in genetics, breeding, and improve-
ment of crop plants, with a particular focus on improving
nutritional quality and host-plant reaction to biotic and abiotic
stress factors.
Dr. Tenkouano worked as a researcher at the Institut de
l’Environnement et de Recherches Agricoles (INERA) and as
a lecturer at the University of Ouagadougou in Burkina Faso
before joining the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT, Mali)
xvii
xviii Editors
as coordinator, West and Central Africa Sorghum Research Network. He then started employment
at the International Institute of Tropical Agriculture (IITA, Nigeria) where he was the research team
coordinator, Plantain and Banana Research, Genetic Improvement Group, and thereafter became
the program coordinator, Plantain and Banana Research.
He moved to Cameroon where he assumed the role of program leader, Diversification of
Agricultural Systems in Humid and Sub-Humid Agro-Ecologies for IITA. He was elected coun-
cilor, Research for Development Council (RDC), at IITA. Dr. Tenkouano has been involved in the
supervision of many MSc and PhD students.
Contributors
Edson Perito Amorim, PhD Markku Anton Häkkinen, PhD
Embrapa Cassava and Tropical Fruits Finnish Museum of Natural History
Cruz das Almas, Bahia, Brazil Botanic Garden, University of Helsinki
Jyrängöntie, Finland
Vanusia Batista de Oliveira Amorim, PhD
Embrapa Cassava and Tropical Fruits Shri Mohan Jain, PhD
Cruz das Almas, Bahia, Brazil Department of Agricultural Sciences
University of Helsinki
Guy Blomme, PhD Helsinki, Finland
Bioversity International
Kampala, Uganda
Deborah Karamura, PhD
Ousmane Coulibaly, PhD Bioversity International
International Institute of Tropical Agriculture Kampala, Uganda
(IITA)
Biological Control Center for Africa Eldad Karamura, PhD
Cotonou, Benin Bioversity International
Kampala, Uganda
Daniel Leigh Coyne, PhD
International Institute of Tropical Agriculture Eli Khayat, PhD
(IITA) Department of R&D
Dar es Salaam, Tanzania Rahan Meristem (1998) Ltd.
Rosh Hanikra, Israel
Christopher Ashley Cullis, PhD
Department of Biology
Mohammed Mustaffa, PhD
Case Western Reserve University
National Research Centre for Banana
Cleveland, Ohio, USA
(ICAR)
Tamil Nadu, India
Thomas Dubois, PhD
International Institute of Tropical Agriculture
(IITA) Cherukatu Kalathil Narayana, PhD
Kampala, Uganda Division of Post Harvest Technology
Indian Institute of Horticultural Research
Simon Eden-Green, PhD Karnataka, India
EG Consulting
Larkfield, Kent, UK Bartholomew Nwauzoma, PhD
Department of Applied & Environmental
Jeanie Anne Fortescue, PhD Biology
School of Plant Biology Faculty of Science
Faculty of Natural and Agricultural Sciences Rivers State University of Science &
University of Western Australia Technology
Crawley, Australia Port Harcourt, Nigeria
xix
xx Contributors
Rodomiro Ortiz, PhD Abdou Tenkouano, PhD

Sociedad Anomia Regional Center for Africa, AVRDC—The
Lima, Peru World Vegetable Center
Duluti, Arusha, Tanzania
Michael Pillay, PhD
Biosciences Department Raman Thangavelu, PhD, PDF
Vaal University of Technology National Research Centre for Banana
Vanderbijlpark, South Africa Tamil Nadu, India
Nicolas Roux, PhD Bradley Till, PhD

Commodities for Livelihoods Programme Plant Breeding Unit
Bioversity International FAO/IAEA Agricultural and Biotechnology
Montpellier, France Laboratory
International Atomic Energy Agency
Sebastião de Oliveira e Silva, PhD Vienna, Austria
Embrapa Cassava and Tropical Fruits
Cruz das Almas, Bahia, Brazil Leena Tripathi, PhD
Marimuthu S. Saraswathi, PhD (IITA)
National Research Center for Banana Kampala, Uganda
(ICAR)
Tamil Nadu, India David William Turner, PhD
School of Plant Biology
Uma Subbaraya, PhD Faculty of Natural and Agricultural Sciences
National Research Center for Banana University of Western Australia
(ICAR) Crawley, Australia
Tamil Nadu, India
Richard Henry Wallace, PhD
Prasnna Suprasanna, PhD Department of Chemistry and Physics
Plant Cell Culture Technology Section Armstrong Atlantic State University
Nuclear Agriculture and Biotechnology Savannah, Georgia, USA
Division
Bhabha Atomic Research Centre
Mumbai, India
David Talengera, MSc

(IITA)
Kampala, Uganda
1 General Plant
Morphology of Musa
Deborah Karamura, Eldad Karamura, and Guy Blomme
Contents
1.1 Introduction...............................................................................................................................1
1.2 Botanical Description................................................................................................................2
1.2.1 The Aerial Shoot............................................................................................................2
1.2.2 The Pseudostem.............................................................................................................2
1.2.3 The Suckers...................................................................................................................3
1.2.4 The Leaves.....................................................................................................................3
1.2.5 The Inflorescence...........................................................................................................5
1.2.6 Bunch Morphology........................................................................................................6
1.2.7 Fruit Morphology..........................................................................................................8
1.3 The Underground System..........................................................................................................9
1.3.1 The Corm.......................................................................................................................9
1.3.2 Root Systems.................................................................................................................9
1.3.2.1 The Considerable Size of the Musa Root System...........................................9
1.3.2.2 The Effect of Soil and Climate on Root Systems......................................... 10
1.3.2.3 Root Branching: The Lateral Roots.............................................................. 11
1.3.2.4 Allocation of Dry Matter to the Shoot and Root System.............................. 11
1.3.2.5 Genetic Variability in Root Growth and Relationships with Shoot
Growth: Refining the Ideotype..................................................................... 13
1.3.2.6 Alternative Methods for Root System Assessment....................................... 14
1.4 Value of Morphological Variation........................................................................................... 15
1.4.1 Introduction................................................................................................................. 15
1.4.2 Sources of Morphological Variation in Cultivated Musa............................................ 15
1.4.2.1 Mutations...................................................................................................... 15
1.4.2.2 Selection........................................................................................................ 16
1.5 Ecological Adaptation............................................................................................................. 16
References......................................................................................................................................... 17
1.1 Introduction
In this chapter, Musa stands for the genus and musa for the crop that includes both bananas and plan-
tains. This chapter describes the morphology of the musa plant and discusses the potential benefits
of morphological diversity. The morphology of Musa has been extensively described by Cheesman
(1947, 1948a, 1948b, 1950), Simmonds and Shepherd (1955), Champion (1961), De Langhe (1964),
Simmonds (1962, 1966), Purseglove (1972), Stover and Simmonds (1987), and Karamura and
Karamura (1995). These authors focused on the aerial parts of the plant, but an understanding of the
root morphology is also critical, given its role in plant nutrition and anchorage. Root architecture
1
2 Banana Breeding: Progress and Challenges
Pseudostem
True stem
(flower stem)
Female
flowers/fruits
Male
flowers/bud
Maiden sucker
Mother plant
Corm
Sword sucker Daughter sucker

(shooting)
Figure 1.1 The morphology of the Musa plant: a mat or stool.
has been studied by Champion (1961, 1963), Swennen et al. (1984), Stover and Simmonds (1987),
Price (1995), Blomme (2000), and Blomme et al. (2003).
The musa plants constitute some of the largest herbaceous perennial plants that range from 2–9
m in height in cultivated plants and 10–15 m in some wild species. Basically, the musa plant consists
of a subterranean stem or corm, an aerial pseudostem, the leaves, and the inflorescence (Figure 1.1).
The corm is the true stem to which are attached developing suckers (that perpetuate the life cycle of
the plant) and roots; the corm supports the pseudostem, the leaves, and the inflorescence that bears
the flowers and subsequently the fruit. The plants are monocarpic, that is, the shoots flower only
once and die after fruiting. Together, the corm and attached structures form a stool or a mat.
1.2 Botanical Description

1.2.1 The Aerial Shoot
The aerial shoot is the above-ground part of the musa plant, consisting of the pseudostem, leaves,
and the inflorescence. The aerial shoot has the greatest utility and value.
1.2.2 The Pseudostem

The pseudostem is made of large overlapping leaf sheaths that are tightly rolled on each other to
form a firm cylindrical structure (Purseglove, 1972; Stover and Simmonds, 1987). The aerial stem
entirely depends on the leaf sheaths for mechanical support and it essentially provides vascular
connection between the leaves, the roots, and the fruit (Stover and Simmonds, 1987). Considerable
variation in height, color, and disposition of the pseudostem occurs and is used to distinguish
banana cultivars. Thus, the pseudostems of Musa AB (e.g., ‘Kisubi’), the AAB (plantains, ‘Silk,’
‘Mysore,’ and ‘Sukali Ndizi’), and the ABB (‘Bluggoes’ and ‘Pisang Awak’) groups are predomi-
nantly yellow-green with little or no pink pigmentation on the undersheaths; the AA (‘Sucrier’) and
the AAA (‘Gros Michel’) groups are generally multicolored (Figure 1.2). The pseudostem of the
General Plant Morphology of Musa 3
AAA-EA AAB Plantain ABB Bluggoe
Figure 1.2 Variation in color of the underlying pseudostem in the different Musa groups.
AA group tends to be rich chocolate-brown at the junction of the pseudostem–petiole region while
the AAA has dull green to green-brown pink-flushed uppersheaths, and a rich mix of green, pink,
and rust-brown background with black mottling along the pseudostem length. The East African
Highland bananas (AAA-EA), also considered as the Lujugira-Mutika subgroup, display a very
variable pseudostem color, the intensity of which varies with environmental conditions. Thus, in the
Lujugira-Mutika subgroup, the pseudostem and leaf petioles tend to get darker as altitude increases.
Pseudostem height also varies across cultivars and agro-ecological conditions, for example, from 4
m on the plains to 8 m in sheltered valleys for the AAA cultivar ‘Gros Michel’ (Purseglove, 1972).
Likewise, Cavendish cultivars may be relatively tall in lowlands where conditions are ideal but
shorter at higher altitudes. In the Lujugira-Mutika, the cultivar ‘Nakyetengu’ usually grows up to 2
m and is early maturing, but it can reach 3 m under some conditions and takes then longer to shoot.
In general short-stemmed cultivars are susceptible to drought while the tall ones are tolerant (Stover
and Simmonds, 1987). Most cultivars have a straight and erect pseudostem, but some cultivars of
the Lujugira-Mutika subgroup, such as ‘Mukazi-aranda,’ distinctly display a “creeping” habit from
which it derives its name (meaning “crawling lady” in Luganda dialect).
1.2.3 The Suckers

The suckers are lateral shoots that emerge from buds located opposite the base of leaves on the corm.
Only 3–4 buds develop into shoots or suckers. Continuous emergence of new suckers perpetuates
the corm’s life, giving musa its perennial status. There are two types of suckers: the broad-leafed
suckers from superficially placed buds (usually due to damaged corms) and the sword-leafed suck-
ers from deep-lying buds. Thus, unless they are removed for propagation purposes, the aerial part
of a plant consists of a large number of shoots forming a clump and the rate of production of shoots
varies from a few to several tens, depending on the clone. The growth of suckers is also greatly
influenced by the stage of maturity reached by the parent plant, and field operations like pruning or
manure/mulch application may also affect sucker production.
1.2.4 The Leaves

The corm also consists of the apical meristem from which the leaves and the flowers are initiated.
At emergence the leaf is more or less vertical, gradually becoming horizontal and drooping as it
ages. The lamina develops as a rolled cylinder (hence the name cigar leaf or heart leaf) during its
passage through the pseudostem, with the right half rolled upon itself and the left half rolled over
the right and the midrib. Purseglove (1972) has attributed the unfurling process of the leaf and other
diurnal lamina movements to growth and turgor changes of specific motor cells of the pulvinar
band exerting pressure against rigid structures. The pulvinar bands are found where the two leaf
blades join the midrib and appear as two pale lines. The leaf blade gradually expands into a large,
oblong lamina with a pronounced supporting midrib and well-marked, pinnately arranged paral-
lel veins. The whole process of leaf blade formation is completed when the leaf sheaths narrow on
both sides to form the petiole (leaf stalk), which is rounded beneath and channeled above, retaining
the crescent-shaped section of the sheath and enlarging at the tip to form the blade. The petiole is
variously colored in many banana cultivars. In the Musa acuminata cultivars, the petiole coloration
has a predominantly reddish to purplish background, with traces of green. Conversely, in cultivars
of M. balbisiana the petioles are largely green and in many cases waxy. Hybrids have mixtures of
green and purplish colors. However, due to somatic mutations, coloration and waxiness may vary
even within the same stool.
Leaf disposition on the plant enables them to receive maximum light for photosynthesis. The
blade is often torn by the wind and hangs in ribbons from the midrib. New leaves are continually
forced up through the center of the pseudostem and expand at the top, where the leaf blades form
a handsome crown or canopy that enables the giant herb to outcompete other herbaceous plants for
light. Banana leaves are light green in color, smooth and glossy, and attain a very large size, often
being used as a temporary shade for other crops. The final leaf to emerge through the pseudostem
is much smaller than the rest and curves to protect the developing inflorescence from direct insula-
tion. In some Lujugira-Mutika cultivars such as ‘Nsowe’ or ‘Kataribwambuzi’ (translated as “that
which cannot be eaten by goats”), the sword suckers continue to produce scale leaves until the plant
is taller than 2 meters. This enables the cultivar to grow without being eaten by herbivores that may
devour the foliage of cultivars that unfurl their leaves at lower heights. The lamina is thickest near
the midrib and thinnest at the edges. The veins of the lamina are parallel as they leave the midrib
but become S-shaped as they approach the margins. Stover and Simmonds (1987), quoting Skutch
(1930), have indicated that there are about 17,000 veins near the midrib in one lamina half of a
large leaf. This venation offers little or no resistance to transverse tearing of the lamina. When this
happens, commissural bundles and some of the ground tissue may be destroyed but the vascular
connection between the midrib and margin remains intact.
Splitting of the lamina along the veins is a normal occurrence, which may increase the surface
area for cooling the plant while reducing transpiration due to suberization of torn surfaces (Taylor,
1969). This results in higher photosynthetic rates in torn leaves because of favorable temperatures
and carbon dioxide exchange (Taylor and Sexton, 1972). However, excessive tearing of the lamina
may reduce photosynthetic rates and cause yield losses in highland agro-ecologies that are prone to
hailstorms (Karamura and Karamura, 1995). Excessive tearing can be prevented with wind breaks,
as is common in Uganda where Ficus trees are prominent on banana farms (Davis, 1995).
Purseglove (1972) observed that the number of functioning leaves remains approximately constant
at 10–15, with a new leaf emerging every 7–10 days. Leaf emergence is influenced by cultivar and
ecological conditions, with a pronounced seasonality effect whereby more leaves are produced in
wet than dry seasons. Likewise, leaf retention is affected by prevailing soil fertility and soil moisture
levels. Air temperature, day length, plantation age, plant density, and plant stature are also known to
influence leaf emergence, notably in the Cavendish and Gros Michel subgroups (Allen et al., 1988).
The posture of the leaf varies with age, gradually shifting from a tightly rolled vertical cylinder at
emergence to an expanded horizontal posture before drooping as the petiole starts to collapse. The
lamina eventually senesces and dies, preceding the decay and shedding of the leaf sheath.
Much variability exists in the color, disposition, and waxiness of musa leaves. In general, dip-
loids tend to have more erect leaves, whereas triploids have broader leaves, more or less drooping,
carried on vigorous stems. In plantains and other interspecific hybrids, the leaves are yellow-green
(or display various shades of greenness). Conversely, within the Lujugira-Mutika subgroup, there
is a wide variability of colors, including the green but variegated leaves of ‘Nasuuna,’ the purplish
leaves of ‘Bitambi,’ the glossy leaves of ‘Namafura,’ and various shades of dirty green of the lamina
with or without the red midrib. In general, however, color variation is more pronounced in M.
acuminata, while M. balbisiana tends to be monochromatic green.
Musa plants have a large leaf area, ranging from 1.27 to 2.80 m2 in dessert bananas (Stover and
Simmonds, 1987; Stover, 1988) and 0.68 to 0.92 m2 in plantains (Anojulu, 1992). The total leaf area
of Cavendish cultivars at flowering is approximately 16.9 to 25 m2, providing a closed canopy that
protects the soil from the impact of rain and oxidative insolation (Stover and Simmonds, 1987).
Leaf pigmentation and posture may affect photosynthetic efficiency and transpiration rates as
described by Brun (1960, 1961a, 1961b, 1962, 1965) and Turner (1972), but there are surprisingly
not many recent studies relating leaf morphology to photosynthetic efficiency or transpiration of the
existing cultivars.
1.2.5 The Inflorescence

The apical meristem and flower initiation have been extensively discussed by Barker and Steward
(1962), Ram and Steward (1962), Fahn et al. (1963), and Stover and Simmonds (1987). The time
of flowering and fruit maturity are both dependent to a large extent on cultivar and environmental
conditions. The following events characterize flowering: elongation of the corm internodes; sup-
pression of leaf development, which is replaced by bract development; and, eventually, initiation
and development of the inflorescence. Floral initiation occurs with the apical meristem ceasing to
produce leaves (about 30–40 leaves in lowland tropical locations) and elongating through the cen-
ter of the pseudostem, until the inflorescence bud emerges. Inflorescence emergence is described
as shooting, at which stage the purplish green/greenish brown inflorescence appears vertical with
a pointed tip and a broader base, but it soon bends over due to its weight. The inflorescence is a
complex spike with a stout peduncle on which flowers are arranged in nodal clusters on transverse
cushions (crown), subtended by large spathe-like bracts that are nearly ovate and usually purple-red
in color (Purseglove, 1972). Each nodal cluster consists of two closely appressed rows of flowers,
one above the other, and enclosed in a large subtending bract. The bracts and their axillary groups
of flowers are arranged spirally around the axis and the bracts closely overlap each other, forming
a tight conical inflorescence. In the cultivar ‘Lwezinga’ (rolled), however, the spiral is continuous
and consequently the bracts form one long strip from the female to the male inflorescences. In other
cultivars the spirally arranged bracts overlap in such a way that all bracts and flowers within are
enclosed tightly and protected in a large bulbous structure. Each bract is a large red/purplish pointed
structure that becomes completely reflexed as the flowers develop and that falls from the axis,
leaving a large scar when the fruits start to mature. Some clones—for example, Musa (AAA-EA)
‘Nakawere’—have yellowish-green bracts with streaks of brown, which makes the emerging inflo-
rescence appear as yellow instead of purple. The flowers are bisexual but mostly unisexual in func-
tion and there are three types of flowers. The lower bracts of the inflorescence axis enclose the
female flowers, the middle few bracts may enclose neutral or hermaphrodite flowers, whilst at the
tip of the inflorescence are the male flowers. The female flower consists of an elongated inferior,
trilocular ovary of three fused carpels, on top of which six tepals (five united and one free) are
implanted, surrounding a thick style and five or six fleshy and nonfunctional stamens (staminodes).
The ovary of female flowers constitutes two thirds of the length of the whole flower, and these are
the flowers that eventually develop into individual fruits or fingers of the bunch. In the male flow-
ers, the ovary constitutes one third of the length of the flower, usually deciduous, and contains one
slender style, five stamens topped by long anthers that do not, however, produce functional pollen.
Neutral or hermaphrodite flowers consist of an ovary that does not develop into fruits but forms
short useless fingers. The ovary is about half the length of the flower. The stamens of neutral flowers
do not produce pollen.
1.2.6 Bunch Morphology

Continued elongation of the main stalk of the inflorescence causes the bunch to hang over, the
bracts open and fall, disclosing the female flower clusters or hands. The female flowers undergo
further development without being pollinated or fertilized. Growth of the inflorescence stalk is
rapid and the hands become separated by several centimeters of stalk. After the emission of a
number of hands of female flowers (1–12 depending on the conditions and cultivar), the bud dif-
ferentiates hands of hermaphrodite and then male flowers. The bracts and the male flowers open
and fall, so that eventually a considerable length of stalk separates the male bud from the bunch
proper. Further rapid growth causes the individual fruits to fill, and their increased weight bends
the main stalk so that the bunch hangs down vertically, although some bananas have upright
bunches. Although the physiology, anatomy, and development of musa cultivars are similar, the
morphology tends to be variable, particularly for the Lujugira-Mutika subgroup. Karamura (1999)
divided this subgroup of bananas into clone sets based on bunch and rachis characteristics (ori-
entation and compactness), persistence of neutral flowers and bracts, and shape and apex of male
bud. The lax pendulous banana bunched clone set is Musakala, whereas the most compacted and
subhorizontal bunched clone set is Nakabululu. ‘Nakitembe’ and ‘Nfuuka’ clone sets have com-
pact medium-sized oblique bunches but ‘Nakitembe’ has persistent floral parts on the rachis and
fruits as well as imbricated male buds (Figure 1.3).
This bunch/inflorescence orientation is common among the widely grown Cavendish and
Plantain subgroups. Within the diploids and other groups, including the highland AAA-EA, two
more inflorescence orientations can be recognized. Firstly there are those that are carried obliquely
(to the pseudostem). The cultivars ‘Nfuuka’ and ‘Mbwazirume’ are examples of obliquely oriented
inflorescences (Figure 1.3). In the “small-bunched” cultivars such as ‘Nabakululu,’ ‘Endirira,’ and
‘Katabunyonyi,’ the orientation is clearly subhorizontal (Figure 1.3). Purseglove (1972) has implied
that geotropism is a factor in determining inflorescence orientation, conferring desirable traits for
the export trade as manifested by the AAA Cavendish and Gros Michel cultivars. The weight of the
inflorescence too appears to play an important role. Among the Lujugira-Mutika, it can be general-
ized that the heaviest-bunch cultivars such as ‘Rumenyamagali’ tend to have pendulous orientation,
while the small-bunched cultivars tend to have subhorizontal inflorescences. The medium-sized
bunch cultivars are carried obliquely on the pseudostem. Another factor that appears to influence
inflorescence orientation relates to the presence or absence of the male bud, the big purple terminal
protuberance on the bunch. It has been observed that cutting off the male bud at shooting makes the
inflorescence peduncle fail to elongate and make the U-downward turn, so that a normally pendu-
lous inflorescence ends up subhorizontal. Among the Lujugira-Mutika, for example, ‘Endiriira,’ the
only AAA cultivar without a male bud, always carries its inflorescence subhorizontally. It would
appear that bananas have a “male-bud factor” that is responsible for inflorescence orientation. In
breeding programs where bunch weight is a consideration, attention should be given to the male
bud factor’s effects on the fruit characters. Cultivars without male buds may be important sources
of traits in breeding programs aimed at controlling Xanthomonas wilt disease of bananas (Biruma
et al., 2007).
The male bud itself varies in many aspects, which include bract arrangement, color, shape and its
apex, and presence and degree of waxiness. In the majority of cultivars, male bud bracts are purple
and purplish green considering the external side of the bracts. In cultivars ‘Nakawere’ and ‘Tereza’
of the Lujugira-Mutika subgroup, however, the male bract is greenish-yellow with vertical brown
streaks. The majority of M. balbisiana clones have a continuous uniform crimson color in the inter-
nal surface of the bracts (Figure 1.4), whereas in the M. acuminata cultivars the crimson color fades
to yellow at the base of the bract (Figure 1.4). Male bracts in many cultivars reflex and roll back
after opening and eventually fall off while still fleshy. In some cultivars such as ‘Mbwazirume,’
‘Namaliga,’ and ‘Nakitembe,’ the bracts persist and form a “protective” cover over the persistent
neutral and male flowers (Figure 1.3). Upon falling, the bracts leave prominent scars. Within the
(a) (b)
(c) (d)
Figure 1.3 Bunch orientation in the Lujugira-Mutika subgroup. (a) Subhorizontal ‘Nakabululu,’ (b)
oblique, with bare rachis ‘Bitambi,’ (c) oblique, rachis with persistent floral parts ‘Mbwazirume,’ (d) pendu-
lous ‘Musakala.’
Lujugira-Mutika subgroup, male bud shape and apex is variable. In general the cultivars with short
and fat fingers, closely packed bunch, and oblique to subhorizontal bunch orientation have obtuse-
angled male bud apices. Conversely, the long-fingered, loosely packed, and pendulous bunch culti-
vars have acute male bud apices.
The compound tepal of the male flower is white to cream but with a flush of pink among the plan-
tains, ‘Silks,’ ‘Mysore,’ ‘Kamaramasenge,’ ‘Bluggoes,’ ‘Pisang Awak,’ and the ‘Red/Green Red.’
The lobes of the compound tepal as well as the stigma range from yellow to orange. The anthers are
pink among the Lujugira-Mutika bananas and the ‘Pisang Awak,’ yellow in the ‘Bluggoes,’ orange
in the Cavendish, and cream in most other groups. The ovule arrangement in the ovary does not
vary much except being either two or four rowed in the different groups. This ovule arrangement
can be clearly seen in the longitudinal sections of fruits.
AA, AAA ABB
AAB Pome AAB Plantain
Figure 1.4 Variation in the internal bract color: Varieties with a B genome have a homogeneous red color
towards the base of the bract.
1.2.7 Fruit Morphology
Both Purseglove (1972) and Stover and Simmonds (1987) provide extensive coverage of the flowers
and fruits of the musa plant, based largely on observations made on the Cavendish subgroup and
French plantains (AAB), with little reference given to the Lujugira-Mutika subgroup. Seedless fruits
develop parthenocarpically from female flowers. However, numerous aborted ovules, carried in an
axile placentation in the ovary, can be seen as small brown objects in the center of the fruit. The
fruit is elongated, curved, and more or less round in cross-section but with the triangular form of the
ovary still visible. At the tip of the fruit, the perianth, androecium, and the style become withered
but persist for a short time, separated from the fruit by a brown corky layer. The fruit is protected
by an epidermis and an underlying parenchyma layer in which the vascular bundles and a series of
latex tubes are found. Inside this “skin” lies the pulp, a tissue of large cells filled with starch that
is partially converted to sugars during the ripening process. As the fruit develops, it slowly curves
under a negative geotropic response.
There is variability in finger length, and consumers generally prefer long fingers to short ones
for all uses. This is true for dessert as well as matooke (green cooking) bananas. The longer fingers
are easily peeled during matooke preparation. Among the Lujugira-Mutika subgroup, the cultivars
with long fingers—‘Musakala,’ ‘Muvubo,’ and ‘Nakibizzi’ (also called ‘Mpologoma’)—are increas-
ingly becoming commercial cultivars in Uganda. Fruits are glabrous, less angular, sharply pointed
or bottle-necked or almost blunt at the apex, without floral relics. Fruit length varies from 5 cm
to more than 30 cm. Within the bunch, fruits vary in their arrangement, position, and number in
a hand, shape of fruit, apex, waxiness, and rows and shapes of ovule they contain. The fruits may
lack ovules like in ‘Kattabunyonyi’ (AAA-EA), or they may have two or four rows of ovules. There
is also variation in the color of fruit skin, pulp (ranging from white, cream, ivory, and beige-pink),
and absence or presence of stalk. Fruit characteristics are increasingly becoming important in the
banana chips industry as well as in the table-fruit markets.
1.3 The Underground System

The underground system of the musa plant consists of a subterranean stem or corm that bears the
root system and developing suckers.
1.3.1 The Corm

The corm is the true stem of the plant, with a cultivar-dependent diameter ranging from 20–25
cm for a typical mature Cavendish and about 15–18 cm for most African plantains. It has short
internodes that are completely covered by tightly appressed leaf scars. The longitudinal section of
the corm looks like an inverted cone; it is differentiated internally into a central parenchymatous
cylinder surrounded by a 1–3 cm thick cortex. The top of the cone is dome shaped, with the apical
meristem at the crest of the dome (Price, 1995). During the vegetative growth stage of the musa
plant, the apical meristem has the form of a flattened dome with little internal differentiation. At
the transition from the vegetative to the floral stage, a profound change occurs in the growing point:
What was more or less a quiescent mass of cells starts showing signs of intense and general mitotic
activity so that the meristematic area becomes convex and rises above the surrounding leaf bases.
The outermost layer of the central cylinder consists of a cambium-like meristematic tissue from
which roots arise.
1.3.2 Root Systems
The Musa root system is a complex structure that supports multiple plant functions. For example,
it ensures the optimal uptake of water and nutrients, provides anchorage to the plant, and produces
plant growth regulators (De Langhe et al., 1983; Swennen et al., 1984; Stover and Simmonds, 1987;
Price, 1995). Research on Musa roots started some 70 years ago (Skutch, 1932) predominantly on
export dessert bananas, such as Gros Michel (Moreau and Le Bourdelles, 1963) and Cavendish cul-
tivars (Beugnon and Champion, 1966; Lassoudière, 1978; Avilan et al., 1982).
Although extensive breeding efforts have been devoted to improve shoot traits of Musa, com-
paratively little has been done for roots, despite the interdependence of shoot growth and root devel-
opment. For example, nematodes reduce root growth, which often results in yield decline in Musa
(Swennen et al., 1988; Gowen and Quénéhervé, 1990). While the nematode pest has been considered
an important priority in breeding programs at the International Institute of Tropical Agriculture
(IITA) (IITA, 1997, 1998), the Centre Africain de Recherches sur Bananiers et Plantains (CARBAP)
(Fogain et al., 1996, 1998), and the Fundación Hondureñea de Investigación Agricola (FHIA) (Rowe,
1991; FHIA, 1998), no systematic effort has been devoted to developing root systems that are less
prone to nematode damage.
A comprehensive study of the root system of a wider gene pool is required to construct an ideo-
type target for the genetic improvement of plantains and bananas. Such studies were carried out at
the International Institute of Tropical Agriculture (IITA) and by Bioversity International in Nigeria
and Uganda, respectively. Experiments focused on elucidating relationships between root and shoot
traits, assessing variability in root system size, assessing the biophysical effects on root develop-
ment, and devising alternative methods for root evaluation (Blomme, 2000).
1.3.2.1 The Considerable Size of the Musa Root System

The Musa root system is adventitious. Cord roots arise, usually in groups of three or four, from a
common primordium (Skutch, 1932; Riopel and Steeves, 1964; Turner, 1970). They grow through
the cortex where cytolysis takes place to give space. Before the roots penetrate the soil, their
tips broaden abruptly (Skutch, 1932). They are 5–10 mm thick (Riopel and Steeves, 1964), ini-
tially white and fleshy before turning somewhat corky with age. The mature roots have prominent
lacunae in the cortex and have large vessels and phloem strands in the central portion of the
stele. Xylem elements are formed at a level in the root at which elongation has ceased (Riopel and
Steeves, 1964).
Champion and Olivier (1961) reported that, for the dessert banana ‘Poyo,’ the zone on the corm on
which roots emerge (i.e., root-bearing zone) is negatively geotropic. Swennen et al. (1988) reported
both a positive and a negative geotropic movement or widening of the root-bearing zone of sucker-
derived plants. However, they found that the center of activity of the root-bearing zone becomes
negatively geotropic with time. The number of cord roots varies considerably depending upon the
health status of the plant. A healthy corm can bear 200 to 400 primary cord roots with a total length
of 230 m (Summerville, 1944; Robin and Champion, 1962; Beugnon and Champion, 1966). Fawcett
(1913) noted that growth rates of the tips may reach 60 cm per month, which is in agreement with
later studies with ‘Poyo’ in Côte d’Ivoire, where growth rates of 2–3.5 cm a day were recorded
(Lassoudière, 1978). Various factors, such as soil properties, climatic conditions, and pest and dis-
eases, influence the root growth rate (Lassoudière, 1971).
Roots generally spread over 2–3 m and may extend up to 5 m from the plant, but most of the
root system occurs within 60 cm of the stem (Avilán et al., 1982; Gousseland, 1983). Root distribu-
tion down the soil profile is strongly influenced by soil type (Irizarry et al., 1981) and drainage.
Compact soils, impermeable soil layers, high clay content, and saturated soil conditions prevent or
reduce root growth (Beugnon and Champion, 1966; Champion and Sioussaram, 1970; Godefroy,
1969; Lassoudière, 1971). Root systems are confined mostly to the upper 40 cm of soil because of
unfavorable subsoil conditions. Araya et al. (1998) found that 65% of the total root weight was in
the upper 30 cm of soil for the clone ‘Valery’ (AAA) growing on a sandy clay loam in Costa Rica.
The same authors found not less than 79% and 88% of the roots in the first 45 and 60 cm of the soil
profile, respectively. Besides the laterally spreading roots, there are a limited number of roots that
grow vertically (Simmonds, 1966; Summerville, 1939).
Cord roots emerge in flushes for sucker-derived plants. Thus, Swennen et al. (1988) reported
that, for the False Horn plantain ‘Agbagba,’ 40 cord roots were formed during the first 3 weeks after
planting, from preformed roots that were present in the cortex of the sucker. The number of primary
roots remained unchanged for the next 4 weeks, at which time another flush of root emergence
was observed (Swennen et al., 1988). Similar emergence flushes were reported by Beugnon and
Champion (1966) for the ‘Poyo’ dessert banana and Lavigne (1987) for a dessert banana grown in a
rhizotron. New roots are formed continuously until flowering occurs (Champion and Olivier, 1961;
Beugnon and Champion, 1966; Turner, 1970; Lavigne, 1987). However, roots may remain alive and
functional beyond fruit maturity. For example, the roots of a harvested mother corm were still alive
(and presumably functioning) during growth and at the harvest of the daughter stem (Walmsey and
Twyford, 1968; Lassoudière, 1980).
The upper surface of corms in aging plantain fields can be seen above soil level (Stover, 1972;
Swennen et al., 1988; Swennen, 1990), a phenomenon called “high mat,” and causing newly formed
roots to only penetrate the topsoil or die off before reaching the soil surface (Moreau and Le
Bourdelles, 1963). The plants become weak and tip over easily because they are no longer firmly
based in the soil (Swennen, 1990). Earthing up (adding soil around the plant) only slightly enhances
root growth and plant vigor. However, mulch protects the roots (which would otherwise dry out) and
improves the ramification and stability of the plants (Swennen, 1990).
1.3.2.2 The Effect of Soil and Climate on Root Systems

Studies on the effects of soil and climate on root growth conducted at IITA stations in Nigeria
revealed significant influences of substrate type and climate on root growth (Blomme, 2000).
Also, field-grown plants had a relatively larger root system (i.e., lower shoot–root ratio) under
low-nutrient conditions. These plants might have invested more dry matter in the root system in
order to explore a larger soil volume to produce a vigorous shoot. In addition, a reduction in soil
bulk density (during the first months after plowing and harrowing) significantly enhanced root and
shoot growth.
1.3.2.3 Root Branching: The Lateral Roots

The banana cord roots bear numerous lateral roots of much smaller diameter. These lateral roots,
bearing root hairs, are believed to be primarily responsible for water and mineral uptake by the plant.
Lateral root growth, however, is highly influenced by microenvironmental conditions (Blomme et
al., 2003) and genotype (Swennen et al., 1986). As a rule, lateral roots are generally abundant on cord
roots growing in organic layers in the topsoil and are absent from those growing deeper in the soil.
The first-order laterals usually emerge 12–15 cm from the tip of a cord root and may be as long
as 15 cm (Laville, 1964). Second-order and third-order laterals may also be present (Riopel, 1966).
When the apex of a primary cord root is damaged, due to biotic or abiotic factors, two or three first-
order laterals may develop into secondary cord roots (Lassoudière, 1977). Lavigne (1987) reported
that no secondary cord roots are formed if the damage occurs more than 20 cm from the apex. In
a study of 10 varieties, Swennen et al. (1986) found lateral roots to account for up to 98% of total
root length, with varietal differences for (1) total root length, (2) relative proportions of cord root
and first-order and second-order laterals, (3) length of first-order laterals, and (4) proportion of cord
roots covered by laterals. This work suggested the existence of genetic variability for these compo-
nents of the root architecture.
In Musa, first-order lateral roots are initiated basipetally in the apical region of the primary axes.
Lateral root primordia can be detected in the pericycle within 450–1200 µm of the apical meristem.
Their circumferential position in the pericycle is adjacent to protoxylem poles and opposed to pro-
tophloem (Riopel and Steeves, 1964; Esau, 1965; Charlton, 1982). Therefore, all laterals arising in asso-
ciation with a given protoxylem form a longitudinal series or rank, the number of ranks per root being
equivalent to that of protoxylem poles, which varies between 28 and 34 (Riopel and Steeves, 1964).
The distribution of lateral root primordia in the root tip has been analyzed on the basis of longitu-
dinal and circumferential distances between successive primordia and between particular primordia
and the apical meristem (Charlton, 1982). Lateral root primordia were rather regularly spaced in a
longitudinal pattern within each rank, each lateral arising at a rather constant proportion of the dis-
tance between the root tip and the previous lateral. They were also less likely to occur close to each
other than would be expected on a random basis. It was concluded that lateral roots are initiated in
a nonrandom dispersed pattern (Riopel, 1966).
Root hairs appear 4–6 cm from the apex of the cord roots and reach their mature size at 8–12 cm
distally from their place of origin. Their length may exceed 2 mm.
1.3.2.4 Allocation of Dry Matter to the Shoot and Root System

The percentage dry matter allocated to the shoot and the root portion of a Musa plant depends on
the developmental stage and planting material (Figure 1.5) (Blomme, 2000). For example, juvenile
in vitro–derived ‘Calcutta 4’ (AA) and ‘FHIA3’ (AAAB) plants allocated up to 45% of dry matter
to the root system just after field establishment. Root dry-matter content declined to about 10–15%
at flowering. In contrast, less than 20% of dry matter was allocated to the roots of juvenile sucker-
derived ‘Mbi Egome’ (AAB) plants, while the corm contained up to 70% of the plant dry matter
(Figure 1.5).
Percentage dry matter invested in the mat root system decreased gradually towards flower emer-
gence (Figure 1.5). For example, there was a clear increase in the shoot–root ratio with increasing
age for the in vitro-derived ‘FHIA3’ mat. The shoot–root ratio of juvenile sucker-derived ‘Mbi
Egome’ plants was relatively high, compared to the in vitro–derived plants and was caused by the
large corm size of the planting material. As for the in vitro–derived plants, the highest shoot–root
ratio was observed during the late vegetative phase.
100%
Calcutta
90%
80%
Percentage dry matter 70%
60%
50%
40%
30%
20%
10%
0%
4 8 12 16 20 24 28 32 36 40 44 48 52 56
WAP
FL
100% Mbi Egome
80%
Leaves
Percentage dry matter
Pseudostem
60% Corm
Roots
40%
20%
0%
4 8 12 16 20 24 28 32 36 40 44 48 52
WAP
FL
100% FHIA
90%
80%
Percentage dry matter
70% Leaves
60% Pseudostem
50% Corm
40% Roots
30%
20%
10%
0%
4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 76 78 82
WAP
FL
Figure 1.5 Dry matter partitioning between roots, corm, pseudostem (including bunch) and leaves in
unthinned mats of ‘Calcutta 4,’ ‘Mbi Egome,’ and ‘FHIA3’ from planting until harvest (FL: flower emer-
gence). (From Blomme, G., 2000, The interdependence of root and shoot development in banana (Musa spp.)
under field conditions and the influence of different biophysical factors on this relationship, PhD diss., K.U.
Leuven. Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen, Belgium.)
1.3.2.5 Genetic Variability in Root Growth and Relationships

with Shoot Growth: Refining the Ideotype
Strong positive correlations between shoot and root development of a mat are observed during the
vegetative and reproductive phases (Blomme, 2000). Thus, vigorous shoot growth in most plantains
and cooking bananas is associated with a large root system. In contrast, the semi-dwarf dessert
banana cultivar ‘Valery’ has a small root system. Despite the observed variability in root system
size between genotypes, shoot–root dry weight ratios are similar formats of a wide variety of geno-
types (Figure 1.6). These results indicate that breeding for an increased root system size, in addition
to being cumbersome, goes along with breeding for a larger aerial part. The study of segregating
populations should, however, be considered as different shoot–root relationships may be observed
at the within-population level.
Strong positive relationships between shoot and root traits are also observed for the lateral shoots
(i.e., suckers) (IITA, 1999; Blomme, 2000). Therefore, breeding for a regulated suckering (two to
three well-developed lateral shoots present during the reproductive phase) may prove to be more
beneficial for plant anchorage and productivity of the crop than breeding for a modified shoot–root
ratio or root system size of the main plant. Under regulated suckering, lateral shoots will have a vig-
orous shoot as well as root development. In addition, their large corm size will give stability to the
mat. For example, plantain hybrids with a regulated suckering will be less susceptible to toppling
compared to plantain landraces, which predominantly exhibit an inhibited suckering.
Studies carried out in Uganda (Kawanda Agricultural Research Institute and Makerere University
Agricultural Research Institute Kabanyolo) also found significant and positive relationships between
root, corm, and aerial growth traits of complete mats in East African Highland bananas (AAA-EA)
during the vegetative and early reproductive stage (Blomme, Ocan, et al., 2004; Bloome, Sebuwufu,
et al., 2004; Sebuwufu et al., 2004a, 2004b). Additionally, on-farm and on-station studies focusing
on the highland bananas ‘Mpologoma,’ ‘Lwadungu,’ ‘Nakitembe,’ ‘Mbwazirume,’ and ‘Kibuzi,’ the
14
12
Shoot-root dry weight ratio
10
0
Calcutta Valery Agbagba Mbi Obino Cardaba TMPx TMPx TMPx
4 Egome l’Ewai 548-9 1658-4 5511-2
Figure 1.6 Shoot–root ratio for the mat of nine Musa spp. genotypes at flower emergence of the plant crop
[‘Calcutta 4’ (AA); ‘Valery’ (AAA); ‘Agbagba,’ ‘Mbi Egome,’ and ‘Obino l’Ewai’ (AAB); ‘Cardaba’ (ABB);
‘TMPx 548-9,’ ‘TMPx 1658-4,’ and ‘TMPx 5511-2’ (tetraploid plantain hybrids)]. (From Blomme, G., 2000,
The interdependence of root and shoot development in banana (Musa spp.) under field conditions and the influ-
ence of different biophysical factors on this relationship, PhD diss., K.U. Leuven. Faculteit Landbouwkundige
en Toegepaste Biologische Wetenschappen, Belgium.)
dessert banana ‘Sukali ndizi’ (AAB), the plantain ‘Gonja’ (AAB), and the beer banana ‘Kayinja’
(ABB) revealed strong relationships between bunch weight, root, corm, and aerial growth traits.
Hence, poor root development will adversely affect shoot and leaf canopy development and as a
result reduce yield. Reciprocally, when leaves are affected by black leaf streak disease, the root
system is reduced.
1.3.2.6 Alternative Methods for Root System Assessment

Two man-days are needed to excavate, wash, and assess the root system of one mature Musa plant.
Also, attempts to correlate root growth of juvenile plants with that of adult plants were inconclu-
sive, indicating that an early assessment of root system growth of juvenile plants may not give suf-
ficient information about the root system size and development of mature plants (Blomme, 2000).
Therefore, methodologies for fast and nondestructive root system assessment were developed (IITA,
1999; Blomme, 2000).
Due to the strong relationships between shoot and root traits, regression models were obtained
to estimate root traits from easily measurable shoot traits (Table 1.1). More than 90% of the vari-
ability in root traits could be explained by the variability observed in shoot traits. However, as the
shoot–root ratio is dependent on the developmental stage of a plant and on environmental conditions
(Blomme, 2000), fine-tuning of these models will be needed when assessing plants grown under
different environments.
Core root samples taken around a plant can also provide adequate information on the variabil-
ity in mat root system size (Blomme, 2000). This method only takes about 5% of the time needed
to excavate and assess the root system of a mature plant. These alternative methods may provide
breeders and especially nematologists with adequate information on the roots of a Musa plant.
Table 1.1
Regression Models to Predict Root System Characteristics
Trait#
Trait # LA ^ PC HS PL R2
DR 0.001628*** 0.596934** – – 0.93
NR 0.001459*** 1.255633*** – – 0.93
LR 0.066704*** 23.476717** – – 0.94
AD – 0.093835*** – 0.681434*** 0.97
TL 0.099478*** – 14.69139*** – 0.92
TD 0.002066*** 0.426590 0.17142* – 0.93
Source: Blomme, G., The Interdependence of Root and Shoot Development in Banana (Musa spp.)
under Field Conditions and the Influence of Different Biophysical Factors on This
Relationship, PhD diss., K. U. Leuven, Faculteit Landbouwkundige en Toegepaste
Biologische Wetenschappen, Belgium, 2000.
Note: Plants are 20 weeks old, using aerial growth characteristics and ploidy level as independent
variables. #: LA: leaf area (cm2); PC: pseudostem circumference (cm); HS: height of the tallest
sucker (cm); PL: ploidy level; DR: root dry weight (g); NR: number of cord roots; LR: cord
root length (cm); AD: average basal diameter of the cord roots (mm); TL: cord root length of
the mat (mother plant and suckers) (cm); TD: root dry weight of the mat (g). *, **, ***
Significant at P < 0.05, 0.01, and 0.001, respectively. ^: independent variables.
1.4 Value of Morphological Variation

1.4.1 Introduction
Musa breeding depends on variations existing within accessions, cultivars, landraces, or wild taxa
related to crops. These genetic resources need to be investigated and evaluated to provide informa-
tion on variation if they are to be utilized efficiently (Pickersgill, 1988). Crop-improvement strat-
egies make use of information on variation with regard to yield, physiology, diseases, and pest
responses as well as morphology.
Morphological variation, which is a prerequisite for breeding, constitutes one of the oldest indi-
cators of diversity. Morphological variation is based on the scoring of numerous descriptors that
can easily be observed and accessed without any special technical skill and at low cost. However,
morphological variation is not a direct expression of the genome and can be influenced by environ-
mental effects so that it expresses more of the phenotype than the genotype (Perrier, 1993).
1.4.2 Sources of Morphological Variation in Cultivated Musa

Variation is the basis of speciation, when acted upon by selection. In nature, variation is a result of
basic evolutionary forces—mutation, recombination, genetic drift, and selection acting in differ-
ent ecologies over time. In clonally propagated populations, however, variation may occur through
mutations although other mechanisms can cause variation in banana (Nyine and Pillay, 2010).
Selection is largely mediated through environmental factors in different areas or habitats.
1.4.2.1 Mutations
Bananas and plantains are clonally propagated and it is generally known that clonal propagation
reduces variation within a population because relatively few genotypes are selected for conserva-
tion and use. However, clonal propagation can conserve heterozygotes as well as homozygotes, and
the only source of variation in these musa plants has been mutations. One example of a group of
bananas is the Lujugira-Mutika subgroup found in East Africa. These bananas were introduced in
the region by Arab traders from India, through Madagascar on the eastern coast during the 15th cen-
tury (Simmonds, 1962). Like all other cultivated bananas, they are vegetatively propagated and there
is hardly any variation in the suckers being produced by the mother plant except through mutations
and the chimerical nature of the plant, a condition that is not yet well understood. When a banana
plant is propagated, it is considered to be genetically the same but in reality, among the AAA-EA
cultivars, not all suckers produced on the same mat are always phenotypically identical (Karamura
et al., 2004). The meristematic tissues of the mother plant may be giving rise to highly variable suck-
ers. Plants composed of two or more genetically distinct tissues have been called chimera (Huxley,
1940; Crane and Laurence, 1956). Although chimerism has been observed in bananas for a long time,
not only is its frequency of occurrence not well understood (De Langhe, 1964; Simmonds, 1966) but
also not many in-depth studies have been attempted. However, the phenomenon is believed to be a
major source of variation within the crop, a process that generates genetic and phenotypic variation
that farmers have continued to exploit through selection breeding.
A number of morphological characters are prone to mutations, including plant stature, bunch
and fruit shapes, and astringency in fruit pulp. The implications of chimerism are not usually obvi-
ous, depending on which traits of the plant have been affected. In the Lujugira-Mutika subgroup,
the phenomenon varies, and its implication for genetic variability and conservation on farm has
not been elucidated. It is not known how chimeric traits are selected by a given community and
subsequently conserved on farm in banana-based farming systems (Karamura et al., 2008). Yet the
farmers’ role in initiating and adjusting the numbers and proportions of cultivars on farm based on
different cultivar traits is fundamentally important to the way landraces should be evaluated and
maintained in ex situ and in situ collections.
All Lujugira-Mutika bananas are resistant to Fusarium wilt races 1 and 2. Conversely, with regard
to black Sigatoka, banana weevils, and nematodes, there is variation in resistance even within the
same clone set. Similarly within the same subgroup, variation with regard to female pollen fertility
exists, with the ‘Nfuuka’ clone set being the most fertile and the ‘Musakala’ clone set the least fer-
tile; ‘Nakitembe’ and ‘Nakabululu’ clone sets fall in between. All these variability options can and
have been exploited for both selection and crossbreeding as well as for commercial purposes.
1.4.2.2 Selection
The process of selection produces patterns of variation in crop populations as well as patterns
of individual variation that would be useful to breeders. The former is of primary importance
in evolution and the latter in identification. The example of the East African Highland bananas
(EAHB) again illustrates this. Since their introduction in the East African region, the EAHB
have diversified in this region, not only because of mutations but also due to continuous natu-
ral and farmers’ selection. The morphological variation pattern existing in these bananas has
been as a result of the action of selective forces on the genotypes, and such variation would
presumably have an evolutionary significance to ensure survival of the selected types and lead
to ultimate improvement and spread of these bananas. Some variation patterns might have little
or no evolutionary significance. Bunch and fruit characters experience great selection pressure,
particularly the compactness and shape of the bunch and fingers, since these are very important
to farmers and other consumers for whom compactness and shape of the bunch in addition to
fruit size and shape are known commercial traits. A compact, medium, shaped bunch can easily
be packed, transported to market, and sold quite easily in that respect. Cylindrical or very lax
bunches or variegated and astringent fruits are not preferred by traders, although they have also
arisen through mutations (Rubaihayo and Gold, 1993). This farmers’ selection breeding has been
based largely on continuously changing morphological characteristics attributed to chimerism,
and this has been the basis for plant identification and nomenclature since cultivar names are
mainly based on the phenotypic characteristics of cultivars. If, for example, a new mat produces
suckers whose characteristics differ from those of the mother mat, farmers can either discard the
“different” suckers, or maintain them as mutants of the mother plant or as a different cultivar
to which they give a name depending on whether the differences between mother and daughter
are major or minor. The major differences with acceptable qualities will readily be selected and
adopted widely by farmers and subsequently by traders and consumers. Hence a daughter plant
may receive a name different from the mother if a major and consistent difference occurs. At the
farm level, the major phenotypic and nomenclature changes are always associated with the floral
parts, probably because these are most used by the farmers. Minor changes that do not normally
alter nomenclature include changes in the color of the pseudostem, petioles, and midribs. In this
case, the daughter plant retains the name of the mother plant but receives a second part of the
name to indicate the minor change. The identities of these cultivars become crucially important
tools in selection breeding and conservation efforts because the value, potential, and limitations
of each clone will very much be influenced by their characters and correct identity. In sum-
mary the structure and morphological variation pattern in a crop varies according to its breeding
system and the amount, intensity, and direction of selection to which they have been subjected
(Pickersgill, 1994).
1.5 Ecological Adaptation
The variability within a clonal population is more phenotypic than genotypic and many times
is caused by slight environmental differences within the habitat. Clonal material is ideal for the
study of morphological and physiological effects of diverse environmental factors on plants from
different areas or habitats. When breeders deal with clones, particularly with those coming out
as new materials from their breeding programs, it is important that they are able to recognize
differences in one cultivar from another. Distinction of one cultivar to another has increasingly
gained emphasis in connection with legislation about breeders’ rights in different countries. The
requirement is further accentuated by traders and buyers who must, in many instances, exercise
care in selecting the correct cultivar for their needs. In addition, different growing conditions
impose different characteristics to essentially the same clone. For example, ‘Sukali Ndizi’ (AAB)
from dry regions is smaller in both finger and bunch sizes than that from wetter regions but the
consumer markets prefer the former to the latter. Furthermore, the farmer wants to get the right
price for the particular cultivar he grows. Thus, primarily for economic reasons, identification
based on morphology is most important at the cultivar level and is only possible when morphologi-
cal variation allows it in various types of environments. Since breeders tend to look much more for
resistance and yields, it is not yet fully understood whether the observed phenotypic variability in
their new materials imparts resistance or tolerance to biotic and edaphic constraints that threaten
the very existence of the crop. Morphological variability seen in a new cultivar may be a reflection
of genetic elasticity, and its understanding will advance efforts to exploit its potential. It therefore
follows that breeders need to focus on the interrelationships between morphological variability on
one hand and the plant’s ability to survive environmental constraints on the other. The following
examples briefly explain such interrelationships between morphological variability, food texture,
and distribution incidences.
The Lujugira-Mutika bananas have been divided into four morphologically distinct clone sets,
and the clone sets bring together clones that share characteristics important to farmers and consum-
ers, though these characteristics were not used in grouping the clone sets (Pickersgill and Karamura,
1999). The clones in ‘Nakabululu’ and ‘Nakitembe’ clone sets sucker profusely, mature quickly,
produce soft-textured/flavored food, and are widely distributed but not rich in diversity. Most clones
in ‘Nfuuka’ are slow to produce suckers, take a long time to mature, and produce hard-textured
food, not evenly distributed but rich in diversity. However, one subcluster of ‘Nfuuka’ shares with
‘Nakabululu’ and ‘Nakitembe’ the characteristics of rapid maturity and production of some tex-
tured food. ‘Nfuuka’ is more heterogeneous than other clone sets and overlaps with most of them.
Its name means “I am changing” and reflects the farmers’ perception that somatic mutations are
particularly frequent in these clones. The final clone set, ‘Musakala,’ contains the higher-yielding
clones (e.g., ‘Lumenyamagali,’ which means “I break bicycles”), grown on a commercial scale to
supply urban markets. Clones in this set tend to be intermediate with regard to time to maturity and
in quality of their food.
In conclusion, morphological variation in clonal crops such as bananas is mediated through
mutation (though there is an increasing number of a crossbreeding program), thought to be somatic
in nature, and through a phenomenon called chimerism. Both chimeric and somatic mutation vari-
ants are then subjected to natural and farmer selection in response to the changing environment and/
or economic conditions to widen the variation and exploit its potential.
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2 Evolution and Genetic
Relationships in Banana
and Plantains
Uma Subbaraya, Marimuthu S. Saraswathi, and Michael Pillay
Contents
2.1 Introduction............................................................................................................................. 21
2.2 Growth of Global Banana Industry......................................................................................... 22
2.3 Consumption and Utilization of Banana................................................................................. 22
2.3.1 Importance of Banana as an Edible Fruit.................................................................... 22
2.3.2 Banana as an Alternative Source of Income............................................................... 23
2.3.3 Indigenous Technical Knowledge (ITK)..................................................................... 23
2.4 Taxonomy................................................................................................................................24
2.5 Origin and History...................................................................................................................24
2.5.1 Origin of Diploid Bananas...........................................................................................26
2.5.2 Origin of Polyploid Bananas (Triploids and Tetraploids)............................................ 27
2.5.3 Development of Bispecific Polyploids......................................................................... 27
2.5.4 AAB Genomic Group..................................................................................................28
2.5.5 ABB Groups................................................................................................................ 29
2.5.6 Australimusa................................................................................................................ 29
2.6 Molecular Characterization..................................................................................................... 29
2.6.1 Isozyme-Based Markers.............................................................................................. 30
2.6.2 DNA-Based Markers................................................................................................... 31
2.6.2.1 Restriction Fragment Length Polymorphism (RFLP).................................. 31
2.6.2.2 Randomly Amplified Polymorphic DNA (RAPD)....................................... 31
2.6.2.3 Retrotransposon-Based Markers................................................................... 32
2.6.2.4 Amplified Fragment Length Polymorphism (AFLP)................................... 32
2.6.2.5 Minisatellites and Microsatellites................................................................. 33
2.6.2.6 DArT Markers............................................................................................... 33
2.6.2.7 Chloroplast and Mitochondrial DNA Polymorphism................................... 33
2.7 Conclusion...............................................................................................................................34
References.........................................................................................................................................34
2.1 Introduction
Banana (Musa spp.) is the world’s most important fresh fruit commodity in terms of volume of
trade. The global banana industry started in the late 1800s as a result of technological advances
like refrigerated shipping and is now more than a century old. Compared to any other agricul-
tural product, banana exhibits colonial economic nationalism and contemporary neoliberal stages
of growth and evolution in the world economy (Wiley, 2008). Banana has grown in its popularity
21
for its versatility and adaptation to many agro-climatic conditions, resilience to climatic changes,
nonspecific seasonal fruit production, year-round availability of fruits, high productivity per unit
area (40–60 tons of fruits per hectare annually), and finally, if well managed, the plantation remains
productive for 10–20 years. The spectrum of cultivar diversity with regards to maturity allows stag-
gered but year-round availability of fruits. The popularity of the fruit led to its adoption and cultiva-
tion in more than 150 countries.
Banana is known by different names in the various countries of the world. These include:
banana (Japanese, Italian, Portuguese, Serbo-Croat), banane (French, German), banaan (Dutch),
banan (Danish), banaani (Finnish), banan (Russian/Polish), banbán (Hungarian), banana/
mpanána (Greek), banana (Hebrew), banema (Guinea), choui (Vietnam), Chiao (Chinese), futo
(New Caledonia), futi (West Polynesia), hnget-pyaw (Burmese), ikindu/kitoke (East African), Kela
(India), klue/klui (Thai), mauz (Turkish), maúz (Arabic/Persian), maso/ndizi (Swahili), pisang
(Malay/Indonesian), plátano/banana (Spanish), saging (Philippines), usi (New Guinea), uch/ut
(Micronesian), and vudi (Fiji).
2.2 Growth of Global Banana Industry

The global area under banana and plantain cultivation is estimated to be 10.2 million hectares (ha)
(FAO STAT, 2008) with the greatest contribution coming from Africa (5.8 million ha) followed by
Asia and Pacific (2.04 million ha) and Latin America (1.31 million ha). During the last 5 years, the
area under cultivation has increased by 6.87% and 0.68% for banana and plantain, respectively, with
African continents (16.5%) followed by Asia (6.5%) contributing mainly to the increase. The global
production is around 125.04 metric tons (mt), with bananas contributing 96.70 mt and plantains
34.30 mt (FAO STAT, 2009). During the last 5 years, banana has witnessed an increase in produc-
tion by 45%, with Asia contributing 38% of the production. India is the largest banana producer
followed by China, the Philippines, Brazil, Ecuador, and others. Although the global productivity
of banana has increased from 15.6 to 18.8 tons/ha, that of plantain has increased marginally from
6.1 to 6.5 tons/ha. Asia, despite its minimum contribution to the increase in global area under
banana and plantain cultivation, has witnessed a giant leap: a 42.3% increase in productivity. Costa
Rica records the highest productivity of 52.54 tons/ha followed by India (35.87 tons/ha), Mexico,
Colombia, and Ecuador. The major exporting countries are Ecuador, the Philippines, China, Costa
Rica, Colombia, Panama, and Honduras, with the export trade worth about US$8.6 billion annually
(FAO STAT, 2008).
2.3 Consumption and Utilization of Banana

Bananas are an important source of carbohydrate and dietary fiber. They contain high amounts
of essential vitamins A1, B1, B2, and C; minerals such as potassium (Chandler, 1995); and sub-
stantial quantities of starch and hemicelluloses (Ketiku, 1973; Mota et al., 2000). Plantains have a
greater amount of vitamins A and C (20 mg/100 g) than bananas. Plantains are revered as a food
equivalent across many African countries, Latin America, the Caribbean, and the Polynesian
islands. Although the average global per capita consumption of banana and plantain is reported
as 5.2 kg/person (Nayar, 2010), it is as high as 239 kg/person in Uganda, 223 in Burundi, 180
in Rwanda, 141 in Gabon, and 131 in Samoa (FAO STAT, 2008), increasing marginally over the
past few years. It is worth noting that statistics provided by the FAO do not distinguish between
plantain and banana.
2.3.1 Importance of Banana as an Edible Fruit

Bananas and plantains are the only group of fruits that also constitute a staple food for millions.
They are consumed fresh, cooked, steamed, roasted, and brewed (Pillay et al., 2002). Besides
Evolution and Genetic Relationships in Banana and Plantains 23
the fruits, the flower buds and inner core of the pseudostem are also used as vegetables as well
as for a wide range of therapeutic uses. Bananas are also processed into puree, juice, fig, jams,
canned banana slices (Thompson, 1995), and beer and wine in Africa (Olaoye et al., 2006) (see
Chapter 14).
Bananas are regarded as nature’s secret of perpetual youth in the practice of traditional medi-
cine in India, China, and ancient Persia (www.articlesbase.com, accessed 19 April 2010). The low
lipid and high energy values make it ideal for obese and geriatric patients (Gasster, 1963). Bananas
are useful for persons with peptic ulcer, infant diarrhea, celiac disease, and colitis (Seelig, 1969).
Being low in salt and high in potassium chloride, it is a recommended dietary supplement to lower
blood pressure (www.herbalextractsplus.com/banana.cfm, accessed 19 April 2010). Bananas can
stimulate the production of hemoglobin in the blood and are useful for anemic patients. Bananas are
rich in tryptophan, a type of protein that the body converts into serotonin, which keeps the mind
in a relaxed state. Fruits are regarded as coolants, which lower both the physical and emotional
temperature of expectant mothers. Bananas are rich in fiber and pectins, which absorb water and
restore bowel movement. Bananas contain benign amino acids that are useful for the removal of
stones in the kidney and gall bladder. Similar functions are also reported for the central core of the
pseudostem, which is either consumed as a salad or cooked with pulses. Bananas are rich in lectins,
which are sugar-binding proteins that can identify foreign invaders like viruses, attach themselves
to pathogens and block their entry into the human body. The roots of banana are considered to have
an antihelmintic effect.
2.3.2 Banana as an Alternative Source of Income

Banana leaves are popular in South India and Africa as hygienic dining plates and wrapping mate-
rial. Leaf production alone is also an income source for small-scale farmers. The underground rhi-
zome is exploited as animal feed in a composite mixture. The banana pseudostem either alone or in
combination with rice straw has proved to be a good substrate for mushroom cultivation. The plant
sap is also used as an indelible ink in industry.
Banana fiber is being used in the treatment of industrial and municipal wastes. Banana fibers
also find use in the pulp industry because of their optimum burst, tear, and tensile indices (Uma et
al., 2002). The fibers are used as a base material in cottage industries for making handicrafts and
for making a wide range of goods, including bags, rope cordage, yarns, abrasive backing paper,
tea bags, shoes, and so forth. Tear and tensile strength of banana fiber derived from plants like M.
textilis make it amenable for printing of Japanese yens, and it is also blended with cotton in various
ratios for use in the textile industry.
2.3.3 Indigenous Technical Knowledge (ITK)

Inhalation of the smoke from burning banana leaves is used to relieve wheezing in asthmatic
patients. Tender leaves smeared with oil are used for dressing of wounds and blistered skin surfaces.
Flower buds boiled with salt and oil are consumed by northeast Indian tribes to cure joint pains and
promote blood circulation. When cooked with pulses or with coconut, flower buds are considered
to be a remedy for heart ailments and for dissolving kidney stones. Banana pulp is added to cereal
beer made of rice, sorghum, and so on to aid in fermentation and providing a fruity flavor. Dried
and powdered pulp of wild cultivars such as ‘Bhimkol’ and ‘Athiakol’ are used as infant food as
they are easily digestible. Both ripe and unripe fruits of M. nagensium are cooked with sorghum
and other cereals and fed to pigs to improve their health. Rhizomes of all wild members of the sec-
tions Eumusa and Rhodochlamys are chopped into pieces and cooked with pulses as animal feed
(Uma, 2006).
2.4 Taxonomy
Botanically banana is classified in the genus Musa, which—together with two other genera, Musella
and Ensete—are placed in the family Musaceae and order Zingiberales (Table 2.1). Members of
this family are large herbs 2–9 m tall with an aerial trunk consisting of compacted leaf sheaths
that grow directly from the top of the corm (Purseglove, 1976). The earliest description of dessert
banana Musa sapientum and ‘Silk’ banana M. paradisiaca was provided by Linnaeus in 1753. Other
monospecific nomenclature such as M. sapientum ssp. paradisiaca by Baker (1893), M. paradi-
siaca ssp. sapientum by Schumann (1900), and M. sapidisiaca by Jacob (1952) were not accepted
by taxonomists. The earliest taxonomic references were made by Baker (1893), while Schumann
(1900) mentioned 42 species in his monograph, of which 10 belonged to the genus Physocaulis, 20
to Eumusa, and 12 to Rhodochlamys. Sagot (1887) and Baker (1893) distinguished three subgenera
in the genus Musa: Physocaulis, Eumusa, and Rhodochlamys. This classification was revised by
Cheesman (1947), whose detailed taxonomic treatment was based on chromosome number, pseu-
dostem stature, inflorescence characters, and seed morphology. Cheesman divided the genus Musa
into four sections: Eumusa (x = 11), Rhodochlamys (x = 11), Australimusa (x = 10), and Callimusa
(x = 10). This classification has been widely accepted, despite subsequent reports of basic chromo-
some numbers n = 7 for M. ingens and n = 9 for M. beccari (Shepherd, 1999). Recently the section
Ingentimusa was added (Argent, 1976; Simmonds and Weatherup, 1990). In fact, several unresolved
issues remain, including the evolutionary relationships among the four sections of the genus Musa,
the separation of sections Eumusa and Rhodochlamys, which has apparently little taxonomic sup-
port, and the relationship between the wild progenitors and the cultivated clones (Simmonds, 1953,
1960; Shepherd, 1999; Ude et al., 2002a; Wong et al., 2003). To date, more than 36 species and
subspecies have been identified and described (Daniells et al., 2001), and more recently 73 species
were described by Häkkinen and Väre (2008), warranting a thorough revision of the genus Musa
and the development of a classification key.
2.5 Origin and History

Banana with its versatile utilities has been recognized as one of the earliest crops to be domesticated
by man. As a wild-seeded plant, banana must have been first recognized for its non-edible purposes
like fiber, roofing, and ropes for navigation purposes. The earliest documentary evidence of banana
is found in the Vedic period (approx. 1700 BCE), during which use of fruits like mangoes, gooseber-
ries, and bananas was mentioned in the Rig-Veda, one of the four volumes of Veda written by the
Indian sages. Further evidence is from the Indian epics, Mahabharata and Ramayana, which date
to approximately 1400 BCE. Aranya Kanda and the forest trek of Valmiki’s Ramayana state that
members of royal heritage were draped in clothes woven of banana fiber.
Suggestions of banana fruits in the contemporary diet in Ramayana perhaps indicate the domes-
ticated status of banana in the Indian subcontinent. Early epics of the Pali Buddhist monks in 500–
600 BCE also describe bananas “as big as an elephant tusk.” This could probably refer to plantains
(Horn plantain) from the southern peninsula of greater India (including Sri Lanka), where plantain
cultivation is in vogue today. It is taken for granted that banana and plantain originated in Southeast
Asia and the Pacific and spread to the rest of the world. However, recent linguistic, anthropological,
and archeological studies have contributed to an alternative migratory theory (Nayar, 2010).
Alexander’s invasion in 327 BCE popularized this “holy fruit” in contemporary writings. This
was followed by Megashthanes (350–290 BCE), the Greek traveler and geographer, in his travel-
ogue, Indica, by Theophrastus (371–287 BCE), and by Pliny (23–79 CE), a Roman naturalist, in his
book Historia Naturalis. Charaka (fourth century) from India has documented the various medici-
nal uses of bananas in his medical compilation, Susrutha Samhita. Reports on early cultivation of
banana in human civilization were obtained from Buddhist sculptures of central India, stupas in
Sanchi, carvings in Nalanda, and paintings in Ajantha and Ellora caves. Archeologists have also
Evolution and Genetic Relationships in Banana and Plantains
Table 2.1
Conspectus of the Family Musaceae, Classification, Distribution, and Uses of Musa Species
Chromosome Uses and
Genus No. (2n) Section Distribution Species Utilization
Ensete 9 – Asia (India, China, Thailand, E. superbum (Roxb.) Cheesman, E. glaucum (Roxb.) Cheesman Fiber, vegetable,
Philippines), Africa E. ventricosum (Welw.) Cheesman, E. livingstoniana, E. proboscidea, E. ornamental
gilletti (De Wild) Cheesman, E. buchanani, E. homblei (Bequaert)
Cheesman, E. perrieri (Claverie) Cheesman
Musa 10 Australimusa Queensland, Polynesia, M. lolodensis Cheesman, M. peekelii Lauterb, M. maclayi von Muell. Ex Fiber, dessert fruit
Philippines, Australia Mikl-Maclay, M. jackeyi W. Hill, M. bukensis Argent, M. textilis Née,
M. Fehi
Callimusa Indo-China, Malaysia, M. coccinea Andrews, M. violascens Ridely, M. gracilis Holttum, Ornamental
Philippines, Indonesia M. beccarii Simmonds, M. erecta, M. borneënsis Beccari, M. coccinea
Andrews, M. exotica R. Valmayor, sp. now M. flavida, M. hotta,
M. gracilis Holttum, M. salaccensis Zoll
11 Eumusa All banana growing countries M. acuminata Colla, M. balbisiana Colla, M. schizocarpa Simmonds, Food, fruit,
and continents M. basjoo Sieb, M. flaviflora Cheesman, M. itinerans Cheesman, vegetable, fiber,
M. flavicarpa, M. sikkimensis Kurz, M. cheesmani Simmonds, and medicinal
M. nagensium Prain, M. halabanensis Meijer, M. ochracea Shepherd applications
11 Rhodochlamys India, Malaysia, Indo-China, M. ornata Roxb., M. rosea Baker, M. rubra Wall. Ex Kurz, M. mannii Ornamental
Myanmar, Cambodia, Vietnam, H. Wendl. Ex Baker, M. velutina H. Wendl. and Drude, M. laterita
Philippines, Thailand Cheesman, M. sanguinea Hook. f., M. aurantiaca Mann ex Baker,
M. rosacea Jacq.
25
found the banana depicted in ancient ruins such as the Buddhist temple of Bharhut, dating from the
second century BCE, and the Javanese monument to Buddha erected in Borobodur in the year 850
CE. Yang Fu, a Chinese official in the T’ang dynasty (618–907), wrote an Encyclopedia of Rare
Things wherein he describes the banana plant, which is possibly the first mention of the banana in
Chinese texts.
Similarly plantains have a long history of domestication in Africa and their entry to the continent
is reported to be about 1500–2000 years ago (Bruce, 1790; Purseglove, 1976; De Langhe et al., 1995;
Doutrelepont et al.,1996; Mbida et al., 2000). Phytoliths of Musa and Ensete unearthed at Nkang in
Cameroon are the first archeological indication of a cultivated crop, dating back 3000 years in Central
Africa and providing a clear indication of an early food economy in Africa (Mbida et al., 2000).
Banana and plantain is a complex crop with vast diversity, utility, and spread, and address-
ing their origin as any other simple crop would be an underestimation. After their simultaneous
and independent evolution across Asia, Polynesia, and Africa, metamorphosis of the earliest wild
banana, a weedy, seeded, nonedible plant into a domesticated, parthenocarpic (nonseeded), edible
tasty fruit occurred in a long evolutionary journey. Archeobotanical and paleoecological studies
combined with radiocarbon dating and stratographic analysis clearly suggest the existence of banana
as early as 6950 to 6440 BCE. Domestication and extensive cultivation was evidenced by increased
phytolith counts in New Guinea. This dating was prior to the influence of Polynesians, suggesting
that bananas and plantains indeed had their independent origin and evolution in the Pacific islands
(Denham et al., 2003, 2004; Lentfer, 2003).
2.5.1 Origin of Diploid Bananas

Wild bananas are all diploids and the ancestry of cultivated bananas originates from two major
species, M. acuminata and M. balbisiana, with occasional mention of the involvement of M. textilis
and M. schizocarpa. Simmonds (1962a) proposed the early domestication of diploid banana—that
is, M. acuminata in its primary center of origin, namely Malaysia, which has the greatest variabil-
ity. A partial occurrence of parthenocarpy in M. acuminata ssp. banksii (Simmonds, 1962a, 1962b)
was suggested, which contributed for the development of parthenocarpy. This was later confirmed
by restriction fragment length polymorphism (RFLP) studies (Carreel, 1994). The development of
parthenocarpy was also reported in M. acuminata ssp. burmannica distributed in Western Ghats of
India and the cultivar ‘Matti’ using morpho-molecular characterization (Uma et al., 2005a). ‘Matti’
is considered to have evolved by continuous human selection for complete parthenocarpy similar to
‘Pisang Rejang,’ the possible seedy progenitor of ‘Pisang Lilin’ (Nasution, 1991).
Unlike M. acuminata, development of parthenocarpy in M. balbisiana has not been investigated
in detail. Occurrence of parthenocarpic diploid and triploid M. balbisiana (BB and BBB) have been
reported from the Philippines and Thailand by several workers, but the true genomic constitution
of these plants still needs confirmation. Valmayor et al. (2002) reported the occurrence of parthe-
nocarpy in M. balbisiana in the natural hybrids like ‘Abuhon’ of the Philippines. This is assumed
to be a cross between seeded forms of M. balbisiana (‘Pacol’) with soft and scanty seeded ‘Chuoi
Hot.’ Similarly, ‘Saba,’ ‘Pisang Kepok,’ ‘Pisang Nipah,’ ‘Klui Lep Chung Kut,’ ‘Chuoi Ngu,’ and
‘Chuoi Mat’ have been recognized as parthenocarpic M. balbisiana clones originating through
chromosome restitution and repeated back-crossing with their parents (Brewbaker and Gorrez,
1956; Danh et al., 1998). Other triploid M. balbisiana (BBB) clones reported from the Philippines
are ‘Saba,’ ‘Cardaba,’ ‘Gubao,’ ‘Bigihan,’ ‘Pa-A Dlaga,’ ‘Saba Sa Hapon,’ ‘Pondol,’ ‘Turankong,’
‘Sabang Puti,’ ‘Mundao,’ ‘Dali-an,’ ‘Kalimpos,’ ‘Binendito,’ and so forth. During a visit by the
senior author to the Field gene bank at Phakchong Banana Research station, Thailand, in 2002,
some accessions reported to be BBB in the gene bank were found to exhibit the traits of M. tex-
tilis. A comprehensive study using morpho- and molecular approaches is warranted to decipher
the actual genomic status of these plants. But edibility in terms of emptiness of seeds, softened
seed coats, and pulpiness of fruits over its mucilaginous pulp has been observed in ‘Bhimkol,’ a
widespread landrace of northeastern India. Human selection for pulpy fruits and fewer seeds is still
practiced in BB landraces.
In general, parthenocarpy in banana is genetically controlled by three complementary genes
with modifiers (Dodds, 1943a, 1945; Dodds and Simmonds, 1946a, 1946b, 1948). Sterility is also
genetically controlled, supplemented by various forms of chromosomal aberrations and abnormal
meiosis, all contributing to edibility in banana. Vegetative propagation favored the perpetuation of
edibility through genetically controlled parthenocarpy and sterility in banana. Evolution for par-
thenocarpy could be expected through landraces like ‘Bhimkol’ over a period. ‘Bhimkol’ derived
hybrids are available at the National Research Center for Banana (NRCB) and are being screened
for parthenocarpy and hardiness.
Edibility in banana occurred over several years with acquisition of parthenocarpy complemented
with the development of sterility. Human interventions for selection of edible diploids and nature’s
intervention to induce sterile mutants resulted in a vast diversity of AA diploids. Another major
milestone was the development of edibility in terms of sweetness. Musa acuminata ssp. banksii
derived diploids have starchier fruits, while sweet-pulped cultivars are suggested to be closer to
M. acuminata ssp. malaccensis (Carreel et al., 1994). Similarly, clones from the different subspe-
cies differed by translocation events in their chromosomes (Shepherd, 1999). Natural introgression
between clones of the different subspecies and human selection for better edible diploids was a vital
step in banana evolution.
2.5.2 Origin of Polyploid Bananas (Triploids and Tetraploids)

Development of polyploid bananas, more specifically triploids, followed the events leading to the
origin of cultivated diploids. In nature, positive selection occurred in favor of heterozygotes that
were nearly or completely seed-sterile (Simmonds, 1962, 1966). Vegetative cultivation and a non-
preference for selection for seed-fertile clones over a long period must have led to accumulation of
structural changes in chromosomes (Simmonds, 1962a). But Faure et al. (1993) indicated that steril-
ity in diploid banana is not due to structural hybridity but is genetically controlled. Female restitu-
tion leading to the production of diploid (unreduced) gametes and hybridization within subspecies
of M. acuminata ssp. or between different banana species led to the production of triploids. It is
reported that production of unreduced diploid gametes is a frequent occurrence in diploid bananas
in both the male and female sides (Dodds and Simmonds, 1946a).
By virtue of their superior traits like parthenocarpy, edible pulp, good bunch, and sturdiness,
triploids were preferred for selection and clonal perpetuation by early man. Naturally occurring
improved edible diploids of a particular subspecies, retaining fertility to some extent, hybridized
with other subspecies, leading to the development of initial M. acuminata triploids. Raboin et al.
(2005) used RFLP markers to trace 2n restitution and n gamete donors of Cavendish and Gros
Michel. They were shown to have a common diploid ancestor from Madagascar and Comoro.
2.5.3 Development of Bispecific Polyploids

Musa balbisiana is the other wild Eumusa species that had broad distribution across South and
Southeast Asia. Although no subspecies have been recognized within the species, recent studies
have revealed sufficient variability within M. balbisiana (Sotto and Rabara, 2000; Ude et al., 2002b;
Uma et al., 2005; Ge et al., 2005). Musa balbisiana has adopted to drier regions but exhibits copious
growth in tropical and subtropical forests of the Philippines, Thailand, New Guinea, India, south
China, Malaysia, Myanmar, Indonesia, and a few other regions (Valmayor et al., 2002; Uma et al.,
2005a). Interspecific crosses between M. acuminata diploids and M. balbisiana occurred either in
their primary origin or on their journey to new locations, resulting in different genomic combina-
tions: AB, AAB, ABB, and ABBB, of which AAB and ABB encompassed a greater diversity.
The AAA genomic group forms the major allopolyploids valued for its commercial status in the
global banana industry. It has various subgroups like Cavendish, Gros Michel, Red, Ibota, Lujugira-
Mutika, and a few unique accessions (Uma and Sathiamoorthy, 2002).
The AB diploids evolved in a specific locality of Southeast Asia, especially southern India and
Sri Lanka (Uma and Sathiamoorthy, 2002), from where it spread to eastern Africa in the recent
past (Shepherd, 1999; De Langhe, 1996; De Langhe and Maret, 1999; Nayar, 2010). The main AB
subgroups in India include ‘Ney Poovan’ and ‘Kunnan,’ while Daniells et al. (2001) recognized two
AB groups as ‘Ney Poovan’ and ‘Kamaramasenge.’ No studies have been carried out to confirm
the genetic relationships between ‘Kamaramasenge’ and ‘Kunnan’ subgroups. Molecular studies
using SSR (simple sequence repeat) markers confirmed the occurrence of only two subgroups in the
AB genome, ‘Kunnan’ and ‘Ney Poovan’ in India (Uma et al., 2010). The AB genotypes specific to
South India—‘Kunnan’ and ‘Safed Velchi’—did not show any evidence of having a M. balbisiana
cytoplasmic genome (Carreel et al., 2002). It is possible that there are M. balbisiana types that
are unique to India. India has a wide diversity of M. balbisiana clones (Uma et al., 2005b, 2006).
Consideration of M. balbisiana from India together with samples from other regions is expected to
provide a holistic picture on the evolution of this species.
2.5.4 AAB Genomic Group

Hybridization between M. acuminata and M. balbisiana led to a broader diversity of genotypes
now classified as Plantain, Silk, Mysore, Pome, and other unique members with AAB genomes.
The plantains are considered to have evolved from M. acuminata ssp. banksii as female parent
and M. balbisiana as male parent (Carreel et al., 2002). Plantains seem to have evolved in New
Guinea where a greater diversity for plantains and cultivated diploids is still present. There is some
evidence to show that plantains also originated in the Philippines due to the natural availability of
French plantains in Luzon (De Langhe and Valmayor, 1980; Valmayor et al., 2002). Although De
Langhe (1996) suggested that southern India could be a possible place of origin for plantains, it is
now accepted that the area is perhaps only a secondary center of diversification. Southern India has
only French plantains and very little diversity has been observed in this group. Therefore, the epics
suggesting the presence of bananas “as big as an elephant tusk” in 500–600 BCE mentioned at the
beginning of this chapter raise doubts about the early occurrence of Horn plantains in India.
The Silk group of bananas is popular in South and South Asia, East Africa, Brazil, and other
Latin American countries. Studies using RFLP (Carreel et al., 2002) reveal a paternal lineage from
M. acuminata ssp. malaccensis for this group, unlike the other AAB subgroups. Subspecies malac-
censis is naturally distributed in Malaysia, Indonesia, and Thailand. It appears that diploids evolved
from this group hybridized with M. balbisiana to give rise to Silk clones (AAB).
The Mysore group (AAB) is a collection of hardy bananas that spread across almost the whole
world. Chloroplast DNA studies shows that it paternally evolved from Musa acuminata ssp. errans
and maternally from a M. acuminata ssp. with type II cpDNA (Carreel et al., 2002). It also has
an AB diploid and diploid M. balbisiana in its ancestry. Subspecies errans is mainly found in the
Philippines. But the natural occurrence of M. swarnaphalya, which morphologically resembles M.
acuminata ssp. banksii/errans in northeast India, could suggest another center of evolution for M.
acuminata ssp. banksii and ssp. errans–derived cultivated bananas. ‘Pisang Kelat,’ a unique AAB
member reported to have a B genome of maternal origin and classified as BAA, is important from
a pathological perspective since it is immune to most of the leaf spot diseases and Fusarium wilt
(Annual Report, 1999).
The Maia Maoli/Popoulu (AAB) group is endemic to Oceania and specifically of Polynesian
origin. The group is recognized for its unusually fat and squat fruits with blunt tips, with the
Maoli group exhibiting longer fruits. Great diversity for fruit shape and size exists in Hawaii, west
Polynesia, and Pohnpei. Popoulu fruits are very characteristic due to their tendency to split at ripe-
ness (Ploetz et al., 2007).
The Iholena subgroup, distinguished by a coppery undersurface of leaves, salmon-colored flesh,

and right-angled fruits, is traditionally grown in the highlands of Hawaii. They are consumed raw
or cooked. Hawaiian diversity is represented by seven Iholena varieties. Iholena fruits are arranged
loosely and at right angles to the bunch. Male flowers have characteristic lavender-colored stamens.
The fruits remain pale yellowish-green throughout their development. The group is also sparsely
represented in New Guinea, Samoa, French Polynesia, Tonga, and others (Ploetz et al., 2007). The
uniqueness of the group and its diversity is attributed to a series of additional somatic mutations in
basic hybrids and cultivars in niche locations of Polynesia (De Langhe and De Maret, 1999). Their
genetic closeness to plantains is suggested by Carreel et al. (1994), with M. acuminata ssp. banksii
contributing the AA genomes in both cases (Horry and Jay, 1988; Lebot et al., 1993; Carreel et al.,
2002).
2.5.5 ABB Groups

All the ABB clones in various subgroups of ‘Bluggoe,’ ‘Pelipita,’ and ‘Ney Mannan’ have M. bal-
bisiana in their lineage (Carreel et al., 2002) and may be referred to as true ABB while ‘Pisang
Awak’ and ‘Peyan,’ the dessert cultivars of ABB, have a cytoplasmic DNA associated with the B
genome and are referred to as BAB. It is debatable whether the subgroup ‘Bontha,’ which is starchy
and primarily cooked and eaten, should be included in the ‘Pisang Awak,’ ‘Peyan’ (dessert bananas),
‘Monthan,’ ‘Bluggoe,’ and ‘Vennutu Mannan’ groups of bananas. Some ‘Bluggoe’ types are com-
mercially used as ripe fruits in South India. Although it is suggested that cultivars with B-rich
genomes are drought tolerant, ‘Monthan’ seems to be a proven exception and others need scientific
confirmation.
2.5.6 Australimusa
The section Australimusa has edible varieties generally referred to as Fe’i banana. It has six recorded
wild species: M. lolodensis, M. peekelii, M. maclayi, M. jackeyi, M. bukensis, M. textilis, and one
cultivated species, M. fehi (Daniells et al., 2001). All are characterized by gigantic stature, red
plant sap, and negatively geotropic bunching habit (erect bunches). Their distribution is restricted
to New Guinea, eastern Indonesia, the Solomon Islands, and other Pacific islands (Argent, 1976;
Nayar, 2010). But early Indian literature on Sunga dynasty (2 BCE) talks about a banana variety
with red sap. Perhaps a banana with red sap did exist in India at some time in the past, which raises
the question whether the Australimusa were present in Southern India. Archeological and archeo-
botanical studies may prove this point. Domestication of Australimusa also started with the occur-
rence of parthenocarpy and seed sterility. Mutations and human selections perpetuated the edible
forms, especially Fe’i bananas. They were passively cultivated and protected in many of the west
Pacific Islands (Mac Daniels, 1947). Morphotaxonomic studies (Cheesman, 1947), RFLP mark-
ers (Jarret et al., 1992), and Carreel (1994) suggested an interspecific origin for the Fe’i bananas
within the Australimusa, with M. maclayi as the most probable ancestor. Musa textilis, a member
of Australimusa, is endemic to Philippines. Several cultivated clones have evolved in nature as
intersectional crosses between M. textilis and M. schizocarpa, and M. textilis and M. balbisiana (for
example, ‘Butuhan,’ ‘Karoina,’ ‘Umbubu,’ and ‘Mayalopa’).
2.6 Molecular Characterization
A classification system for banana and plantains was first developed by Simmonds and Shepherd
(1955), who used a simple numerical system by scoring 15 morphological characters on a score
from 1 to 5. A modified and revised scoring system was developed by Silayoi and Chomchalow
(1987) and Singh and Uma (1996). The latter system incorporates the genetic variability available
especially in India. A general key to identifying the various groups was based on 121 morphological
characters using the high degree of variability for characters such as pseudostem blotching, pigmen-
tation, and leaf length: breadth ratio, flowering and fruiting duration, number of hands and fingers,
peduncle nature, pedicel length, fruit size, taste and nature of ripe fruit flesh, and so forth (IPGRI-
INIBAP/CIRAD, 1996). User-friendly software was developed for classifying a banana genotype/
clone by using a pair-wise discriminant function for the five genomes with 10 algorithms and 19
morphological characters.
Morphotaxonomic characterization and genomic classification (Simmonds and Shepherd, 1955)
for identification of unique accessions, synonyms, and mutants in Indian germplasm identified
81 distinct clones and 23 mutants in the gene pool of 240 accessions at Tamil Nadu Agricultural
University, Coimbatore. A data base has been created for 545 accessions that were characterized at
the NRCB using the modified score card and detailed morphotaxonomic characterization using the
“Musa descriptor.” The morphotaxonomic classification system failed to distinguish 45 ABB acces-
sions that appeared to be synonyms, and they were later confirmed with molecular characterization
(Saraswathi et al., 2009b).
Four categories—that is, group, subgroup, clone set, and clone—were adopted in the classi-
fication of East African Highland bananas based on qualitative characters (Karamura, 1998).
Complementing morphotaxonomic characterization with molecular characterization is more useful
in drawing meaningful conclusions on banana diversity.
Molecular markers have been used widely for diversity analysis, characterization of germplasm,
and fingerprinting of genotypes in Musa. Precise knowledge on the extent of diversity in a germ-
plasm is a prerequisite for a strategic breeding program in a crop like Musa that is recalcitrant to
breeding owing to parthenocarpy, sterility, and polyploidy.
2.6.1 Isozyme-Based Markers
A few studies were carried out to assess genetic diversity in Musa using isozyme markers (Bonner
et al., 1974; Horry, 1993; Jarret and Litz, 1986a, 1986b). Jarret and Litz (1986a) identified polymor-
phisms in banana and plantains representing various ploidy levels for enzymes: malate dehydroge-
nase (MDH), phospho glucomutase (PGM), glutamate oxaloacetate transaminase (GOT), shikimate
dehydrogenase (SKDH), and peroxidase. Bhat et al. (1992a, 1992b) observed a high degree of poly-
morphism for peroxidase, superoxide dismutase, esterases, and acid phosphatases in bananas and
plantains. Espino and Pimentel (1990) used SKDH and MDH and found species-specific markers as
well as markers for identification of interspecific hybrids.
While some enzymes were of little use in discriminating genomic groups (Espino and Pimentel,
1990; Bhat et al., 1992b), others like MDH were useful in differentiating the ABB and AAB from
BB/BBB group of cultivars (Espino and Pimentel, 1990). Isozyme polymorphism was able to distin-
guish between the ‘Saba’ and ‘Bluggoe’ subgroups (Rivera, 1983) but failed to differentiate cultivars
within the Cavendish subgroup (Jarret and Litz, 1986a).
The isozyme data of Lebot et al. (1993, 1994) suggested that the genes contributed by M. acumi-
nata to the Pacific plantains are similar to those of the M. acuminata × banksii complex of Papua
New Guinea. The Pacific plantains may have originated in Papua New Guinea rather than in Asia
or the Malayan archipelago. Analysis of the anthocyanin composition of bracts in wild and culti-
vated forms by Horry and Jay (1988) suggested two independent centers of domestication for M.
acuminata, one in Southeast Asia and the other in Papua New Guinea. They also suggested that
the A genome of AAB plantains is more allied to Papuan M. acuminata ssp. banksii than Asian
M. acuminata. Isozyme polymorphism among Indian and exotic germplasm showed that the exotic
introductions were distinctly different from indigenous diploids. However, an indigenous red diploid
(‘Sannachenkadali’) grouped with ‘Pisang Berlin,’ an exotic introduction, needs further investiga-
tion. Selvarajan et al. (2002) were able to differentiate the Sigatoka leaf-spot-resistant and suscep-
tible varieties using SKDH.
2.6.2 DNA-Based Markers
2.6.2.1 Restriction Fragment Length Polymorphism (RFLP)
RFLP markers individually or in combination with other techniques have been used in the clas-
sification of Musa to amend the genome formula and subspecies/subgroup classification of some
varieties. Delineation of the four sections of the genus Musa using RFLPs was reported by Gawal
et al. (1992). The study grouped Eumusa and Rhodochlamys in one clade and Australimusa and
Callimusa in the other. Phylogenetic analysis of species and subspecies of Musa showed that M.
schizocarpa is very close to M. acuminata (Jarret et al., 1992). Fe’i bananas were distinct from the
five species of Australimusa—including M. maclayi, its presumed ancestor—but showed that M.
lododensis was closest to the Fe’i bananas. In a similar study conducted at the Centre de Coopération
Internationale en Recherche Agronomique pour le Développement (CIRAD), nuclear probes were
used to identify the alleles specific to M. acuminata, M. balbisiana, and M. schizocarpa. Starchy
cultivars were found to be closely associated with M. acuminata ssp. banksii while sweet-pulped
cultivars were closer to M. acuminata ssp. malaccensis. RFLP studies also confirmed the involve-
ment of M. acuminata in the origin of parthenocarpy, as all the diploid parthenocarpic bananas
contained M. acuminata alleles. Studies by Carreel et al. (2002) revealed that the parthenocarpic
varieties are linked to M. acuminata ssp. banksii and M. acuminata ssp. errans. RFLP analysis
provided evidence for a strong bias towards maternal transmission of chloroplast DNA (cpDNA)
and paternal transmission of mitochondrial DNA in M. acuminata, suggesting two separate mecha-
nisms of organelle transmission and selection. Knowledge of the organelle mode of inheritance
constitutes an important point for phylogeny analyses in bananas and may offer a powerful tool to
confirm the origin of hybrids.
RFLP of cpDNA among the M. acuminata–derived clones displayed a range of variation (Gawel
and Jarret, 1991). The resultant cladogram showed clear clustering of the M. acuminata subspecies
and differentiated M. acuminata and M. balbisiana cytoplasm. On the basis of cytosolic RFLP
probes, the triploid Cavendish group (AAA) was found to be related to M. acuminata ssp. errans
and M. acuminata ssp. malaccensis, whereas RFLP data from the nuclear genome did not show any
association with either subspecies. Carreel et al. (2002) used RFLP in combination with heterolo-
gous mitochondrial and chloroplast probes to investigate the contribution of other Musa spp. besides
M. acuminata and M. balbisiana to the origin of cultivated bananas. Generally, beer bananas could
not be distinguished from similar AAA types used for cooking (Simmonds, 1966) but apparently
their chloroplast genome was quite distinct for several cleavage sites when compared to dessert
bananas.
2.6.2.2 Randomly Amplified Polymorphic DNA (RAPD)

RAPD studies have been widely used in Musa for varietal identification (Bhat and Jarret, 1995),
species identification (Pillay et al., 2000), identification of genomic groups (Kaemmer et al., 1992;
Howell et al., 1994; Pillay et al., 2000; Visser, 2000; Rekha et al., 2001), identification of genomes
(Pillay et al., 2000), and genetic diversity (Bhat and Jarret, 1995; Pillay et al., 2001). Multivariate
analysis of RAPD bands identified similar patterns of variation that matched the morphological
variation in Musa (Howell et al., 1994). RAPD polymorphism was also able to differentiate Musa
cultivars of Kenyan origin that were closely related (Kahangi et al., 2002; Onguso et al., 2004;
Mizutani et al., 2004).
RAPDs was used to investigate the genetic variation and phylogenetic relationships among
‘Silk’ group representatives of both indigenous and exotic origin (Uma et al., 2004). RAPDs
used to compare the genetic relationships in M. balbisiana accessions collected from the Indian
mainland and Andaman and Nicobar Island (Uma et al., 2004) produced two distinct clusters
based on geographical origin. Wild types from the Indian mainland were distinct from those
collected from Andaman and Nicobar Islands. Further intraspecific relationships among M. bal-
bisiana from different regions of India indicated the existence of considerable variation with a
polymorphism of 74.6% (Uma et al., 2006). RAPDs were shown to detect differences between
closely related organisms such as cultivars of plantain grown in Jamaica and Nigeria (Agoreyo et
al., 2008).
Duplex RAPDs were used to identify clones of ‘Thella Chakkarakeli’ (AAA) at NRCB
(unpublished).
2.6.2.3 Retrotransposon-Based Markers
The inter-retrotransposon amplified polymorphism (IRAP) markers were able to identify presence
of the B genome in natural hybrids (Nair et al., 2005). Microarray analysis of long terminal repeat
(LTR) retrotransposons showed that the copy number varies with the subgroup and genome com-
position (Teo et al., 2005). Therefore, it could used as potential marker in Musa for characterization
of genome composition, identification of ancestral genomes (Teo et al., 2005), and classification
of varieties. The IRAP primer designed based on the LTR sequence of banana Ty3-gypsy-like
retroelement (M. acuminata Monkey retrotransposon, AF 1433332) was used by Nair et al. (2005)
to identify the B genome in banana cultivars. A second primer pair designed from the B-specific
bands of M. balbisiana ‘Pisang Gala’ was used to classify AAB and ABB cultivars. Among the 36
cultivars tested with this primer, the B-specific band was absent in the AA and AAA cultivars but
present in all interspecific hybrids with the B genome, including AB, AAB, and ABB cultivars. In
ABB genomes, the band intensity was high when compared to those observed in AAB genomes.
B-genome dosage was also reported by Pillay et al. (2000) and Nwakanma et al., (2003a). IRAP has
also been used by Asif and Othman (2005) to discriminate the Fusarium wilt-resistant and suscep-
tible somaclones of cultivar ‘Rasthali.’
Preliminary molecular characterization of M. swarnaphalya was carried out using RAPD and
IRAP markers to confirm its uniqueness among wild M. acuminata and M. balbisiana that was
observed from its morphological characterization. Three distinct groupings were observed. In one
group, M. swarnaphalya grouped with M. itinerans and M. nagensium into a distinct cluster, sug-
gesting its unique species status within section Eumusa. The three wild acuminata species formed
one cluster, while all three M. balbisiana species (both wild and cultivated) grouped into the third
cluster. Molecular characterization using retroelements (IRAP markers) also exhibited similar
results, with M. swarnaphalya sharing 58% similarity with members of Eumusa (M. acuminata
ssp. burmannica and M. acuminata ssp. bumannicoides) and was distinct from Rhodochlamys and
Ensete members (Anon, 2005). IRAP was found to be more robust than RAPD in studying the
intragroup diversity within Cavendish clones (Saraswathi et al., 2009a).
2.6.2.4 Amplified Fragment Length Polymorphism (AFLP)

AFLP has been used to determine the degree of intra- and intersectional and intra- and interspecific
genetic variation in Musa (Ude et al., 2002a, 2003; Tugume et al., 2002; Wong et al., 2002). Studies
of Ude et al. (2002b) indicated that there is much more genetic diversity within M. balbisiana than
those suggested using morphological descriptors.
Bhat et al. (2004) showed that it was possible to identify differences between two accessions
of the AAB genome cultivar ‘Rasthali,’ indicating the presence of intracultivar genetic variation.
Significant genetic diversity was also present among AA, AB, and ABB Indian banana cultivars,
supporting the notion that India, along with other neighboring Southeast Asian countries, is the
center of diversity for cultivars of banana and plantain (Bhat et al., 2004). AFLP proved to be a
useful tool in the identification of clones and related cultivars, as well as for distinguishing between
banana cultivars from China (Wong et al., 2002). Molecular phylogeny using AFLP suggested that
species of section Rhodochlamys had a close affinity with species of section Eumusa, and that sec-
tion Australimusa could be merged with those of section Callimusa (Wong et al., 2002). Sequence
related amplified polymorphism (SRAP) was found to be comparatively better than AFLP in ana-
lyzing the genetic diversity of banana and plantain (Youssef et al., 2010).
2.6.2.5 Minisatellites and Microsatellites

Kaemmer et al. (1992) used oligoprobes to differentiate various genomic groups in Musa.
Subsequently, Bhat et al. (1995) used oligoprobes for cultivar identification and overall genome
analysis to establish relatedness among Musa germplasm accessions. The frequency of microsat-
ellites expected in bananas is one sequence-tagged microsatellite site (STMS) per 30 kp, as that
of humans (Lagoda et al., 1998). Weising et al. (1996) were the first to use microsatellite-based
markers to characterize banana and plantain. Later, the discrimination potential of STMS markers
was explored by Grapin et al. (1998) to confirm subspecies organization and clarify some clonal
relationships. Oriero et al. (2006) were able to distinguish diploid from triploid cultivars through
hierarchical cluster analysis. Creste et al. (2003, 2005) used microsatellite markers to classify the
genotypes according to their genomic group and subgroups. However, separation of wild diploid
genotypes from the cultivated ones was difficult, indicating a common origin of these genotypes
(Creste et al., 2004). Sotto and Volkaert (2004) reported that M. acuminata microsatellites are usu-
ally less polymorphic in M. balbisiana than in M. acuminata.
In a comparative analysis made by Crouch et al. (1999), all three assays—namely, RAPD, the
variable number of tandem repeats (VNTR), and AFLP—detected a high level of polymorphism
between parental genotypes and within progeny populations. However, AFLP had the highest mul-
tiplex ratio while VNTR detected the highest levels of polymorphism. Further VNTR and RAPD
analyses suggested a high frequency of homologous recombination, which is quite useful in the
introgression of useful traits from exotic germplasm.
De Langhe et al. (2005) tried to integrate the morphotaxonomic and molecular data while classify-
ing the African plantains. The mismatch observed implies that either morphological characters are
being influenced by the environment or the markers have not sampled critical regions of the genome.
Genetic diversity and phylogenetic studies were undertaken to identify possible progenitors of
‘Silk’ banana varieties (Uma et al., 2008). Microsatellite markers distinguished acuminata wild,
balbisiana wild, and bispecific cultivated ‘Silk’ accessions. The results suggest that, irrespective
of geographical locations of origin, the ‘Silk’ group of bananas has a narrow genetic base with
more than 80% similarity and that its origin could be from a single genotype. The same study also
clustered ‘Khungsong’ wild, a wild M. balbisiana with ‘Silk’ and M. acuminata instead of M. bal-
bisiana as expected, suggesting that it may be involved in evolution of the ‘Silk’ group (Uma et al.,
2008). Microsatellite markers were used to characterize 41 accessions from sections Eumusa and
Rhodochlamys (Durai, 2008). The two groups were distinct, with Rhodochlamys showing more
affinity with species of M. acuminata rather than those of M. balbisiana. Microsatellites were useful
in identifying duplicates, synonyms, mutants, and unique accessions in the ABB group of Indian
origin. This study proposed ‘Bontha’ as a new subgroup in ABB cooking bananas (Saraswathi et
al., 2009b).
2.6.2.6 DArT Markers
Genetic diversity of carotenoid-rich bananas was evaluated by diversity arrays technology (DArT)
(Amorim et al., 2009) towards the development of novel cultivars with functional properties.
2.6.2.7 Chloroplast and Mitochondrial DNA Polymorphism

Genetic information present in plant mitochondrial and chloroplast DNA is widely used in phylogenetic
studies and population genetics because of its complementarities to nuclear Mendelian segregation.
Nwakanma et al. (2003a) were also able to define two lines of evolution in Musa using six chloro-
plast and two mitochondrial DNA probes. One lineage comprised of the sections Australimusa and
Callimusa (with basic chromosome number x = 10) while species of section Rhodochlamys formed
the other lineage. Species of Eumusa were distributed in both lineages. Section Rhodochlamys
appeared as a sister group of section Eumusa, with M. laterita having a genome that was identical
to some subspecies of the M. acuminata complex. The progenitors of the present-day bananas were
evolutionarily distant from each other. M. balbisiana occupied a basal position in the cladogram,
indicating its primitive status in the evolutionary pattern.
Nwakanma et al. (2003b) also amplified the intergenic spacer (ITS) region of seven M. acumi-
nata and five M. balbisiana accessions using specific primers. A 700 bp fragment produced by all
accessions was then digested with 10 restriction enzymes. Digestion with EcoRV produced markers
for the A and B genomes of Musa. The non-coding cpDNA sequences were utilized by Swangpol et
al. (2007) for assessing the lineage of Musa interspecific hybrids from Thailand.
The population structure of wild M. balbisiana in China was assessed with cpDNA polymerase
chain reaction–restriction fragment length polymorphism (PCR-RFLP). A chloroplast DNA gene-
alogy of 21 haplophytes identified two major clades that correspond to two geographical regions
separated by the Beijiang and Xijiang rivers. Nuclear SSR data also revealed significant geographi-
cal structuring in banana populations (Ge et al., 2005).
2.7 Conclusion
Banana and plantain are ancient fruits domesticated by man to become an important commodity in
terms of trade. Banana has been a versatile tropical crop with buoyancy to adapt to diverse climatic
conditions, season-independent production, with nutritional opulence and high productivity, mak-
ing it popular across 150 countries.
The history of banana cultivation is well documented. Its domestication has been proven
through advanced archeobotanical and paleoecological studies. New studies are revealing infor-
mation about its evolution. Banana taxonomy has undergone a number of revisions, but the devel-
opment of classification keys to identify the greater diversity and variability of new accessions
from previous inaccessible areas need revision. There is a need for more detailed studies on the
parental contribution at the subspecies level. Further research on the diversity in M. balbisiana,
its evolution, and the existence of subspecies is required. The existence of triploid M. balbisiana
remains to be solved. The involvement of other sections, especially Rhodochlamys, which has a
close genetic proximity to Eumusa, their coexistence, and the intersectional introgression, and
natural occurrence of intersectional hybrids can provide a new dimension for the evolution of
extant bananas. Compared to the genus Musa, Ensete is the least researched in spite of its impor-
tance as a food-fiber crop, especially in Ethiopia. Its possible role in the evolution of bananas
needs investigation, especially with the identification of a new species M. kuppiana (Anon, 2005)
in the forests of northeast states that exhibits a transitional status between Musa and Ensete.
Morphotaxonomic characterization has been an important tool in studying diversity and phylog-
eny and, complemented with molecular markers, has provided better resolution for the evolutionary
studies. The application of molecular markers has more benefits in banana compared to seeded plants
due to inherent barriers like parthenocarpy, sterility, polyploidy, and so forth. Molecular analysis of
various genomes across global gene banks more specifically from the areas of Musa origin should
lead to a better understanding and knowledge on Musa origin and evolution. But compared to other
commercial crops, genomic research in banana and plantain is still gaining momentum.
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3 Genetic Resources for
Banana Improvement
Markku Häkkinen and Richard Wallace
Contents
3.1 Introduction............................................................................................................................. 41
3.2 Wild Seeded Musa Species..................................................................................................... 42
3.3 Classification of the Wild Species........................................................................................... 42
3.3.1 Musa Section Australimusa......................................................................................... 42
3.3.2 Musa Section Callimusa Cheesman............................................................................ 43
3.3.3 Musa Section Musa L..................................................................................................44
3.3.4 Musa Section Rhodochlamys (Baker) Cheesman....................................................... 45
3.3.5 Musa Section Ingentimusa Argent.............................................................................. 47
3.4 Conclusion............................................................................................................................... 48
References......................................................................................................................................... 49
3.1 Introduction
Bananas (inclusive of plantains) represent a major source of carbohydrates in the diets of a large
percentage of the world’s population. In terms of worldwide production of food crops, bananas
and plantains rank fourth behind rice, wheat, and corn. The majority of the fruit produced is
consumed locally with only a small percentage (~15%) of the worldwide production entering the
export market (Panis et al., 2007). The development of new edible banana cultivars is required
as a result of the existing parthenocarpic varieties’ susceptibility. Diseases and pests that attack
bananas are discussed in detail in other chapters of this book. Although progress has been
made in the development of new varieties of edible bananas by (employing both traditional and
non-traditional methods of breeding) urgency associated with the development of additional
improved cultivars.
Most of the edible bananas known today are derived from two wild-seeded species, Musa
acuminata Colla and M. balbisiana Colla (Colla, 1820). The fruits of these plants are barely
edible and contain numerous seeds with only a small amount of edible pulp (Simmonds, 1962).
The edible bananas grown today belong to one of three categories. One of the groups displays the
characteristics of M. acuminata, another displays the characteristics of M. balbisiana, and a third
group displays characteristics of both (Simmonds, 1962, 1966). Diploids of M. acuminata gave
rise to seedless fruit when they became parthenocarpic and sterile (Simmonds, 1962). These par-
thenocarpic fruit-producing plants were vegetatively propagated via divisions known as “pups” or
“suckers.” As the production scale was increased and large numbers of these sterile cultivars were
grown in close proximity, pathogens began to attack the plants (Simmonds, 1966). Employing
these parthenocarpic plants has resulted in them becoming susceptible to diseases and pests due
to a lack of genetic diversity. This chapter focuses on the wild-seeded bananas in the genus Musa
that represent some of the best sources of genetic diversity that can be used in the breeding of
new edible bananas.
41
3.2 Wild Seeded Musa Species

Over the last 150 years, various botanists have categorized wild bananas into sections or subgenera.
Linnaeus was the first to assign scientific nomenclature to bananas in his book Species Plantarum,
published in 1753. He then established the modern botanical nomenclature that is still in wide use
today. Sagot (1887) and Baker (1893) distinguished three subgenera for the genus Musa: M. subgen.
Physocaulis, M. subgen. Eumusa, and M. subgen. Rhodochlamys. Cheesman (1947, publ. 1948)
developed a clear and coherent classification system for the genus Musa. His original grouping of
the species in the genus Musa into four sections proved to be very useful and it has, therefore, been
widely accepted. However, he indicated that “the groups have deliberately been called sections
rather than subgenera in an attempt to avoid the implication that they are of equal rank.” He further
pointed out that this work “may stimulate investigation of a genus that is difficult to collect and
study, but sufficiently interesting and important in both economic and its more strictly botanical
aspects to repay the investigators.” In Cheesman’s classification, the genus was divided into four
sections: Australimusa 2n = 20, Callimusa 2n = 20, Musa (Eumusa) 2n = 22, and Rhodochlamys 2n
= 22 (Cheesman, 1947, 1948a, 1948b, 1949a, 1949b, 1950; Shepherd, 1959, 1999; Champion, 1967;
Simmonds and Weatherup, 1990; Häkkinen and Sharrock, 2002; Häkkinen, 2004a, 2007, 2009a,
2009b; Häkkinen et al., 2007; Häkkinen and Väre, 2008c). Argent (1976) added one more section,
Ingentimusa 2n = 14, comprised of a single species, Musa ingens Simmonds.
As evidenced from the above discussion, the genus Musa contains a number of members with
various characteristics. Some of the members of the different sections are compatible in a conven-
tional breeding sense and some intersectional hybrids have been reported (vide infra). The incor-
poration of genetic traits (disease and pest resistance, drought and cold tolerance, etc.) from the
sections listed above and described in the subsequent paragraphs will play an important role in the
development of new, improved hybrid bananas for use by future generations.
3.3 Classification of the Wild Species

3.3.1 Musa Section Australimusa
Musa section Australimusa (originally reported in Kew Bulletin 2:11 in 1947 by Cheesman; type:
Musa textilis and by Née, published in Anales de Ciencias Naturales 4:123 in 1801) originated from
the Philippines through the Indonesian archipelago to Irian Jaya, Papua New Guinea, and northern
Australia. The section Australimusa consists of nine described species:
M. boman Argent (Argent, 1976)

M. bukensis Argent (Argent, 1976)
M. fitzalanii M. Muell. (Mueller, 1875)
M. jackeyi W. Hill (Hill, 1874)
M. johnsii Argent (Argent, 2001)
M. lolodensis Cheesman (Chessman, 1950)
M. maclayi M. Muell (Mueller, 1885; Argent, 1976)
M. peekelii Lauterb (Lauterbach 1914; Häkkinen and Väre, 2009)
M. textilis Née (Nee, 1801)
There is also a group of parthenocarpic edible types of Australimusa that are known as Fe’i bananas
(Musa fehi Bertero, in Viellard, 1862; MacDaniels, 1947). These cultivars are distinguished by their
erect bunches and red sap and are found almost exclusively in the Pacific region. The red sap from
this group of plants is unusually stable to light exposure and has been employed as dye and ink. The
fruit from this group of plants must be cooked prior to eating as it is very astringent when raw. As
is the case with other members of the genus Musa, various parts (leaves, fiber, pseudostems, etc.)
Genetic Resources for Banana Improvement 43
Figure 3.1 Musa textilis Née. (Courtesy of Markku Häkkinen.)
of plants from the Australimusa section are used in a variety of applications in the region where
they occur. Hybrids between the members of Australimusa and Musa (formerly known as Eumusa;
Simmonds, 1956, publ. 1957) sections have also been identified (Carreel, 1994).
3.3.2 Musa Section Callimusa Cheesman

Musa section Callimusa Cheesman (originally published by Cheesman in 1947 in Kew Bulletin
2:112; type: Musa Coccinea, and by Andrews in Botanist’s Repository 1, plate 47, in 1797) origi-
nated on the Asian continent from southern China to northern Vietnam, peninsular Malaysia,
Sumatra, and Borneo. The section Callimusa consists of 22 described species:
M. azizii Häkkinen (Häkkinen, 2005a)

M. barioensis Häkkinen (Häkkinen, 2006a)
M. bauensis Häkkinen and Meekiong (Häkkinen and Meekiong, 2005a)
M. beccarii N. W. Simmonds (Simmonds, 1960; Häkkinen et al., 2005)
M. borneensis Becc. (Beccari, 1902; Häkkinen and Meekiong, 2005b)
M. campestris Becc. (Beccari, 1902; Häkkinen, 2003b, 2004a, 2004b)
M. coccinea Andrews (Andrews, 1797)
M. exotica R. V. Valmayor (Valmayor, 2001)
M. gracilis Holttum (Cheesman, 1950)
M. hirta Becc. (Beccari 1902, Häkkinen 2004b)
M. lawitiensis Nasution and Supard (Nasution and Supardiyono, 1998; Häkkinen, 2006b)
M. lokok Geri and Ng (Geri and Ng, 2005)
M. lutea R. V. Valmayor (Valmayor, Danh, and Häkkinen, 2004)
M. monticola Argent (Argent, 2000)
M. muluensis M. Hotta (Hotta, 1967)
M. paracoccinea A. Z. Liu and D. Z. Li (Liu and Li, 2002)
M. sakaiana Meekiong, Ipor, and Tawan (Meekiong, Ipor, and Tawan, 2005)
M. salaccensis Zoll. ex Backer 1924 (Backer, 1924; Häkkinen and Väre, 2009)
M. tuberculata M. Hotta (Hotta, 1967)
M. violascens Ridl. (Ridley, 1893)
M. viridis R. V. Valmayor, L. D. Danh and Häkkinen (Valmayor, Danh and Häkkinen,
2004)
M. voonii Häkkinen (Häkkinen, 2004a)
Figure 3.2 Musa campestris Becc. (Courtesy of Markku Häkkinen.)
Ornamental bananas of the section Callimusa (Cheesman, 1947; Häkkinen, 2004a; Häkkinen and Väre,
2008c) are very attractive plants of the genus Musa L. that are largely grown for their brightly colored
inflorescences. Some of the colors found in the bracts of the members of this section include pink, red,
orange, yellow, purple, and white. In some cases, the fruit itself is colored, which further increases the
ornamental potential of the plants. The fruits from plants in this section are unsuitable for eating due to
the large number of seeds and small amount of edible pulp. The members of the Callimusa section are
smaller plants (in both height and diameter) than the members of the section Musa.
3.3.3 Musa Section Musa L.

Musa section Musa (originally published in Species Plantarom 1043 in 1753; Type: Musa paradi-
siaca L. Autonym Musa [Eumusa] and by Cheesman in Kew Bulletin 2:108 in 1947) originated in
southern India through the Asian continent to the Philippines, Sumatra, Borneo Indonesia, Papua
New Guinea, and northern Australia. The section Musa consists of 15 described species:
M. acuminata Colla (Colla, 1820; Cheesman, 1948; Simmonds, 1956; Häkkinen and De
Langhe, 2001)
M. balbisiana Colla (Colla, 1820; Cheesman, 1948; Simmonds, 1956; Argent, 1976)
M. banksii F. Muell. (Mueller, 1864)
M. basjoo Iinuma (Iinuma, 1874)
M. celebica Warb. (Warburg, 1900)
Figure 3.3 Musa balbisiana Colla. (Courtesy of Markku Häkkinen.)
M. cheesmanii N. W. Simmonds (Simmonds, 1956)

M. griersonii Noltie (Noltie, 1994)
M. itinerans Cheesman (Cheesman, 1949; Häkkinen et al., 2008)
M. lanceolata Warb. (Warburg, 1900)
M. nagensium Prain (Prain, 1904; Häkkinen, 2008)
M. ochracea K. Sheph. (Shepherd, 1964)
M. schizocarpa N. W. Simmonds (Simmonds, 1956)
M. sikkimensis Kurz (Kurz, 1877)
M. thomsonii (King) Baker (Baker, 1893; Cheesman, 1948; Cowan and Cowan, 1929)
M. yunnanensis Häkkinen and H. Wang (Häkkinen and Wang, 2007, 2008b)
The vast majority of the banana and plantain cultivars grown worldwide for fruit production are
contained in this section of the genus Musa.
Although the majority of the members of this section are well known, the most recently discov-
ered member of this section, M. yunnanensis, has interesting characteristics that may warrant its
further exploration in banana breeding research. The plant was originally discovered in the Mekong
River watershed at elevations of 500–1800 m (Häkkinen and Wang, 2007). It tolerates seasonal
frost damage (and snow), which occurs during January and February in its habitat. The plant is
also grown at higher elevations (up to 2100 m) where the stems and leaves are used as animal fod-
der. Three additional varieties of M. yunnanensis have also recently been reported (Häkkinen and
Wang, 2008b). In addition to the resistance to cold displayed by M. yunnanensis and its varieties, it
also appears to be disease resistant, as no common banana diseases have been observed in the dif-
ferent populations studied to date (M. Häkkinen, personal observation).
3.3.4 Musa Section Rhodochlamys (Baker) Cheesman

Musa section Rhodochlamys (originally reported by Cheesman in Kew Bulletin 2:110 in 1948; type:
Musa ornata Roxb Fl Ind 2:488 in 1824, Basionym: Musa spp. Rhodochlamys Baker in Annals of
Botany [Oxford] 7:204 in 1893) originated on the Asian continent from Tibet to Cambodia, including
western Yunnan. The members of this section are unique within the genus Musa in that they are tol-
erant to drought. In their native environments, plants in this section go dormant during the dry season
but grow very quickly when the rainy season arrives. They then reach maturity and produce fruit and
seeds prior to the return of the dry season when they again go dormant. The section Rhodochlamys
consists of 12 described species from continental Asia ranging from Tibet to Cambodia:
M. aurantiaca Baker (Baker, 1893; Häkkinen and Väre, 2008a)

M. chunii Häkkinen (Häkkinen, 2009a)
M. dasycarpa Kurz (Kurz, 1867; Häkkinen and Väre, 2008b)
M. laterita Cheesman (Cheesman, 1949b; Häkkinen, 2001)
M. mannii Baker (Baker, 1892; Hooker, 1893; Häkkinen, 2007)
M. ornata Roxb. (Roxburgh, 1824; Häkkinen and Väre, 2008c)
M. rosea Baker (Baker, 1893; Häkkinen, 2006c)
M. rubinea Häkkinen and Teo (Häkkinen and Teo, 2008)
M. rubra Kurz (Kurz, 1867; Hooker, 1895; Häkkinen, 2003a)
M. sanguinea Hook. f. (Hooker, 1872; Häkkinen, 2007)
M. siamensis Häkkinen and Rich. H. Wallace (Häkkinen and Wallace, 2007)
M. zaifui Häkkinen and H. Wang (Häkkinen and Wang, 2008a)
The majority of the members of this section possess erect inflorescences with brightly colored
bracts. Like the members of the section Callimusa, most of the members of this section are grown
largely for their ornamental value (Hakkinen, 2005b). Some intersectional hybrids between the
Figure 3.4 Musa siamensis Häkkinen and Rich. H. Wallace. (Courtesy of Richard Wallace.)
Figure 3.5 Musa x georgiana Rich. H. Wallace. (Courtesy of Richard Wallace.)
members of the sections Musa and Rhodochlamys have been reported in the past (Simmonds, 1962;
Shepherd 1999; Uma et al., 2006), but these hybrids were not described or characterized in detail by
the authors. A new intersectional hybrid, Musa x georgiana Rich. H. Wallace (Figure 3.5) has been
recently described and illustrated (Wallace and Häkkinen, 2009). The hybrid has M. balbisiana
(one of the seeded members of the section Musa and an ancestor of edible bananas) as its female
parent and M. velutina (a brightly colored member of the section Rhodochlamys) as its male parent.
Due to the ever-increasing pest and disease pressure edible bananas are experiencing, incorporating
valuable genetic characteristics (drought tolerance, disease and/or pest resistance, etc.) from other
members of the Musa genus may represent an important component to the development of new
hybrid edible bananas. In this respect, intersectional hybrids such as M. x georgiana may provide
useful information for banana breeding programs.
3.3.5 Musa Section Ingentimusa Argent

The sap from this plant (originally reported in Notes of the Royal Botanical Gardens of Edinburgh
35(1):111 in 1976; type: Musa ingens, and by N. W. Simmons in Kew Bulletin 14:198 in 1960) is
white-milky in young suckers and watery in older parts. The bracts are dull cream becoming gray-
brown on lifting, with the fruit ripening yellow to coppery brown. Simmonds (1960) notes after
describing M. ingens: “It will probably prove to be best placed in a new section of the genus.” This
seems to be the only logical course of action since it shows no obvious morphological affinity with
any other banana species, and the chromosome number is still unique in the genus, confirming its
isolated position.
In Papua New Guinea, M. ingens occurs in the Western Highlands District: Kubor range,
Kamang, Minj valley, above the Tsau River north of Banz (observed by Argent); Madang District:
Bundi Kara near Bundi Patrol Post (obs.); Eastern Highlands District: Kassam Pass (obs.); east-
ern slopes of Mt. Piora (obs.); Morobe District: eastern slopes of Mt Shungol (obs.); Sarawaket
Mountains near Kasonombe (obs.); above Mindik (obs.); and the Northern District: northeast slopes
of Mt. Victoria above Kokoda [(obs.), Argent 1976]. This species is common in several parts of the
Figure 3.6 Musa ingens N. W. Simmonds. (Courtesy of Jeff Daniells.)
highlands and undoubtedly will be found in many more localities. Variation in male bud shape may
have geographical basis but too little information is at present available. The altitudinal range of the
species is from 1000 to 2100 m, although fruiting specimens appear to be restricted to the lower 600
m of the altitudinal range. The seed germinated at sea level in Lae (6°44'0" S, 147°0'0" E), Papua
New Guinea, but did not establish; suckers brought down to the lowlands became progressively
more emaciated and died unless placed in air conditioning at least during the night. The species thus
seems intolerant of continuous high temperatures.
3.4 Conclusion
The genus Musa is composed of five sections—Australimusa, Callimusa, Musa, Rhodochlamys,
and Ingentimusa—that possess a rich diversity of genetic traits and morphological characteristics.
Contained within the section Musa (Eumusa) are the edible bananas and plantains, which are of
great importance both as an export crop and as a staple carbohydrate to a large portion of the world’s
population. Due to the ever-increasing pest and disease pressure edible bananas and plantains are
experiencing, incorporating valuable genetic characteristics (drought tolerance, disease and/or pest
resistance, etc.) from other members of the genus Musa will continue to represent an important
component in the development of new hybrid edible bananas for use by future generations.
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4 Genomes, Cytogenetics, and
Flow Cytometry of Musa
Michael Pillay and Abdou Tenkouano
Contents
4.1 Introduction............................................................................................................................. 53
4.2 Polyploidy, Origin, and Variation in Cultivated Musa............................................................ 54
4.3 Genomes Identified in Musa.................................................................................................... 55
4.3.1 In Situ Hybridization................................................................................................... 56
4.3.2 Flow Cytometry........................................................................................................... 57
4.3.3 Isozymes and Retrotransposons.................................................................................. 57
4.3.4 Molecular Markers for A and B Genome Sequences.................................................. 57
4.4 Cytogenetics of Musa.............................................................................................................. 58
4.4.1 Mitotic versus Meiotic Chromosomes......................................................................... 58
4.4.2 Molecular Cytogenetics............................................................................................... 59
4.5 Flow Cytometry in Musa.........................................................................................................60
4.5.1 Ploidy Analysis in Musa.............................................................................................. 61
4.5.2 DNA Content, Genome Size, and Base Composition.................................................. 62
4.5.3 Gene Content and Density........................................................................................... 63
4.6 Cytogenetic and Fertility Relationships in Musa.................................................................... 63
4.7 Conclusion...............................................................................................................................64
References.........................................................................................................................................64
4.1 Introduction
Banana and plantain (Musa spp.) are vegetatively propagated crops that are both essential compo-
nents of the diet and important sources of income for about 400 million people in over 120 countries
in the tropical and subtropical zones (Jones, 2000). In this chapter the term banana is used collec-
tively for both bananas and plantains.
Banana production in many countries is seriously threatened by a complex of fungal and bac-
terial diseases, nematodes, viruses, and insect pests (Pillay et al., 2002). Banana breeding pro-
grams use diverse techniques, from conventional (Pillay et al., 2002; Pillay and Tripathi, 2006,
2007) to mutation breeding (http://mvgs.iaea.org/, accessed 23 April 2010) and asexual biotechno-
logical approaches such as somaclonal variation, protoplast engineering, and somatic hybridization
(Hwang, 2002; Hwang and Ko, 1988, 1989, 2002, 2004; Assani et al., 1996, 2005; Matsumoto et
al., 2002) to create improved varieties. Plant breeding is multidisciplinary in nature, making use
of knowledge from several scientific disciplines, including genetics, agronomy, horticulture, crop
protection, cytogenetics, and many others (Fehr, 1987). Chromosomes have a special importance
in plant breeding since they harbor the genes that constitute genetic linkage groups. Therefore,
changes in chromosome structure and function are important determinants of trait inheritance
and expression. Thus, segregation distortions in the construction of linkage maps in banana were
explained by chromosomal structural rearrangements (Fauré et al., 1993). Meiotic analysis of Musa
53
revealed chromosome inversions and translocations (Wilson, 1946) and other meiotic abnormalities
(Adeleke et al., 2004). Therefore, better knowledge of chromosome structure and evolution in Musa
is required.
Over the last 20 years, chromosome studies have shifted from a purely cytogenetic level and
dying science to the molecular level ushering in an era of molecular cytogenetics. The development
of new techniques for genome analysis has made it possible to examine in greater detail the struc-
ture of plant genomes. These techniques include restriction fragment length polymorphism (RFLP),
the various methods using polymerase chain reaction (PCR) such as random amplified polymorphic
DNA (RAPD), microsatellites or simple sequence repeats (SSR) or variable number of tandem
repeats (VNTR), amplified fragment length polymorphism (AFLP), fluorescent and genomic in
situ hybridization (FISH and GISH), diversity array techniques (DArT; Jaccoud et al., 2001), and
high-resolution DNA melting (high resolution DNA melting [HRM]) (de Koeyer et al., 2009). High-
throughput technologies such as single nucleotide polymorphisms (SNPs) or small-scale insertions/
deletions (indels) allow easy and unambiguous identification of alleles or haplotypes (Bhattramakki
and Rafalski, 2001).
The exploitation of plant genomes and genes for plant breeding is now becoming better recog-
nized with the advent of molecular markers, marker-assisted selection, and molecular and cytoge-
netic maps. Although the genus Musa has benefited immensely from such studies, there is scope for
greater in-depth research into the cytogenetics and molecular genetics of the genus. Some progress
has been made since our last treatment of this topic (Pillay et al., 2004). This chapter provides a
summary of our current understanding of cytogenetics, genomes, and flow cytometry in Musa and
their applications in genome analysis and breeding. This chapter first considers the physical char-
acteristics of the genomes in Musa and the methods used for their identification, then examines the
current status of conventional and molecular cytogenetics, and finally discusses the role of flow
cytometry in genome analysis.
4.2 Polyploidy, Origin, and Variation in Cultivated Musa

Banana is a polyploid crop with over 50 species, not all of which are edible. Wild species are dip-
loid (2n = 2x = 22), while the cultivated varieties are primarily triploid (2n = 3x = 33) with a few
tetraploids (2n = 4x = 44) that are mainly derived either from natural or artificial hybridization
(Robinson, 1996). Musa is divided into five sections (Australimusa, Callimusa, Rhodochlamys,
Eumusa, and Ingentimusa) that vary in the basic number of chromosomes (Stover and Simmonds,
1987; Purseglove, 1988). The Callimusa and Australimusa have a basic chromosome number of x =
10, while Eumusa and Rhodochlamys have a basic chromosome number of x = 11. Ingentimusa with
a single species, M. ingens, has a chromosome number of 2n = 14. The Callimusa and Rhodochlamys
consist of nonparthenocarpic (seed-bearing) species that have no nutritional value but are impor-
tant as ornamentals. Australimusa consists of parthenocarpic edible types, collectively known as
Fe’i bananas, with erect fruit bunches and a red sap that is diagnostic for the section. The section
is important for food and fiber, and a valuable dark red dye is obtained from the pseudostems.
The Australimusa has the highest vitamin A content among all bananas (Englberger et al., 2003a,
2003b, 2003c). Eumusa is the largest, most widely distributed and diversified, and most important
section containing all edible bananas (Horry et al., 1997). Musa acuminata (A genome) is the most
widespread of the Eumusa species and is found throughout the range of the section, with Malaysia
(Simmonds, 1962) or Indonesia (Nasution, 1991; Horry et al., 1997) as the center of diversity. New
molecular data show that the first center of domestication for Musa was in the Philippines–New
Guinea area (Carreel et al., 2002). Musa balbisiana is the other wild Eumusa species that had
broad distribution across South and Southeast Asia. Musa balbisiana has adopted to drier regions
but exhibits copious growth in tropical and subtropical forests of the Philippines, Thailand, New
Guinea, India, South China, Malaysia, Myanmar, Indonesia, and a few other regions (Valmayor et
al., 2002; Uma et al., 2005).
Genomes, Cytogenetics, and Flow Cytometry of Musa 55
Intraspecific hybridization between and among the various subspecies of M. acuminata pro-
duced a range of diploid cultivars with AA genomes. Diploid AAs produced triploid AAA types by
mechanisms such as chromosome restitution (Shepherd, 1999). Interspecific hybridization between
AA diploids and M. balbisiana (BB) gave rise to the many AAB and ABB types (Robinson,
1996). Other genomic groups including AB, ABBB, AAAB, and AABB also exist (Simmonds and
Shepherd, 1955).
It is possible that a range of diverse M. balbisiana genotypes were involved in the hybridizations,
creating the variability that is observed in extant B genome containing banana clones. Isoenzyme
analysis (Lebot et al., 1993), sequence tagged microsatellite analyses (Kaemmer et al., 1997), ampli-
fied fragment length polymorphisms (Ude et al., 2002a; Wang et al., 2005), and chloroplast DNA
analysis (Ge et al., 2005) coupled with morphological variation (Shepherd, 1988; Hari, 1989; Sotto
and Rabara, 2000) revealed that M. balbisiana is a polymorphic species. Although variability in
Musa cultivars is mainly attributed to natural mutations (de Langhe, 1964), other mechanisms such
as somaclonal variation, transposable element activity, new genome combinations, polyploidy,
genome duplications, mitotic recombination, and recombination of novel alleles could have played
a role in this process (Nyine and Pillay, 2010). Data on the rate of natural mutations in crop plants
are scarce. Pillay et al. (2003a) assessed the rate of natural mutation in the banana cultivar ‘Sukali
Ndizi’ to be F = 0.0003. At this mutation rate, it is expected that greater variation should be pres-
ent in extant banana cultivars. Although somaclonal variation is defined as the genetic variation in
plants regenerated from cultured somatic cells (Larkin and Scowcroft, 1981), Cullis (2005) indi-
cated that somaclonal variation may not be due only to in vitro propagation but may occur natu-
rally in plant somatic and reproductive tissues. Much of the natural morphological variability that
was observed in plantains was also observed in somaclonal variants in plantains (Vuylsteke et al.,
1991). Somaclonal variation was cited as the reason for the change in bunch morphology observed
in tissue-cultured ‘Agbagba’ variants that also showed increased fertility (Vuylsteke et al., 1991).
Somaclonal variation has been associated with genomic changes in Musa. Thus, Oh et al. (2007)
found at least one labile portion of the banana genome that is highly reactive to stress induced
during tissue culture, suggesting that other mechanisms may also be responsible for variation in
banana. Transposable elements have been identified in Musa (Balint-Kurti et al., 2000) although
no direct links have been established, as yet, between variation and the transposable elements. It is
known that transposable elements bring about variation in plants and animals (Kidwell and Lisch,
1997), and retrotransposons of rice were involved in mutations induced by tissue culture (Hirochika
et al., 1996).
4.3 Genomes Identified in Musa

Genome composition has played an important role in the classification of bananas (Simmonds and
Shepherd, 1955). Four genomes—A, B, S, and T—are presently known in cultivated bananas (Pillay
et al., 2004). The A, B, and S genomes are present in section Eumusa, with the S genome character-
istic of M. schizocarpa, and the T genome confined to the section Australimusa. Most modern cul-
tivars of banana have one or more copies of the A and B genomes (Simmonds and Shepherd, 1955),
whereas the S and T genomes occur in only a few (du Montcel, 1989; Sharrock, 1989; Carreel, 1995).
Classification of edible bananas into genomic groups is based on a system created by Simmonds
and Shepherd (1955). This system assigns a score of 1 to 5 for 15 selected morphological features
that differentiate M. acuminata from M. balbisiana. To determine the genome composition of a
cultivar, the 15 morphological characters are scored, with the total score determining the relative
contribution of the A and B genomes to the constitution of a clone. The system is complicated
by polyploidy and requires chromosome counts of a clone before its genome constitution can be
determined. The classification system described above was revised by Silayoi and Chomchalow
(1987) who introduced the new BBB clones in the system. The existence of pure BBB clones in
Musa remains questionable and is not supported by molecular methods (Pillay et al., 2000) or field
observations (Buddenhagen, personal communication). Thus, Valmayor et al. (1981) endorsed the
original scheme proposed by Simmonds and Shepherd (1955).
Although Simmonds and Shepherd’s (1955) classification system is useful and reliable and agrees
with recent molecular methods, it has some limitations. It can only be applied to diploid, triploid,
and tetraploid clones with the A and B genomes and does not cater for cultivars with the S and T
genome characteristics. It is also difficult to study the evolution and taxonomy of the genus only by
means of morphological markers because of the wide range of genetic variation existing in Musa
(Robinson, 1996; Ortiz, 1997).
Banana clones that share similar characteristics and are considered to have arisen from a single
clone by mutation are classified as subgroups. A list of the subgroups and some important clones
that constitute each genomic group in Musa can be found in Robinson (1996) and Jones (2000). It is
worth noting that banana clones with similar genomes may share similar characteristics but can be
very different. For example, the AAA group contains the sweet dessert bananas as well as the cook-
ing bananas of the East African highlands. The latter is starchy even in the ripe stage and is generally
cooked before eating. This may suggest the presence of different A genomes in Musa (Ude et al.,
2002a). Various combinations of the different A genomes produce different characteristics. The identi-
fication of different A genomes remains an interesting research topic. Sequencing of the Musa genome
may be able to provide some answers to this question. Similarly, the AAB group includes the plantains
that are cooked before becoming palatable and the ‘Pome’ subgroup (AAB) that is widely used as
dessert bananas in some countries. Although the clones of the ABB group are rather homogeneous
because all clones are cooked, some are also used as dessert banana, when overripe, in Malawi.
Cultivars with the S and T genomes have not spread throughout the world as did those with the A
and B genomes. They have been identified only in Papua New Guinea (Shepherd and Ferreira, 1984;
Arnaud and Horry, 1997). Genomic groups with the S genome include diploid AS, triploid AAS,
and tetraploid ABBS, while T genome groups include AAT, AAAT, and ABBT. Molecular marker
methods were used to show that M. schizocarpa and one or more species of section Australimusa
played a role in the origin of some New Guinea cultivars (Carreel, 1995). Further confirmation for
the involvement of the S and T genomes in some of these cultivars was elucidated by genomic in situ
hybridization (D’Hont et al., 2000).
Several different methods have been used either directly or indirectly to identify the genomes
in Musa.
4.3.1 In Situ Hybridization

Osuji et al. (1997a) were the first to apply molecular cytogenetic techniques for genome identifica-
tion in Musa. This was followed by physical mapping of the 18S-25S and 5S ribosomal RNA genes
of banana (Dolezelova et al., 1998). These studies used GISH and FISH, respectively, to differenti-
ate the chromosomes present in different genome combinations. Samples used in the above studies
were comprised of only the A and B genomes and/or their combinations. The study by D’Hont et
al. (2000) included accessions with the A, B, S, and T genomes and some of their combinations.
Genomic DNA from species representing the different genomes (M. acuminata, AA; M. balbisi-
ana, BB; M. schizocarpa, SS; and M. augustigemma, TT) were used as probes to identify chromo-
somes from the A, B, S, and T genomes, respectively. Together, these studies made it possible to
differentiate the four genomes in banana cultivars and hybrids using different fluorochromes. For
example, plantains (AAB) were shown to have 22 A genome chromosomes and 11 chromosomes
of B genome origin. Similarly, ‘Wompa’ (AS) displayed 11 chromosomes each from the A and S
genomes, respectively (D’Hont et al., 2000). More interesting and for practical breeding reasons
was the identification of genomes in synthetic hybrids of Musa. In a diploid hybrid (‘TMP2x’) from
the cross ‘Obino l’Ewai’ (AAB) × ‘Calcutta 4’ (AA), GISH experiments showed that there were
22 A genome chromosomes, strongly suggesting that each parent contributed one set (11 chromo-
somes) of the A genome. Perhaps more interesting was the observation of 33 A genome and 11 B
genome chromosomes in the tetraploid hybrid ‘TMPx 4698-1’ from the same cross. This result
arose from a normal gamete (A) from ‘Calcutta 4’ and a 2n = 3x egg (AAB) from ‘Obino l’Ewai.’
GISH results were also useful in settling cases of disputed genomic constitution. For example, the
cultivar ‘Karoina’ considered to be either ATT or AAT from morphological descriptors and molecu-
lar markers was found to be AAT. However, exceptions were also identified. For example, ‘Pelipita’
(ABB) was shown to have 8 A chromosomes and 25 B chromosomes instead of the expected 11 A
and 22 B. Similarly, 21 A and 12 B chromosomes were identified in two plantains from Cameroon,
‘M. bouroukou’ and ‘Nyombe’ (D’Hont et al., 2000).
FISH studies showed a high degree of cross-hybridization between the A and B genomes of
banana, suggesting that the two genomes are incompletely differentiated and share common DNA
sequences (Osuji et al., 1997a). D’Hont et al. (2000) reported greater cross hybridization between
the A and B genomes than between the A and S genomes, while the least cross hybridization was
observed between the T and the A or B genomes. The intensity of cross hybridization is, perhaps, a
reflection of the sequence homologies and affinities between the genomes. It also reflects the genetic
distances between cultivars representing the different genomes and corresponds with morphologi-
cal (Simmonds and Weatherup, 1990) and molecular markers analyses (Carreel, 1995; Ude et al.,
2002a, 2002b).
4.3.2 Flow Cytometry
The sizes of the A, B, S, and T genomes were shown to be significantly different from each other
by flow cytometric analysis of DNA content (D’Hont et al., 1999). The S and T genomes are larger
than the A and B genomes, while the T genome has the highest DNA content. This information was
used as a diagnostic tool to identify the different genomes in Musa. Genome size variation was also
used to determine the species involved in interspecific diploid clones as well as the ploidy of banana
clones. However, caution has to be exercised when using DNA content per se to identify and cluster
similar genomes in Musa since the study by Kamaté et al. (2001) showed that DNA content is quite
variable in cultivars with similar genomes. For example, the DNA content for AAB clones ranged
from 1.61 pg to 1.79 pg, while those for ABB clones ranged from 1.70 pg to 2.23 pg.
4.3.3 Isozymes and Retrotransposons

Isozyme patterns were used to distinguish the A and B genomes in Musa (Espino and Pimentel,
1989). Hybridization of the monkey retrotransposon to a HindIII digest of genomic DNA of Musa
produced bands that were specific to the A and B genomes (Balint-Kurti et al., 2000). The only
exception was the cultivar ‘Chakrakeli’ (AAB), which did not show the B-specific band. It was
suggested that ‘Chakrakeli’ is probably of AAA constitution. This assertion is probably true since
‘Chakkarakeli’ is classified as AAA in Stover and Simmonds (1987). Due to differences in spell-
ing of the names in the two literature sources, proper identification of the cultivar is necessary to
confirm these results. Mandal et al. (2001) used isoenzymes to identify banana varieties in India.
Amongst four isozymes, esterase was found to be the most efficient in identifying eight cultivars
out of ten. Teo et al. (2005) used retrotransposons to determine the genome constitution of hybrid
bananas. One of the criticisms that can be leveled against the above studies is the small sample
size. Consequently, a more comprehensive study with a variety of accessions representing different
genomic combinations may be needed to determine the ultimate value of isozymes and retrotrans-
posons as genome markers.
4.3.4 Molecular Markers for A and B Genome Sequences

Identification of PCR markers for detection of A and B genome sequences in Musa was reported
by Pillay et al. (2000). Unique banding profiles for the differentiation of M. acuminata (A genome)
and M. balbisiana (B genome) were obtained with three RAPD primers (A17, A18, D10) from
OPERON Technologies (Alameda, Calif., USA). An interesting aspect of the B genome markers
was that one of the RAPD fragments was diagnostic for clones with two B genomes. These prim-
ers were tested on a sample of 40 accessions representing landraces and hybrids of different ploidy
and genome combinations. PCR assays made it possible to elucidate the genome composition of all
the plants and were generally in agreement with genomic designations obtained from phenotypic
descriptors. Some exceptions were observed. The clones ‘Monthan Saba’ and ‘Bluggoe’ were previ-
ously classified as BBB by Vakili (1967) and BBB and ABB, respectively, by Valmayor et al. (1981).
Both ‘Monthan Saba’ and ‘Bluggoe’ were shown to be ABB with the RAPD marker system. Using
morphological markers, ‘Klue Tiparot’ was considered to be a tetraploid but was reclassified as a
triploid (Jenny et al. 1997; Horry et al., 1998). The RAPD marker system showed that ‘Klue Tiparot’
was a triploid ABB and resolved the conflicting classifications derived from morphological mark-
ers. In later experiments we found that ‘Lep Chung Kut’ usually classified as BBB in Jones (2000)
and Stover and Simmonds (1987) is an ABB clone. This result puts into question the existence of
naturally occurring BBB clones. As shown in Lysak et al. (1999), ‘Red Dacca’ (AAA) clustered
with the AAB clones on the basis of nuclear DNA content. The same study also reported that the
DNA content of ‘Red Dacca’ was slightly lower than expected for other AAA clones. Our RAPD
marker system showed that ‘Red Dacca’ has an AAB genome constitution. It is known that RAPD
can produce varying results in different laboratories. Researchers planning to use the RAPD marker
system are encouraged to use the ‘Red Hot’ Taq polymerase from AB Gene (UK) to obtain com-
parable results.
The internal transcribed spacer (ITS) regions of the ribosomal RNA genes also provided mark-
ers for the A and B genomes in Musa (Nwakanma et al., 2003). A 530 bp fragment unique to the
A genome and two fragments of 350 bp and 180 bp specific for the B genome were identified.
Interspecific cultivars with both A and B genomes possessed all three fragments. A dosage effect
was observed for the B genomes since the staining intensity of accessions with two B genomes was
approximately two times that of accessions with a single B genome.
Nair et al. (2005) used the IRAP primer to identify the B genome in banana cultivars. The study
was able to identify misidentification of some samples.
4.4 Cytogenetics of Musa
4.4.1 Mitotic versus Meiotic Chromosomes
Banana chromosomes are very small, with size in the range of 1–2 μm, and stain poorly with con-
ventional stains (Dolezel et al., 1998). Even with improved staining techniques (Pillay and Adeleke,
2001; Adeleke et al., 2002) the small chromosomes are difficult to characterize because they con-
dense tightly, making measurements of the long and short arms very difficult, if not impossible.
The mitotic metaphase chromosomes of bananas appear as small dotted structures without any
cytological markers in the fully contracted state. The chromosomes are also homogeneous, mak-
ing it difficult to identify individual chromosomes and impeding the development of karyotypes.
A karyotype of Musa was reported by Dantas et al. (1993). The only chromosomes that are easily
identifiable are the pair of satellite chromosomes that are also the longest chromosomes. Wang et al.
(1993) also reported a single pair of chromosomes with satellites in wild AA bananas. The rest of
the chromosomes appear to be metacentric to submetacentric.
Since the 1960s chromosome banding techniques were used to provide cytological markers
along the length of chromosomes, enabling easy identification of homologous pairs and individual
chromosomes of a karyotype. These techniques exploited the variation of staining intensity along
chromosomes based on chromatin condensation in euchromatic versus heterochromatic regions
(Danilova and Birchler, 2008).
Many different banding techniques are available, each giving a different banding pattern based
on the specific biochemical consequences of each method (Stace, 1980). The common types applied
to plants include C-, G-, Q-, R-, and Hy-banding. Banding techniques of banana chromosomes were
not successful in the authors’ laboratory and in other leading cytogenetics laboratories (Dolezelova
et al., 1998). Perhaps a refinement of current techniques and more extensive studies may be required
to determine the potential of banding techniques in Musa and its role in Musa breeding.
Most cytological studies in Musa have been restricted to mitotic metaphase chromosomes. It
appears that either mitotic prometaphase or pachytene chromosomes may be more promising for
producing karyotypes in Musa (Adeleke et al., 2002). Our unpublished work showed that mitotic
prometaphase chromosomes are generally about three to five times longer than those at the fully
condensed metaphase stage. In such chromosomes the primary and secondary constrictions are
more easily distinguished than in the overcondensed metaphase stage. In most plants with very
small chromosomes, researchers are shifting to the pachytene stage of meiosis for studying chro-
mosome morphology. Compared with metaphase chromosomes, pachytene chromosomes present
higher axial resolution because the chromosomes are much longer (Wang et al., 2009). Axial reso-
lution is defined as the smallest physical distance that can be detected by microscopy (De Jong et
al., 1999). The extent of compaction of mitotic and meiotic chromosomes varies in plant species:
25 times in Arabidopsis (Fransz et al., 2000), 6.2 times in rye (Zoller et al., 2004), and 15.9 times
in maize (Wang et al., 2006). Further advantages of pachytene analysis are useful landmarks for
identifying individual chromosomes, including the visibility of centromeres, chromomeres, telom-
eres, knobs, nucleoli, and the possibility to distinguish eu- and heterochromatin (Schulz-Schaeffer,
1980). A technique to analyze pachytene chromosome in Musa has been reported by Adeleke et al.
(2002). With further studies of pachytene chromosomes, it may be possible to identify chromosomal
structural changes that occur during evolution and breeding. New techniques such as FISH using
chromosomes’ specific DNA sequences may also be useful in identifying banana chromosomes
(Hribova et al., 2008).
4.4.2 Molecular Cytogenetics
Over the last two decades molecular cytogenetics has considerably contributed to a better under-
standing of plant genome structure and evolution (Valarik et al., 2004). FISH and GISH research
has facilitated identification of parental genomes in hybrids and individual chromosomes in some
plants (Taketa et al., 2000), physical mapping of genes on chromosomes (Dolezelova et al., 1998;
Fuchs et al., 1998), integration of genetic and physical maps with marker-tagged bacterial artificial
chromosomes (BAC) clones (Fahrenkrug et al., 2001; Yuan et al., 2000), analysis of genomic dis-
tribution of mobile genetic elements (Balint-Kurti et al., 2000), and other repetitive DNA sequences
(Gortner et al., 1998; Heslop-Harrison, 1996).
In addition to genome identification, FISH studies in banana were useful in providing some
degree of chromosome identification (Osuji et al., 1997a; Dolezelova et al., 1998). The very
strong FISH signals in the centromeric regions of some banana chromosomes (Osuji et al.,
1997a) could be useful in identifying individual Musa chromosomes since stronger hybridiza-
tion signals were observed only in some chromosomes. The differential hybridization signals
along the length of the chromosome may be due to the organization of the different classes of
repetitive DNA. Our unpublished research has shown that the chromosomes of Musa contract
differentially during prometaphase and metaphase. The centromeric regions appear to be more
highly condensed than the distal regions, suggesting that condensation of chromosomes in
Musa begins in the centromeric region. The distal regions lag behind in this process and often
appear as lightly stained “tails.” This may provide another reason for the stronger hybrid-
ization signals observed in the centromeric regions. Uneven staining patterns are considered
characteristic of prometaphase chromosomes, especially in plants with small chromosomes
(Fukui, 1996). They are caused by differential condensation of the chromatin fiber and are
called condensation patterns. Condensed regions at the proximal regions are called primary
condensations, whereas small condensations at the interstitial or terminal regions are termed
faint, unstable, or small condensations. Centromeric regions in other species with small chro-
mosomes such as Arabidopsis are also known to stain more brightly (Maluszynska and Heslop-
Harrison, 1993).
FISH studies identified a single pair of sites for the 18S-25S rDNA in Musa (Dolezelova et al.,
1998; Osuji et al., 1998) whereas the 5S rDNA genes were localized on two to four different chro-
mosomes (Bartos et al., 2005). All known banana repetitive DNA sequences were not useful in
chromosome identification since they were dispersed on all chromosomes of M. acuminata. The
monkey transposons were concentrated in the nucleolar organizer regions and appeared dispersed
throughout the genome (Balint-Kurti et al., 2000). The only exception was a part of the retrotrans-
poson monkey and the repetitive DNA clone Radka14 that localized to the secondary constriction
(Balint-Kurti et al., 2000; Valarik et al., 2002). Chromosomes with secondary constrictions are even
identifiable without FISH. Other useful cytogenetic markers are DNA sequences from large insert
libraries such as BAC (Hribova et al., 2008). In order to position BACs and other DNA sequences
along the chromosomes, molecular cytogenetic maps are needed. Cytogenetic maps can be highly
informative to support the construction of physical maps and map-based cloning projects and to
position genes in large heterochromatic regions where linkage distances are inaccurate due to low
levels of meiotic recombination (Lambie and Roeder, 1986; Zhong et al., 1999).
Several BAC libraries are available for banana, including a BAC library from M. acuminata cv
Calcutta 4 (Vilarinhos et al., 2003, 2006), binary BAC (BIBAC) library of M. acuminata cv Tuu
Gia (Ortiz-Vanquez et al., 2005), and a BAC library of M. balbisiana cv Pisang Klutuk Wulung
(Safar et al., 2004). Although BAC-FISH was used successfully to localize chromosome specific
BAC clones in other plants, such as potato, rice, and cotton, localization of BAC clones on mitotic
chromosomes of M. acuminata using FISH was not very successful (Hribova et al., 2008). Only one
of eight BAC clones tested produced a single locus signal on the chromosomes of M. acuminata.
The clone signal corresponded with the location of the 5S rDNA genes. The study by Hribova et al.
(2008) showed that BAC libraries appear to have low efficiency for developing cytogenetic markers
in banana.
Similar problems were encountered in cotton (Wang et al., 2009). Cotton contains high levels
of secondary compounds, including polysaccharides, phenolics, and other organic constituents that
could have prevented the availability of target DNA sequences to the hybridization probe during
BAC-FISH analysis (Wang et al., 2009). Similar reasons may be provided for the poor BAC-FISH
resolution of banana chromosomes.
Highly condensed mitotic metaphase chromosomes are not useful for FISH since they limit the
optical resolution of adjacent FISH targets (Kulikova et al., 2001).
The optical axial resolution of FISH targets in metaphase chromosomes is limited to 1–3 Mbp
(Lichter et al., 1990; Cheng et al., 2002). For higher resolution, pachytene chromosomes are more
appropriate because their complements measure 10–40 times longer and display a differenti-
ated pattern of euchromatic and heterochromatic regions (Zhong et al., 1996; Cheng et al., 2002).
Although the production of high-quality pachytene spreads is technically more demanding than
mitotic metaphase spreads (De Jong et al., 1999), it appears that this may be the only way to map
BACs on banana chromosomes. High-resolution pachytene chromosome mapping of BACs has
been achieved in many crops, such as in rice (Cheng et al., 2001) and cotton (Wang et al., 2009).
FISH-based mapping will be valuable to align the physical and cytogenetic maps in banana.
4.5 Flow Cytometry in Musa

Flow cytometry is extensively used in basic and applied research. Flow cytometry has been used in
ploidy analysis, nuclear genome composition, and nuclear DNA content in Musa.
4.5.1 Ploidy Analysis in Musa

Since ploidy manipulation is critical to banana breeding, ploidy determination is an essential com-
ponent of any Musa improvement program. For example, crossing the triploid plantain with diploid
accessions generates diploid, triploid, tetraploid, aneuploid, and hyperploid progeny (Vuylsteke et
al., 1993; Osuji et al., 1997b). Oselebe et al. (2006a) also found that progenies of 2x-2x crosses were
predominantly diploid (99.7%), those of 2x-4x crosses were mainly diploid (96.2%), while the 4x-2x
crosses produced predominantly triploid progenies (94.1%). Ploidy polymorphism has been attrib-
uted to abnormal meiosis (Asker, 1980), selective affinity of chromosomes of the A and B genomes,
and complex microsporogenesis leading to the formation of gametes with varying chromosome
constitutions. For selection of a hybrid of a desired ploidy level, there is need for a rapid and precise
method for ploidy screening at an early stage of plant development (Dolezel et al., 1997). Ploidy
determination in Musa was done primarily by examining plants at the morphological level. The
characters of the plant were scored and compared with its resemblance to the progenitor species,
M. acuminata and M. balbisiana (Stover and Simmonds, 1987). Although this system was quite
accurate, it had some difficulties. For example, only mature plants could be used and banana takes
between 12 and 24 months to reach full maturity when the characters are fully expressed and can be
measured. In addition, morphological characters can be greatly influenced by the environment and
this method can lead to inconsistent results. Conventional root-tip methods for ploidy determination
are labor intensive and difficult due to the small size of Musa chromosomes and are not practical
when thousands of plants are involved. Alternative methods for rapid ploidy determination in Musa
including gametophytic and sporophytic characters such as pollen size, stomatal size, and density
have been reported (Vandenhout et al., 1995; Tenkouano et al., 1998). These methods also have their
limitations. Determination of pollen size cannot be used for early screening since plants require 9 to
12 months before flowering (Tenkouano et al., 1998). Tenkouano et al. (1998) found that the average
number of chloroplasts in stomatal guard cells of triploid and tetraploids was, respectively, 1.30 and
1.53 times higher than in diploids. Consequently, the number of chloroplasts in stomatal guard cells
can be used to determine ploidy levels in Musa germplasm. Flow cytometry is still a quicker and
easier method for ploidy determination in banana. Many studies have used flow cytometry to mea-
sure ploidy in banana (Dolezel et al., 1997; Pillay et al., 2000, 2006; Oselebe et al. 2006a, 2006b).
These studies have tested the validity of some of the ploidy levels determined entirely from mor-
phological characters. For example, ‘Klue Tiparot’ classified as a natural tetraploid (ABBB) is now
known to be a triploid ABB with 2n = 3x = 33 chromosomes both by classical microscopy and flow
cytometry (Jenny et al., 1997; Horry et al., 1998; Pillay et al., 2000). Similarly, Horry et al. (1998)
found that ‘Pisang Jambe,’ classified as a tetraploid AAAA, is actually a triploid AAA (2n = 3x =
33), while the triploid ‘(Kluai) Ngoen’ (AAB) from Malaysia is a tetraploid AAAB (2n = 4x = 44).
Nsabimana and van Staden (2006) were the first to use frozen banana tissue to determine
ploidy of the banana. Their study revealed that the clones ‘Pomme,’ ‘Kamaramasenge,’ and
‘Gisubi Kayinja’ that were previously thought to be diploid were actually triploid. Likewise,
‘Gisubi Kagongo’ and ‘Dibis’ that were considered to be triploid and tetraploid, respectively,
were found to be diploid and triploid, respectively. There appears to be some confusion on the
ploidy of ‘Kamaramasenge,’ which was reported to be an AB diploid (Pillay et al., 2001) and as
a triploid in the above study and by Dolezelova et al. (2005). This anomaly can best be solved by
confirming the identity of the plant and counting its chromosomes using conventional root-tip
techniques. One has to be cautious when assigning new ploidy levels to banana clones and ensure
that no transcription errors were committed when plants are transferred from one germplasm col-
lection to another with regards to the names of plants and the ploidy. Records of the germplasm
collection at the Onne Station of the International Institute of Tropical Agriculture in Nigeria
indicated a clone named ‘Diby 2’ that was recorded as a triploid. Whether the clone called ‘Dibis’
in the study by Nsabimana and van Staden (2006) is the same as ‘Diby 2’ requires confirmation.
Similarly, the word “kayinja” is typically used for beer banana in Uganda and it is well known
that it is a triploid. In all likelihood, ‘Gisubi Kayinja’ is a triploid and was perhaps mislabeled as
a diploid.
Flow cytometry was used to confirm the ploidy level of the dessert banana cultivar ‘Sukali Ndizi’
that was known to be a diploid with an AB genome composition since its introduction into Uganda
in 1903 (Pillay et al., 2003a). Flow cytometry and conventional chromosome analysis together with
molecular marker technology showed that ‘Sukali Ndizi’ is a triploid with AAB genomes. Likewise,
‘Pisang Awak’ was known to be triploid (Robinson, 1996). However, Pillay et al. (2006) used flow
cytometry and conventional root-tip chromosome counts to determine the tetraploid status of ‘Pisang
Awak’ and two other cultivars (‘Foulah 4’ and ‘Nzizi’) that had been previously classified as trip-
loids. Perhaps different cytotypes of ‘Pisang Awak’ exist. Similarly, cultivars that were previously
classified as diploids, including ‘Too’ and ‘Toowoolee,’ were found to be triploids. Careful screening
of Musa genotypes with flow cytometric and molecular techniques will undoubtedly help to resolve
similar problems and provide a more consistent classification system. Flow cytometric methods for
determining ploidy should allow breeders to screen in the early stages of development for euploids
in segregating populations of banana hybrids (Dolezel et al., 1994), thus saving valuable space, time,
and resources currently being used to grow out aneuploids and hyperpolyploids.
It appears that a detailed morphological description of field-grown plants, conventional chromo-
some counting, and flow cytometry is essential to confirm the status of plants making up the AB
and ABB genome groups in Musa.
Breeding schemes that depend on mutation, somaclonal variation, protoplast fusion, cell suspen-
sion cultures, anther and microspore culture, protoplast engineering, and colchicine doubling of
chromosomes have used flow cytometry as an indispensable tool (Novak et al., 1989; Megia et al.,
1993; Panis et al., 1993; Cote et al., 1996; Matsumoto and Oka, 1998; Gimenez et al., 2001; Assani
et al., 2003, 2005; Xiao et al., 2008, 2009).
4.5.2 DNA Content, Genome Size, and Base Composition

Flow cytometric analysis of the nuclear DNA content is based on the analysis of the relative fluores-
cence intensity of nuclei stained with a DNA fluorochrome (Dolezel, 1991; Dolezel et al., 1997). The
nuclear DNA content of many plant species, including Musa, has been estimated by flow cytometry.
However, relatively few studies have been conducted in Musa to estimate the DNA content and
genome size (Arumuganathan and Earle, 1991; Lysak et al., 1999; D’Hont et al., 1999; Kamate et
al., 2001). One of the major shortcomings of these studies is the small sample size representing
the different genomic groups. While two of these studies (Lysak et al., 1999; Kamate et al., 2001)
involved accessions with only the A and B genomes, only one study included plants with the S and
T genomes in its analysis (D’Hont et al., 1999). However, there is a large difference in the DNA
content estimates obtained by D’Hont et al. (1999) when compared with the studies by Lysak et al.
(1999) and Kamate et al. (2001). The first report of the total DNA content and genome size in Musa
was provided by Arumuganathan and Earle (1991). A 2C DNA content of 1.81 pg with a genome
size of 873 Mbp was reported. The term “2C” represents the DNA content of a diploid nucleus and
is represented by the G1 peak in flow cytometric analysis. Afza et al. (1993) reported a 2C value of
1.23 pg for M. acuminata subsp. banksii; 2C = 1.26 pg for M. acuminata subsp. Errans, and 2C =
1.26 for the AA diploid cultivar ‘Pisang Mas.’ They estimated that 2C = 1.14pg in M. balbisiana.
The DNA content for AA genome accessions ranges from 1.22 to 1.27 pg (Lysak et al., 1999)
and 1.20 to 1.33 pg (Kamate et al., 2001). This corresponds to the estimate of Afza et al. (1993).
Similarly, genome size estimates of Musa ranges from 534 to 615 Mbp (Lysak et al., 1999) and 560
to 610 Mbp (Kamate et al., 2001). The estimate of DNA content by Arumuganathan and Earle (1991)
is similar to those reported for triploid accessions in other studies. It is likely that Arumuganathan
and Earle (1991) measured a triploid rather than a diploid accession.
With regards to individual genomes in Musa, the B genome is considered to have the smallest
nuclear DNA content, with estimates ranging from 1.03 pg/2C to 1.16 pg/2C in different studies
(Lysak et al., 1999; D’Hont et al., 1999; Kamate et al., 2001). Estimates for the average size of the
A genome have been placed at 1.25 pg/2C and 1.27 pg/2C (Kamate et al., 2001) while D’Hont et al.
(1999) reported that the A genome is 1.11 pg/2C. The S and T genomes are reported to contain 1.18
pg/2C and 1.27 pg/2C DNA, respectively (D’Hont et al., 1999). As indicated earlier, the DNA esti-
mates of D’Hont et al. (1999) are significantly lower, and a meaningful comparison of DNA content
in the A, B, S, and T genomes is not possible. However, the DNA content of the S and T genomes
is reported to be greater than those of the A and B genomes (D’Hont et al., 1999). Nevertheless,
it is clear that there are significant differences of 12–15% in DNA content between the A and B
genomes. The size difference is probably due to the presence of repetitive sequences that were found
to be in greater abundance (10.5 Mbp) in M. acuminata (Valarik et al., 2002). The expectation is
that a triploid accession with two B genomes (ABB) will invariably have less DNA than a triploid
accession with one B genome (AAB). However, this is not the case, as shown by the results of Lysak
et al. (1999) and Kamate et al. (2001). The bigger sample size used in Kamate et al. (2001) showed
a greater number of outliers with respect to genome designation and DNA content. For example,
the genome size of ‘Simili Radjah’ (ABB) was typical of that of a tetraploid. Further, an 11% dif-
ference in DNA content was noted within subspecies of the M. acuminata complex. This contrasts
with the 3.9% variation in DNA content among the AA accessions reported by Lysak et al. (1999).
Differences in the estimation of DNA content have been reported for other crops such as Pisum
sativum (Cavallini and Natali, 1990; Cavallini et al., 1993) and Glycine max (Graham et al., 1994).
However, both the P. sativum and G. max results were not supported by the studies of Baranyi and
Greilhuber (1996) and Greilhuber and Obermayer (1997), respectively. Consequently, interpreting
DNA content data in Musa must be approached with great caution, until more accurate methods for
DNA determination become available.
Lysak et al. (1999) used DNA content to cluster the clones with different genome combina-
tions. With the exception of one clone, ‘Red Dacca,’ all the clones with similar genomes clustered
together. The clustering corresponded with accepted taxonomic classification of Musa based on
other characters.
4.5.3 Gene Content and Density

Gene content and density have not been widely studied in banana. The gene content and density of
two BAC clones from M. acuminata was revealed by sequencing the BAC clones (Aert et al., 2004).
One of the clones (MuH9) had a gene density of one gene per 6.9 kb while the other (MuG9) had a
gene density of one gene per 10.5 kb. The genomic base composition of Musa is estimated to have
a median value of 40.8% GC.
4.6 Cytogenetic and Fertility Relationships in Musa

Most of the widely grown banana cultivars are characterized by either low male and female fertility
or complete male and female sterility (Pillay et al., 2002). The high level of sterility is due to pro-
duction of gametes with uneven chromosome numbers. The development of silver staining for Musa
chromosomes made it possible to visualize the meiotic chromosomes in Musa (Pillay and Adeleke,
2002). Cytological studies of microsporogenesis in Musa highlighted a number of meiotic anoma-
lies that showed direct relationships between meiotic behavior and fertility (Adeleke et al., 2002).
Sporad configurations for a number of accessions were shown to exceed the usual tetrad in both
diploids and triploids. These unusual sporad configurations were related to abnormal chromosome
movements during sporogenesis. Lagging chromosomes and univalents were observed in anaphase
I of many cultivars. These chromosomes were excluded from the nuclear material of the daughter
cells as they proceed into the resting phase. There was a strong relationship between tetrad forma-
tion and pollen fertility. For example, the AA diploids ‘Calcutta 4’ and ‘Long Tavoy’ showed very
high pollen fertility levels that corresponded strongly with the high percentage of tetrads observed
in these accessions. Interestingly, the diploids ‘Pitu’ and ‘SF265’ had lower pollen fertility although
they did not manifest any irregular sporad formation. In general, pollen fertility decreased in the
triploids, with the plantains (AAB) showing a very marked decline in pollen fertility. Univalents
were observed frequently in Musa, indicating the erratic chromosome movements observed gener-
ally in this genus at anaphase. Further studies are required to show the full extent in which chromo-
some behavior and sterility in Musa are correlated.
4.7 Conclusion
New techniques in cytogenetics are constantly evolving. While serious efforts have been made
in developing genetic maps for many crops species, much less effort is being devoted to physical
mapping. The majority of linkage maps developed in plant species is not integrated with any type
of physical map (Cheng et al., 2001). In plants with both large and small chromosomes, recombina-
tion is mainly distributed along the distal half of the chromosomes. Many studies have shown that
recombination in the centromeric regions, which may account for almost 50% of the length of the
chromosomes, is suppressed (Werner et al., 1992; Gill et al., 1993; Delaney et al., 1995, 1995b).
Therefore there is need to align linkage and physical maps. Physical maps are being developed for
many crops mainly by direct visualization of DNA sequences on chromosomes by FISH. In plants
like banana with small chromosomes, it seems that FISH on pachytene chromosomes may be the
route to follow. Application of BAC-FISH on pachytene complements can contribute significantly
to the construction of physical maps and to map base cloning by confirming the physical location of
markers on the linkage groups (Tang et al., 2009).
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5 Genetics of Important
Traits in Musa
Eli Khayat and Rodomiro Ortiz
Contents
5.1 Introduction............................................................................................................................. 71
5.2 Components of Yield............................................................................................................... 72
5.3 Plant Architecture.................................................................................................................... 73
5.4 Fruit Parthenocarpy................................................................................................................. 74
5.5 Fruit Ripening and Senescence............................................................................................... 75
5.6 Nematode Resistance............................................................................................................... 77
5.7 Resistance to Black Leaf Streak Disease................................................................................ 78
5.8 Concluding Remarks............................................................................................................... 79
References.........................................................................................................................................80
5.1 Introduction
Despite parthenocarpy and female sterility, the genus Musa portrays a wealth of alleles that govern
important agronomic as well as consumer traits. Initially, cultivars were selected by plant vigor,
yield, and hardiness—traits closely linked to higher ploidy levels (Simmonds, 1962). However, while
most triploid cultivars are useless to breeders due to the high occurrence of female sterility, tetra-
ploids are undesirable due to early senescence, fruit drop, short shelf life, and a weak pseudostem
(Pillay et al., 2002). Nearly all domesticated bananas and plantains are inter- or intraspecific triploid
hybrids that constitute all possible combinations of the A and B genomes that are represented by
Musa acuminata Colla and M. balbisiana Colla, respectively. In addition to the growth character-
istics, triploids were preferred by farmers due to their seedless phenotype. In this regard, cultivated
bananas differ from their wild diploid ancestors (AA, BB genomes), which bear large hard seeds.
Hybridization of diploid Musa species, followed by recombination events and mutations, gener-
ated enough genetic diversity to allow domestication of diploid cultivars bearing parthenocarpic
fruit (Simmonds, 1962). Sexual polyploidization (2n × n) resulted in almost-sterile triploid cultivars,
which multiplied through sucker propagules in the centers of origin. During the long history of cul-
tivation of triploid cultivars, new mutants originated and farmers selected among them for specific
genetic combinations that showed desirable phenotypes. Domestication occurred after vegetative
propagation of a few selected high-yielding phenotypes and linkages containing favorable combina-
tions. Given that parthenocarpy was a fundamental precondition, the gene pool of cultivated clones
remained stagnant throughout the history of banana domestication. Ironically, the abundance of
alleles for each trait in the genus Musa is not resourceful enough to alter specific characteristics in
commercial cultivars.
In the 80 years of banana and plantain breeding, very few qualitative or quantitative traits have
been improved in commercially important cultivars when compared to other crop species. However,
recent advancements in banana structural and functional genomics have opened new horizons
for banana breeding through genetic engineering (Crouch et al., 1998; Khayat et al., 2004a). The
71
technology’s impressive contributions to insights in population and molecular genetics have been
fueled by advances in computational methodology (Khayat et al., 2004a), and indeed these insights
and methodologies have spurred developments in the understanding of the banana genome and to
some extent the mapping of genes that control specific traits on virtual chromosomes. Still, the
complexity of positional cloning in banana, combined with the lack of saturated linkage maps, hin-
ders gene-targeted hybridization breeding. The inability of researchers to use map-based cloning
is a serious limitation to identifying strategic loci in segregating populations. However, the recent
progress made in banana genomics (Cheung and Town, 2007) will undoubtedly assist breeders to
identify interesting loci and genes that govern important traits. To live up to such expectations will
be a tall order due to considerable obstacles remaining to be surmounted. The main barriers are a
long-cycling time span from seed to seed in the triploid lines, and the low fertility of most polyploid
cultivars.
Through domestication, several morphological and physiological traits were emphasized to
distinguish domesticated crops from their wild ancestors. These characteristics are referred to as
the “domestication syndrome” (Poncet et al., 1998). Normally, syndrome characteristics include a
more compact growth habit, increased earliness, reduction/loss of seed dispersal and dormancy,
gigantism, and increased morphological diversity in the consumed portion of the plant (Frary and
Doganlar, 2003). Studies on the domestication syndrome and domestication process have revealed
that numerous traits that distinguish crop plants from their wild relatives are often governed by a
relatively small number of loci having effects of unequal magnitude (Frary and Doganlar, 2003).
The differences between domesticated bananas and their wild relatives involve parameters that
govern yield and fruit characteristics. Above all, farmers tended to select hybrids exhibiting parthe-
nocarpic fruit.
5.2 Components of Yield
The components of yield are well defined by banana and plantain producers and breeders. These
include bunch weight, number of marketable hands in a bunch, number of fingers in a hand, finger
size, cycling time, and number of stems per hectare. In the export industry (mainly Cavendish), the
most commonly used yield parameter is box per stem ratio. This appraisal combines weight and
fruit quality. These parameters are widely used for selection of hybrids (Osuji et al., 1997; Krikorian
et al., 1993; Crouch et al., 2000) and somaclones (Khayat et al., 2004b).
The merit of selecting yield components is limited due to masking of the traits by diseases and pests.
It was therefore argued that subtle quantitative traits are best selected for in subtropical regions that
are devoid of diseases and pests (Khayat et al., 2004). To this end, somaclonal selections of Cavendish
were highly productive in one of the most marginal climates for banana growth: northern Israel. The
yield performance of these selections was proven superior not only in subtropical areas such as in
Israel and South Africa (Eckstein et al., 1998) but also for tropical climates such as the Davao region
of the Philippines (Khayat et al., 2004), Africa, and Latin America (unpublished results).
The shift from diploid to polyploid karyotypes greatly increased the bunch weight and fruit size
of both banana and plantain hybrids (Ortiz and Vuylsteke, 1995a). The combination of A and B
genomes contributes to productivity.
Banana and plantain breeders should quantify additive and nonadditive components of genetic
variance to choose appropriate selection methods to improve quantitative traits. Furthermore, nar-
row sense heritability, which is the ratio between the additive genetic variance and the total pheno-
typic variance, is a scale-independent quantity that plays an important role in the theory of selection
methods because the response to selection can be predicted if the breeder knows the target trait heri-
tability. Triploid parent-offspring regression analysis suggests that bunch weight, number of fruits,
and fruit length had intermediate to low heritability in tetraploid plantain-banana hybrids from
3x–2x crosses (Ortiz, 2000). This indicates that they could not be predicted using the phenotype of
the triploid parent and that progeny testing will be required to assess and select the best parents for
Genetics of Important Traits in Musa 73
population improvement of such traits. Progeny testing in secondary triploid hybrids (from 4x–2x
crosses) shows, however, that general combining ability was much greater than specific combining
ability for yield and associated traits (Tenkouano et al., 1998). This result suggests therefore that
the 4x–2x breeding scheme should aim to accumulate favorable alleles in potential tetraploid and
diploid parents through recurrent selection.
5.3 Plant Architecture
Due to cultural impacts, banana breeders emphasized structural characteristics of the pseudostem,
the bunch, and roots. Plant size, from rhizome to the petiole, greatly fluctuates in many cultivars.
Cavendish populations (M. acuminata Cavendish, AAA), particularly plants produced by tissue cul-
ture, are notoriously known to mutate to either “dwarf” or “tall” phenotypes. In general, the dwarf
mutants develop a small deformed bunch (Reuveni et al., 1996; Khayat et al., 2004). For practical
reasons, lower stature plants are considered advantageous. However, in most cultivars, taller plants
are positively correlated with a larger bunch. For instance, short Cavendish cultivars ‘Buena Vista’
and ‘Dwarf Cavendish’ showed a yield reduction of approximately 11% in comparison to ‘Grand
Nain’ (Eckstein et al., 1998). An unusual exception to this rule was recently selected in Western
Galilee (Israel). A somaclonal mutant of ‘Zelig’ (a Cavendish selection) registered under the name
‘Adi’ was selected for a combined low stature and large bunch (Figure 5.1). The average plant height
of the selected clone is approximately 230 cm, compared to 315 cm of its originator ‘Zelig.’ The
average bunch weight and finger size of ‘Adi’ are both larger than ‘Zelig’ (Khayat, 2009).
The “dwarf” and “tall” mutants in Cavendish have been characterized as under- or oversensi-
tive to the hormone gibberellic acid, respectively. An in vitro bioassay was developed where a low
concentration of gibberelic acid 3 (GA3) was included in the growth medium during the elonga-
tion stage of the tissue culture. Wild-type plants in the GA3 containing medium elongated above
the plants that were placed on a substrate devoid of the hormone (control treatment). “Dwarf”
mutants on the hormone medium grew below the height of the control while oversensitive mutants
grew above (Khayat et al., 2004). A mutant that was depicted as “Long and Narrow Leaves”
(LNL) was created by exhaustive cycling of tissue culture, leading to induction of retrotransposon
expression and transposition. These experiments (Khayat et al., 2004b) led to the conclusion that
the long-internode mutation is caused by insertions of retro-elements in a chromosomal locus
having an effect on plant height. An extensive number of cycles in tissue culture enhances the
activity of retrotransposable elements in the genome. Similar results were reported in rice where
‘Adi’ ‘Zelig’ ‘Jaﬀa’
Figure 5.1 ‘Adi,’ a new Cavendish selection. Comparison of height between the selections ‘Adi,’ ‘Zelig,’
and ‘Jaffa.’
specific genes were mutated by transposition of Tos 17, a rice retrotransposon activated by tissue
culture (Hirochika et al., 1996). Interestingly, in rice at least 50% of the Tos 17 landing sites were
detected as coding regions. The “Landing Pad” hypothesis of retrotransposable elements may
explain the high frequency of “dwarf” mutants in Musa. The conserved Ty1-copia group of ret-
rotransposons, highly expressed in banana (Khayat et al., 2004), is ubiquitous to various dicoty-
ledonous and monocotyledonous plant species, including potatoes, onions, fava beans, barley, and
rye (Amar et al., 1997; Kumar et al., 1997).
The process of tissue culture also induces mutations mediated by DNA methylation (Peraza-
Echeverria et al., 2001). It was pointed out that the source of explants had an effect on the degree
of polymorphism. In vitro plants produced from floral meristems showed greater frequencies of
methylation in comparison to sucker meristems.
Other mechanisms of point mutations, such as deletions or nucleotide replacements, could not
account for the high incidence of this mutation. “Semi-dwarf” mutants were isolated by Tang (2005)
in Cavendish. The author describes the Cavendish selection ‘TC1–229’ as having a stature of 70
cm lower than its originator, ‘Tai-Chiao No 3.’ The dwarf French plantain ‘Njock Kon’ was pos-
tulated by Swennen and Vuylsteke (1987) to be a mutation of a giant French cultivar. This finding
was later confirmed by Osuji et al. (1997). The height of the pseudostem—that is, the distance
between the soil and the petioles of the highest leaves—is used for subgrouping plantain cultivars
into giant, medium, and small. Musa clones with short internodes are called “dwarf” cultivars
(Ortiz and Vuylsteke, 1998a). Such a dwarfism is controlled by the single recessive gene dw (Ortiz
and Vuylsteke, 1995b), which appears to be close to the centromere and shows a dosage effect at the
tetraploid level.
The inhibition of lateral bud growth due to substances released by the terminal bud is a limiting
factor for plantain perennial productivity. The inheritance of apical dominance is controlled by the
major recessive gene ad (Ortiz and Vuylsteke, 1994a). The dominant Ad allele improves the suck-
ering of the crop as measured by the height of the tallest sucker in plantain-banana hybrids. The
Ad gene may regulate gibberellic acid production, but this allele shows incomplete penetrance and
variable expressivity.
Another important morphological characteristic is the form of the fruit bunch. For the export
industry, a large cylindrical bunch is highly valued for its characteristic to reduce abrasion of the
fingers with each other (Robinson et al., 1993). A formula was proposed to characterize the bunch
conformation. To assess conicalness of the bunch, Champion (1967) proposed the formula (D1 –
D2)/L, where D1 is the diameter of the largest (basal hand), D2 is the diameter of the smallest (apical
hand), and L is the length of the bunch. Ortiz and Vuylsteke (1998b) indicate that bunch orientation
might be an oligogenic trait regulated by the epistatic effects of at least three dominant loci.
Completion of the vegetative state and flower initiation and development are critical components
of yield. Stover and Simmonds (1987) proposed that bunch size is affected by the timing of the
transition from vegetative to reproductive growth as well as by the timing of transition from male to
female organs in the developing inflorescence (Stover and Simmonds, 1987).
5.4 Fruit Parthenocarpy

Parthenocarpy entails the development of ovaries into fruit without fertilization. The mechanism of
the parthenocarpic trait was extensively studied in various fruit crops. Genes for parthenocarpy are
thought to express changes in the pattern of hormone production, transport, and/or metabolism. In
addition, parthenocarpy is also implemented as a strategy to overcome a growth substance concen-
tration threshold during the critical period of anthesis and to promote ovary growth in such a way
that pollination and fertilization are no longer needed or possible (Nitsch, 1970).
By far, most cultivated banana and plantain cultivars are parthenocarpic and sterile, and con-
sequently the trait is well documented in the literature (Ortiz and Vuylsteke, 1992; Ortiz and
Vuylsteke, 1995a, 1995b). Over 50 years ago it was discovered by Simmonds that the parthenocarpy
trait in banana is governed by three complementary genes, and that the diploid variety ‘Calcutta
4’ is deficient in one of the three, while the other two are homozygous and dominant (Simmonds,
1953). Ortiz and Vuylsteke (1995a) reported a wide variation of yield components in euploid seg-
regants of F1 hybrids between the French plantain ‘Obino Ewai’ (AAB) × ‘Bobby Tannap’ and the
diploid ‘Calcutta 4’ (AA). The segregating population for one of the three elements (P1) is correlated
to bunch weight, hands per bunch, fruit weight, fruit length, and fruit circumference but not to fruit
filling. Unfortunately, as of today the loci harboring the genes for fruit parthenocarpy have not yet
been mapped. Moreover, the biochemical mechanism in banana remains obscure.
5.5 Fruit Ripening and Senescence

The genetic control of fruit ripening and senescence is relatively well understood due to the resem-
blance of physiological and biochemical processes in climacteric fruits of other better-studied
species. Tomatoes serve as a model species in which both the genetic and biochemical changes dur-
ing ripening were studied in great detail (Barry and Giovannoni, 2006; Giovannoni, 2007). Long
shelf life is among the most important marketing parameters of fruit quality. In fact, the choice of
Cavendish as the main export cultivar was largely based on its extended shelf life compared to other
triploid and diploid cultivars (Serano et al., 2003).
The majority of ripening-related enzymes have been identified and cloned either by comparing
the protein sequences to orthologs from other plant species or by using a differential display of gene
expression between ripe and unripe fruit (Clendennen and May, 1997; Khayat et al., 2004). Among
these are enzymes in the pathways leading to ethylene biosynthesis, starch hydrolysis, sucrose bio-
synthesis, tissue softening, chlorophyll degradation, and lipid degradation.
The significant variation in fruit ripening time among plantain and banana hybrids and landraces
suggests that this characteristic could be improved through crossbreeding (Ferris et al., 1999).
Banana and plantain differ largely in traits that relate to the conversion of starch to sugar in the pulp.
The conversion of starch to soluble sugars—sucrose, glucose, and fructose—entails the catalysis of
a series of chemical reactions. It is well established that plantain and cooking bananas (especially
cultivars containing at least one copy of the B genome) degrade starch slower and less completely in
comparison to dessert bananas that are composed of the A genome (Cordenunsi and Lajolo, 1995;
Robinson, 1996). However, there are several exceptions to the rule. The cultivar ‘Populou CMR,’
which is an AAB hybrid, contains a significant amount of sugars; the same holds for the cultivars
‘French Clair’ and ‘Batard’ (Ngalani et al., 1999).
Starch formation in the fruit is largely mediated by the enzyme starch synthase. The gene
encoding this enzyme was isolated from banana (Clendennen and May, 1997; Medina-Suarez et
al., 1997) and its expression was analyzed in different stages of fruit ripening. Very little infor-
mation can be extracted from the literature about the catalytic characteristics of starch synthase
in banana fruit. The question of whether the difference in starch accumulation between A and B
genomes in the preclimacteric stage is due to differences in the catalytic activity of starch syn-
thase remains unresolved. Degradation of starch in the fruit is mediated by at least three classes
of enzymes: namely, starch phosphorylase responsible for the phospholitic pathway and α and β
amylases that mediate the hydrolytic pathway. Da Mota et al. (2002) reported two forms of starch
phosphorylases in banana fruit: type I with high affinity to branched glucan chains and type II
with low affinity to glucan chains. Clearly bananas and plantains express both the hydrolytic as
well as the phosphorolytic pathway (Seymour, 1993). Another unresolved issue is related to phos-
phorylation versus hydrolysis of starch breakdown in the different banana and plantain cultivars.
At the cell level, type I phosphorylase is highly correlated to starch degradation and phosphoryla-
tion of glucose, suggesting a coarse control of the rate of starch degradation.
Following starch degradation, bananas and plantains synthesize and store sucrose as an end
product. Sucrose biosynthesis in higher plants can be mediated either by sucrose phosphate syn-
thase (SPS), which uses uridinated glucose and fructose 6-phosphate as substrates, or by sucrose
synthase (SuS) that uses uridinated glucose and fructose as substrates. While SPS is unidirec-
tional, SuS is bidirectional, and in various tissues it competes with invertases to catabolize
sucrose. SPS is considered a rate-limiting enzyme in many plant tissues, including banana fruit
(Nascimento et al., 1997). In photosynthetic and storage tissues of various crop species, the reac-
tion of SPS is regulated allosterically by sucrose, and by post-translational modification of serine
phosphorylation (Huber and Huber, 1996). Surprisingly, in banana fruit, the enzyme was shown
to be transcriptionally regulated. SPS transcripts are up-regulated in concert with the physiologi-
cal events associated with the climacteric rise (Nascimento et al., 1997). There are no indications
that the transcription of banana fruit SPS is activated by ethylene. Given that sucrose is the main
constituent that contributes to fruit sweetness, it is highly conceivable that the enzyme plays a
significant role in the trait of fruit sweetness.
Sink activity and mobilization of carbohydrates to the developing fruit were not extensively
studied in bananas. Sugar transporters across the cell membranes, hydrolysis of sucrose, and mobi-
lization of carbohydrates to the plastids are envisioned to have a strong impact on yield. These
metabolic processes were shown to institute the activity of the sink, and consequently have a strong
impact on yield. In tomatoes, it was realized that sink activity was mapped to a quantitative trait loci
(QTL) harboring cell-wall-bound acid invertase. This gene was correlated with high yield (Fridman
et al., 2004).
Ethylene gas is considered to be the trigger of transition of all climacteric fruit from the pre-
climacteric stage to the climacteric rise (Stover and Simmonds, 1987). Consequently, inhibition of
ethylene synthesis is expected to delay ripening and extend the shelf life of the green fruit. This
was confirmed by using tools of genetic engineering in tomatoes (Hamilton et al., 1990). The rate-
limiting step in the pathway leading to ethylene synthesis in plants is the formation of its precursor
molecule, the amino acid ACC from AdoMet. The reaction is catalyzed by the enzyme ACC syn-
thase (ACCsyn). The final step is mediated by ACC oxidase (ACCox) that catalyzes the reaction in
which ACC is converted to ethylene (Yang and Hoffman, 1984). Hamilton et al. (1990) inhibited
ethylene biosynthesis in transgenic tomatoes by silencing the gene encoding ACC oxidase. The
transgenic plants with an antisense construct to ACC oxidase produced approximately 3% of the
ethylene produced in the wild type. Similar results were achieved in tomatoes by antisense silenc-
ing of ACC synthase (Oeller et al., 1991). Both constructs improved the shelf life of the transgenic
tomato fruit. However, since ethylene is a regulator of other ripening processes, total abolishment
of ethylene synthesis resulted in fruit with poor aroma and deficient in pigments. These traits could
be reversed by exposure of the harvested fruit to exogenous ethylene.
Genes encoding ACC synthase and ACC oxidase were isolated from banana (Lopez-Gomez et
al., 1997; Huang et al., 2006). While ACC synthase is a multigene family represented by at least
eight members in the banana genome, ACC appears in a single locus suggesting a single copy. The
pattern of expression of the ripening-related transcript isoform of ACCsyn as well as ACCox cor-
relates well with the pattern of ethylene synthesis. In banana, unlike most climacteric fruit, soon
after the climacteric rise there is a sharp decline in ethylene evolution, which is correlated to the in
vivo activity of ACC oxidase (Liu et al., 1999). Mutants in ACCsyn1 or ACC oxidase are expected to
have an extended shelf life along with matching phenotypes that are related to fruit ripening—that
is, delayed chlorophyll degradation, inhibition of synthesis of aroma compounds, delayed conver-
sion of starch to soluble sugars, and inhibition of fruit softening. These biochemical processes could
be induced by application of exogenous ethylene. Exploring tools of biotechnology, gene silencing,
using RNA interference (RNAi) or antisense technologies is expected to prolong the green life of
the fruit. Given that ACCox is a single-copy gene, silencing it may hamper biological processes not
associated with fruit ripening. On the other hand, given that ACCsyn1 is specifically associated
with fruit ripening, it would presumably be a better candidate for post-transcriptional silencing of
fruit ripening.
Transcription factors that act upstream of the ethylene as well as non-ethylene-mediated regu-
lation of fruit ripening were identified and cloned in tomatoes by positional cloning of a mutated
locus mapped to chromosome 5. The gene product was identified as two MADS-box genes residing
side by side in the same locus (Vrebalov et al., 2002). These factors cosegregate with the well-
studied Rin mutant in tomatoes. The Rin mutation is widely used in hybrid tomatoes for extension
of the shelf life of tomato fruit. The heterozygous mutant for the Rin allele exhibits firm fruit with
normal pigmentation. A MADS-box gene (MADS:MuMADS1) was recently isolated from banana
(Liu et al., 2009). Homology data suggest that the transcript encoding MuMADS1 is related to the
AGAMOUS subfamily. Expression was shown in the stamen and pistil, as well as in the rhizome.
The expression levels were enhanced in fruit that were exposed to external ethylene. The analysis
performed by Liu et al. (2009) suggests that MuMADS1 is involved in fruit ripening.
5.6 Nematode Resistance
Nematodes cause massive damage to cultivated banana and plantains. The damages to commercial
plantations can reach up to 50% of yield loss in untreated soils. The susceptibility of banana and
plantains to nematodes is facilitated by the roots’ soft texture and shallow penetration in the soil.
The problem is accentuated in large-scale banana plantations where monocropping is a common
practice. Banning of nematicides like methyl bromide in various parts of the world exacerbated
the problem and left farmers with inappropriate and unreliable alternatives (for more details, see
Chapter 7 in this book). Although the Musa genus contains a wide range of resistant alleles for both
horizontal (nonspecific to a certain nematode) as well as vertical resistance (specific to a certain
nematode), for the most part natural hybrids are considered susceptible. However, several natural
diploid Musa AA lines were reported to have resistance to the most virulent banana nematode in the
tropics, Radopholus similis (Pinochet and Rowe, 1978). Screening methods were instituted to deter-
mine resistance levels of banana germplasm to nematodes, in particular for resistance to R. similis
and Pratylenchus coffeae (Collingborn and Gowen, 1997). Several cultivars of diploid and triploid
bananas were demonstrated to exhibit various degrees of resistance to nematodes. For instance,
the mixed (AB) diploid ‘Kunanis’ as well as the triploid ‘Yangambi Km5’ (AAA) were found to be
highly resistant to R. similis (Sarah et al., 1992; Collingborn and Gowen, 1997; Fogain and Gowen,
1997). The resistance in ‘Yangambi Km5’ is incomplete as the roots of this cultivar develop lesions,
but the number of nematodes is rather low. The phenomenon may stem from exhibiting the defense
mechanism of the plant to the nematodes.
Very limited information exists regarding the inheritance of resistance in commercial culti-
vars. For the most part, “race” specific (pathotype) resistance of the host is governed by a single
dominant gene, while resistance to a wider range of nematodes (horizontal resistance) is due to
more than a single allele (oligo or polygenic mode of resistance). Dochez et al. (2009) reported on
crosses between the two diploid genotypes: ‘TMB2x 6142-1’ susceptible to R. similis and ‘TMB2x
8075-7,’ which is highly resistant. Approximately 40% of the segregants expressed a resistant phe-
notype, 34% exhibited susceptibility, and 14% exhibited partial resistance, while the remainder
was inconclusive. The heterozygous nature of both parental lines contributed to the complexity of
interpretation of the results. The authors concluded that resistance in the segregants was controlled
by two separate alleles. According to the model developed by Dochez et al. (2009), resistance is
conferred by both genes in concert. Double-recessive alleles of one of the genes suppress the other
gene, resulting in susceptibility. Interestingly, resistance of the ‘TMB2x 8075-7’ is inherited from
its parental line ‘Calcutta 4’ (‘C-4’). However the resistance of ‘C-4’ to R. similis is unequivocal
(Moens et al., 2002). This parental line is considered highly resistant to black leaf streak disease.
It would be interesting to determine the genetic source of the diverse resistance and the mode of
inheritance to hybrid progenies of which ‘C-4’ was used as a parental line.
A novel biotechnological approach was used by Michaeli et al. (2004) to confer resistance to a
Cavendish (AAA) selection by expressing a silencing double-stranded RNA (for RNA interference)
transcript encoding part of the nematode Collagen 5 gene (Col 5). When the nematode feeds on the
plant, the double-stranded RNA is absorbed by the nematode and the RNAi silencing mechanism
is activated in the reproductive organs of the nematode, resulting in defective embryos. Plants were
tested in a potted experiment as well as in a field trial for resistance to nematodes. Various trans-
genic lines proved to be resistant to a range of nematodes sharing a consensus sequence in the Col
5 gene. RNase protection assays revealed that the double-stranded RNA molecules remained active
despite their mobilization through the digestive tract of the nematodes.
5.7 Resistance to Black Leaf Streak Disease

Black leaf streak disease (BLSD) is a fungal disease causing the most serious economic, environ-
mental, and public health issues in banana-growing areas (Hernandez and Witter, 1996). BLSD is
caused by the ascomycete fungus Mycosphaerella fijiensis Morelet. The fungus was first reported in
Fiji in 1964, but according to Stover (1978) the center of origin of the pathogen is Papua New Guinea
and the Solomon Islands. Transfer of germplasm from Southeast Asia to Latin America and Africa
spread the disease to these regions. The disease became an epidemic in most of the world’s tropical
banana-growing countries. Gradually, M. fijiensis replaced a former epidemic in Musa caused by
the related fungus M. musicola, which was first identified in Java in 1902 (Jones, 1990). The dif-
ferences between M. fijiensis and M. musicola were observed in the rate of lesion development on
the surface of the leaves of infected plants (Moulion-Pefura et al., 1996). In addition to the smaller
lesions in M. musicola compared with M. fijiensis, the spread of tissue oxidation in the leaf lamina
is limited to the vicinity of the site of infection. Advanced strains of M. fijiensis developed an evo-
lutionary capacity to rapidly adapt to a necrotrophic phase transition. The dead tissue can offer no
effective defense against the pathogen.
Effective activation of defense mechanisms against fungal pathogens largely relies on the plant’s
rapid recognition that the pathogen is present. When a plant contains genetic resistance to a par-
ticular disease, the pathogen will elicit a cascade of responses in the plant that induce the appro-
priate mechanism of defense in the plant. Some defense mechanisms initiated by R (resistance)
genes, to name a few, including hypersensitive responses that lead to a programmed death of plant
cells (Goodman and Novacky, 1994; Jones and Dangl, 1996), induced synthesis of antimicrobial
compounds (for example, phytoalexins), synthesis of antimicrobial proteins (pathogenesis-related
[PR] proteins, defensins, and so forth), cell-wall reinforcement in the infected areas, and block-
age of vessels (reviewed by Dixon et al., 1994). Importantly, resistant and susceptible plants both
contain this basic defense machinery. What is lacking in the susceptible plants is recognition of
the disease mediated by specific R genes and other elicitor receptor proteins. The vast majority of
R genes isolated to date contain nucleotide-binding sites (NBS) and leucine-rich repeats (LRR).
The latter motif probably mediates the aspect of disease specificity (reviewed by Baker et al., 1997;
Hammond-Kosack and Jones, 1997).
In recent years, knowledge of genes regulating plant disease resistance has increased consider-
ably (Chisholm et al., 2006). However, identification of genes conferring susceptibility is narrow
(Van Damme et al., 2005; Yang et al., 2006; Consonni et al., 2006). The information gap between
the nature of resistance and that of susceptibility is likely due to differences in their genetic
tractability. These findings demonstrate that NBS-LRR genes can condition disease susceptibil-
ity and resistance and may have implications for R gene deployment (Lorang et al., 2007). This
postulation may shed a new light on susceptibility as a trait, particularly in parthenocarpic spe-
cies like bananas and plantains that ceased to evolve early in evolutionary history, due to lack of
ability to cross hybridize.
“Gene-for-gene” type resistance (Flor, 1946) is commonly triggered by enabling of a genetically
dominant resistance R gene product by a dominant, pathogen-derived, avirulence (Avr) gene prod-
uct. Given that recognition of the Avr product is crucial, a single allele of the receptor product is
sufficient for activation of the cascade reaction of the downstream defense mechanism. Recognition
of pathogen signaling plays a key role in the activation. In 1995, Fullerton and Olsen reported 63
distinct virulent strains of M. fijiensis. Consequently, resistance through introgression of a single

receptor protein that activates a downstream defense mechanism is unlikely. In fact, a segregation
analysis for the resistance to BLSD in a cross between two triploid plantains and ‘C-4’ revealed
that the trait is governed by no fewer than three different loci (Ortiz and Vuylsteke, 1994b). As a
consequence of the oligogenic nature of the trait, availability of fully resistant material is scarce.
Analyzing the genetic inheritance of resistance, Ortiz and Vuylsteke (1994b) suggested a major
recessive gene (bs1) and two minor additive modifier genes (bsri). These authors distinguish between
severity of symptoms on the leaves and the delay in the infection as the youngest leaf exhibiting
spots expresses it. It was suggested that the youngest spotted leaf is an indication of a gene for gene
response. Craenen and Ortiz (1997) showed further that the intralocus interaction in the bs1 locus
apparently regulates the appearance of symptoms on the leaf surface, whereas the additive effect
and the intralocus interaction of the bsi locus affect disease development in the host plant. An exten-
sive search of banana R genes belonging to the subfamily containing NBS-LRR motifs revealed 52
contiguous sequences in the ‘C-4’ genome (Miller et al., 2008).
Another class of R genes belongs to the kinase superfamily. To this end, 13 different serine/thre-
onin kinase proteins, classified as Pto type, were isolated from bananas and characterized (Peraza-
Echeverria et al., 2007). Seven of these were classified as Pto-type genes based on their similarity
to tomato Pto-type resistance genes. This class of R-gene candidates is unusual in the sense that
it recognizes more than one Avr protein and as such may confer resistance against more than one
pathogen (Kim et al., 2002).
It has been noted that the climatic conditions of high temperature and humidity enhance the
disease and delay the defense mechanism employed by the plant. Under these climatic conditions
banana cultivars rarely express complete resistance to M. fijiensis. In highly resistant cultivars, the
disease development is delayed to a point where the impact on yield is negligent, but nevertheless
the defense is inadequate (Carlier et al., 2000). This resistance has also been described as nonspe-
cific with no interactions between signals from the pathogen and its host (Fullerton, 2002). These
modes of resistance are defined as “innate immunity traits.” Examples of innate traits that confer
banana resistance to BLSD were suggested by Craenen and Ortiz (1997) and include stomata den-
sity and pseudostem waxiness.
Finally, true and complete resistance of banana germplasm to M. fijiensis has not been shown
to exist, although a level of tolerance/resistance occurs in the resistant diploids and their progenies.
The precise denotation of the terms resistance and tolerance as used in reports is not always well
defined. The term resistance is appropriately used in less-severe diseases due to any cause, tissue
susceptibility included. Tolerance, in the sense of a relatively small yield reduction arising from
relatively severe infections, has been reported in various hybrids containing introgressions from
‘C-4’ or other “resistant” diploid material. Tetraploid cultivars that exhibit high tolerance to the dis-
ease under experimental conditions, including field trials, are often disappointing when exposed to
a wide range of isolates. In some cases the susceptibility is amplified in older plants under the stress
of harsh environmental conditions.
5.8 Concluding Remarks
The discipline of banana genetics is expected to undertake major changes in the next few years. The
prospects of elucidating the biochemical and genetic mechanisms of genes that govern important
traits in the Musa genome will undoubtedly accelerate breeding in the postgenomic era. Elucidation
of the complete Musa genomes (A and B) will open opportunities that did not exist for breeders until
now. Novel bioinformatic tools for inter- and intragenome comparisons will assist Musa breeders
to overcome the lack of high-resolution physical and genetic maps. Modern computation tools for
synteny projection of chromosome profiles will circumvent the need of positional cloning to identify
strategic QTL. Finally, if accepted by the public, genetic engineering may be very resourceful for
the creation of new and improved traits in the Musa genome.
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6 Major Diseases of Banana
Guy Blomme, Simon Eden-Green, Mohammed Mustaffa,
Bartholemew Nwauzoma, and Raman Thangavelu
Contents
6.1 General Introduction................................................................................................................ 86
6.2 Sigatoka Diseases of Banana................................................................................................... 86
6.2.1 Introduction................................................................................................................. 86
6.2.2 Origin and Geographical Distribution of Sigatoka Diseases...................................... 87
6.2.3 Symptom Description.................................................................................................. 88
6.2.4 Epidemiology............................................................................................................... 89
6.2.5 Disease Management................................................................................................... 89
6.3 Fusarium Wilt Disease in Banana...........................................................................................90
6.3.1 Introduction.................................................................................................................90
6.3.2 General Symptoms......................................................................................................90
6.3.3 Causal Organism......................................................................................................... 91
6.3.4 Infection Processes of Foc...........................................................................................92
6.3.5 Survival and Spread.....................................................................................................92
6.3.6 Classification................................................................................................................92
6.3.7 Disease Management................................................................................................... 93
6.3.8 Host Resistance............................................................................................................94
6.3.9 Conclusion................................................................................................................... 95
6.4 Banana Bacterial Wilt Diseases.............................................................................................. 95
6.4.1 Introduction................................................................................................................. 95
6.4.2 The Diseases and Their Distribution........................................................................... 95
6.4.3 Symptoms and Disease Cycle......................................................................................97
6.4.4 Disease Cycle and Implications for Control................................................................ 98
6.4.4.1 Infection via Soil and Roots.......................................................................... 98
6.4.4.2 Spread on Cutting and Cultivation Tools......................................................99
6.4.4.3 Inflorescence Infection................................................................................ 100
6.4.4.4 Planting Materials....................................................................................... 101
6.4.4.5 Other Modes of Infection............................................................................ 101
6.4.5 Screening and Breeding for Resistance..................................................................... 101
6.4.6 Impact and Future Outlook........................................................................................ 102
6.5 Banana Viruses...................................................................................................................... 103
6.5.1 Introduction............................................................................................................... 103
6.5.2 Banana Streak Virus................................................................................................... 103
6.5.2.1 Distribution................................................................................................. 103
6.5.2.2 Structure and Composition......................................................................... 103
6.5.2.3 Host Range and Viral Transmission........................................................... 104
6.5.2.4 Symptom Expression.................................................................................. 104
6.5.2.5 Diagnosis and Detection............................................................................. 105
6.5.2.6 Economic Importance................................................................................. 105
6.5.2.7 Control and Elimination............................................................................. 105
85
6.5.3 Banana Bunchy Top Virus (BBTV).......................................................................... 106

6.5.3.1 Distribution................................................................................................. 106
6.5.3.3 Transmission and Host Range..................................................................... 107
6.5.3.4 Disease Symptoms...................................................................................... 107
6.5.3.5 Detection and Control................................................................................. 107
6.5.4 Banana Bract Mosaic Virus (BBrMV)...................................................................... 108
6.5.4.2 Disease Symptoms...................................................................................... 108
6.5.4.3 Transmission and Detection........................................................................ 108
6.5.4.4 Economic Importance and Control............................................................. 108
6.5.5 Banana Mosaic or Cucumber Mosaic Virus (CMV)................................................. 108
6.5.5.2 Disease Symptoms and Host Range........................................................... 109
6.5.5.3 Viral Transmission...................................................................................... 109
6.5.5.4 Disease Management.................................................................................. 109
References....................................................................................................................................... 110
6.1 General Introduction
Banana is produced in over 100 countries and used in various ways. While large plantations owned
by multinational companies are grown mainly for export, small-scale farmers rely on banana for
food and income. Although the former cultivation systems are monocultures of a single cultivar,
banana cultivation for local consumption relies on a multitude of cultivars suited to different farming
systems and uses (Aurore et al., 2008). The tropical and subtropical environments that are ideal for
growing bananas and plantains are at the same time regions with high disease and pest pressure. The
creation of disease- and pest-resistant varieties is the priority for genetic improvement programs.
Bananas are affected by a number of fungal, bacterial, and viral diseases. The most important fungal
diseases are the Sigatoka diseases (yellow and black) and Fusarium wilt. Banana Xanthomonas wilt,
a relatively new bacterial disease, is becoming a serious threat to bananas in East Africa in much the
same way that blood disease posed in Indonesia and Moko in Central America. A large number of
banana viruses affect banana in different regions, with some becoming more predominant in some
areas. This chapter discusses the major fungal, bacterial, and viral diseases of banana. The inter-
ested reader in diseases of banana should also consult the treatment of this topic by Jones (2000a).
6.2 Sigatoka Diseases of Banana

6.2.1 Introduction
A wide range of leaf spot diseases affect banana (Musa spp.) in different parts of the globe, many
of them of minor economic importance, but some cause severe reduction in production and quality,
leading to important economic loss. Among the economically important leaf spot diseases are the
fungal diseases black leaf streak (black Sigatoka), caused by Mycosphaerella fijiensis Morelet; yel-
low Sigatoka, also known as Sigatoka, caused by M. musicola Leach; and Mycosphaerella speckle,
caused by M. musae (Spreg.) Syd. Symptoms of black and yellow Sigatoka are sometimes difficult to
differentiate. In general, the first symptom is the appearance on the upper leaf surface of pale yellow
streaks (yellow Sigatoka) or dark brown streaks on the lower surface (black Sigatoka), each 1–2 mm
long, that enlarge to form necrotic lesions with yellow halos and light gray centers (Mourichon et
al., 1997). The initial symptoms of Mycosphaerella speckle are light brown or tan-colored irregular
blotches on the lower leaf surface that may show as smoky patches on the upper surface. The three
Major Diseases of Banana 87
diseases can be recognized by the primary lesions. This stage of the disease is the most recognizable
and can be used to distinguish between the three Mycosphaerella leaf spot diseases. As the disease
progresses, spots become gray in the center but keep a brown border (Crous and Mourichon, 2002).
6.2.2 Origin and Geographical Distribution of Sigatoka Diseases

Southeast Asia is not only the place of origin of Musa, but it is also the place of origin of the Sigatoka
disease complex. Thus, yellow Sigatoka was first identified in the Java island of Indonesia in 1902
(Zimmerman, 1902). However, the disease became an epidemic in the Sigatoka valley of Fiji in
1903, from where the disease took its name. It is in the Fiji islands that black Sigatoka was first iden-
tified in 1963 (Rhodes, 1964), but the disease was probably not widespread in Southeast Asia and the
Southern Pacific region at that time (Ploetz, 2001). Both diseases have since become pandemic. In
Asia, yellow Sigatoka has been recorded in China, Malaya, Taiwan, the Philippines, Indonesia, and
India, where it is prevalent in the following states: Assam, Bihar, West Bengal, Kerala, Tamil Nadu,
Karnataka, Maharashtra, and Gujarat (Rawal, 2000). The first record of M. musicola in Africa was
in Uganda in 1932, but the disease was not widely distributed until the 1950s (Simmonds, 1962).
The disease has not been recorded in Egypt, Israel, and the Canary Islands (Rawal, 2000). Black
Sigatoka has become the most important foliar disease of banana and plantain in Latin America,
Asia, Africa, and the Pacific Islands (Meredith and Firman, 1970; Stover, 1978; Graham, 1969). In
Latin America, black Sigatoka was observed for the first time in Honduras in 1972 (Stover, 1983). It
now occurs in south central Mexico and Bolivia, as well as northwestern Brazil. In the Caribbean,
it is found in Cuba, Jamaica, the Dominican Republic, and southern Florida (Ploetz, 2001). Black
Sigatoka was accidentally introduced into Africa and was first recorded in Gabon in 1978 (Frossard,
1980). A suspected occurrence in Zambia in the early 1970s (Raemaekers, 1975) was shown not to
be associated with M. fijiensis (Tushemereirwe and Waller, 1993). From Gabon, black Sigatoka rap-
idly spread into Cameroon in 1980 (Fouré, 1985, 1987) and Congo in 1985 (Mourichon, 1986). The
disease reached Nigeria in 1986 (Wilson and Buddenhagen, 1986). In East Africa, black Sigatoka
was first reported from the island of Pemba (Tanzania) in 1987 (Dabek and Waller, 1990), Kenya
(Kung’u et al., 1992), and Malawi (Ploetz et al., 1992). Mobambo and Naku (1993) reported the
presence of the disease in Eastern Zaire (Democratic Republic of Congo) and other Central African
countries. Mourichon and Fullerton (1990) gave a comprehensive account on the global distribution
of both diseases.
Mycosphaerella eumusae was discovered in the mid 1990s as a member of the Sigatoka disease
complex of banana (Beveraggi et al., 1995; Burt et al., 1999). The disease originated from Southeast
Asia and parts of Africa, where it affects varieties that are resistant to both M. fijiensis and M. musi-
cola (Mourichon et al., 1997). So far, there has been no official confirmation of M. fijiensis in India,
but M. musicola remains a major production constraint (Nwauzoma et al., 2008). All commercial
cultivars such as ‘Rasthali’ (AAB), synonym ‘Silk,’ ‘Karpuravalli’ (ABB), synonym ‘Pisang Awak,’
‘Monthan’ (ABB), and those of the AAA Cavendish subgroup exhibit different levels of suscepti-
bility. The actual distribution and epidemiology of Sigatoka leaf spot diseases remain ambiguous
due to similarity in symptoms and life cycle of the pathogens. However, rapid and robust species-
specific molecular-based diagnostic tools have been developed to detect and quantify the pathogens
(Arzanlou et al., 2007).
Mycosphaerella fijiensis is considered a quarantine pathogen in Musa-producing areas and con-
tinues to occupy new ecological niches. Mycosphaerella musicola exists in colder environments
compared to M. fijiensis, and warmer temperatures favor faster development of ascospores and germ
tubes in M. fijiensis. Moreover, M. fijiensis is more vigorous and aggressive than M. musicola, pro-
ducing four times as many ascospores in the same period (Peterson et al., 2005), causing premature
fruit ripening. Yield losses of between 20 and 90% have been reported in Nigeria (Mobambo et al.,
1993) and elsewhere (Jones, 2002a, 2000b; Marin et al., 2003; Fouré, 1985).
6.2.3 Symptom Description

Symptoms of black and yellow Sigatoka diseases are very similar and often difficult to distinguish
with the naked eye, which hampers disease management. Tiny, chlorotic spots at the lower (abaxial)
surface of third and fourth unrolled or opened leaves are the first symptoms of black Sigatoka dis-
ease, about 2–3 weeks after infection. These spots grow into tiny brown streaks and later change
into streaks darker in color and sometimes with a purple tinge that appear on the upper (adaxial)
surface of the leaf. The lesions later coalesce, becoming fusiform or elliptical and darker, to give
the characteristic black streaking of the leaves. Fouré (1982, 1987) divided the disease development
into six stages as seen in AAB plantain cultivars:
1. Stage 1: Presence of minute specks yellowish in color and less than 1 mm in length only
present on the lower or abaxial surface of the leaf. They are often more abundant near the
left side of the leaf, particularly towards the tip but not visible in transmitted light.
2. Stage 2: There is the transition from specks into streaks, 3–4 mm long and 1 mm wide.
They are reddish-brown in color and more visible on the lower surface of the leaf than on
the upper part.
3. Stage 3: The streaks coalesce (2–3 cm in length) and the color changes from reddish-brown
to dark brown. When the streaks are many and more or less evenly distributed, the entire
leaf surface blackens.
4. Stage 4: The transition from streak to spot is characterized by the development of light
brown, water-soaked, elliptical spots, very much visible early in the morning with the pres-
ence of dew or rainfall.
5. Stage 5: The round, elliptical spots become completely black and are surrounded by water-
soaked border.
6. Stage 6: The center of the spot dries, turns light gray, and becomes surrounded by a nar-
row, well-defined black ring that in turn is encircled by a bright yellow halo. These spots
remain visible even after the leaf has dried out completely.
There is no sharp distinction between one symptom stage and the next. Fouré’s (1982, 1987) system
of classification is convenient for disease assessment procedures. The rate at which initial specks
change into streaks depends on climatic conditions, plant age, vigor, and cultivar as well as disease
development and severity (Meredith and Lawrence, 1969). On resistant varieties, symptom devel-
opment is very slow and may not progress to stage 4 (appearance of spots) until leaf senescence
(Jacome and Schuh, 1992; Fullerton, 1994). Resistance of Musa species to M. fijiensis seems to
be more related to postinfection production and activation of pathogen-related proteins (Lepoivre
et al., 1993) and phytoalexins (Luis et al., 1993) than to small changes in structure and preformed
substances. According to Beveraggi et al. (1995), partial resistance in the cultivar ‘Fougamou’ was
linked to a preexisting antifungal plant phenolic compound, while in the case of the highly resistant
cultivar ‘Yagambi Km 5,’ an active mechanism was induced following penetration of stomata by
the pathogen.
Lesions caused by yellow Sigatoka may look similar to those caused by black Sigatoka, but one
can distinguish yellow from black Sigatoka by examining the fungal hyphae producing asexual
spores. For example, the conidiophores of M. fijiensis occur in clumps (sporodochia) with basal
scars at the points of attachment while M. musicola has no scars. Conidia (asexual spores) and the
structures producing male sexual spores (spermagonia) occur on the underside of the leaf in infec-
tions caused by M. fijiensis, whereas M. musicola produces conidia mostly on the upper surface of
the leaf. Symptom development is slower in M. musicola than in M. fijiensis. Recently, polymerase
chain reaction (PCR)–based diagnostics have become available for the diagnosis and detection of
the three major causal agents of Sigatoka diseases (Arzanlou et al., 2007).
6.2.4 Epidemiology
Epidemiology involves studying the factors that influence disease development such as host–
pathogen interactions and the environmental and edaphic factors. A thorough understanding of
disease epidemiology is a prerequisite for the development of management strategies, including
durable host resistance (Craenen, 1998). The main factors that influence the production, release,
and spread of black and yellow Sigatoka inoculum are rainfall, temperature, dew, and wind. Well-
defined seasonal and day-to-day trends in inoculum production can be related to availability of
free moisture on the surface of the leaf and to minimum temperature. Maximum temperatures are
really limiting factors, provided free moisture is present (Stover, 1972). However, the principal
factor in the release of ascospores is rain. Short and violent rains, separated by sunny periods,
are the most favorable periods for the release of ascospores. Gauhl and Pasberg-Gauhl (1994a,
1994b) and Gauhl (1994) described seasonal variation in production of ascospores in Nigeria and
conidia in Costa Rica. Both black and yellow Sigatoka are spread by ascospores, with conidia
being an additional source of inoculums for yellow Sigatoka. Conidia are only produced in black
Sigakota in the absence of rainy weather. Conidia are produced on young leaves in the early streak
stage in black Sigatoka and in the mature spot stage in yellow Sigatoka. About 30,000 and 1,200
conidia per spot are produced by M. musicola and M. fijiensis, respectively. The conidia are sepa-
rated from the conidiophores by water or wind. Mature ascospores are produced 4 weeks after
the appearance of streaks in M. musicola and 2 weeks in M. fijiensis, and are actively released
to young, actively growing leaves mainly by wind and water. Germination of spores occurs on
the leaf when a film of water is present, after about 2–6 hours, depending on the temperature.
Following germination, stomatal penetration occurs after about 48–72 hours favored by tem-
perature above 20°C (Stover, 1980) and fluctuations in humidity (Goods and Tsirch, 1963). Once
infection is established, hyphae emerge from the stomata and either develop into conidiophores or
grow across the surface to infect adjacent stomata. Marin et al. (2003) stated that M. fijiensis has
greater penetration ability into the stomata and greater spotting than M. musicola.
6.2.5 Disease Management
Although plant diseases can hardly be completely eradicated, their impact can be reduced to
acceptable economic levels. A combination of cultural and chemical practices is recommended
for the management of Sigatoka disease complex: field sanitation, host nutrition, and sound cul-
tural practices; fungicides; detrashing (deleafing); pruning; ensuring good drainage and canopy
aeration; resistant cultivars. Chemical control is achieved by alternating protectant fungicides like
mancozeb or chlorothalonil with systemic fungicides such as benzimidazole or triazole. Systemic
fungicides are more suitable than protectant fungicides even though they pose the risk of potential
resistance in pathogen population (Marin et al., 2003). This, in addition to concern for the envi-
ronment, has resulted in the development of biological control methods using epiphytic bacteria
isolated from banana leaves against M. fijiensis (Jiménez et al., 1987). Some of the bacterial species
used in the biological control of Sigatoka disease complex are Pseudomonas spp., Bacillus spp.,
and Serratia marcescens.
The simplest way for the home gardener to control the disease is to destroy the severely diseased
leaves or remove them and place them top-side down on the ground to reduce the chance of spore
dispersal into the banana canopy. However, breeding for resistant cultivars has been credited as the
most appropriate long-term control method. One of the strategies to overcome leaf spot diseases in
Musa is through the hybridization of resistant AA diploids with triploids to obtain tetraploids that
combine disease resistance with good agronomic traits (Ortiz et al., 1995). Several tetraploid hybrids
have been developed by the International Institute for Tropical Agriculture (IITA), Nigeria, and the
Fundación Hondureña de Investigación Agrícola (FHIA) in Honduras (Rowe and Rosales, 2000),
as well as other international breeding programs in Brazil, Cameroon, and Guadeloupe (Fullerton,
1994; Carlier et al., 2000). Genetic transformation and somaclonal variation have also been used
to incorporate or identify durable disease resistance (Trujillo and Garcia, 1996; Sági et al., 1997;
Crouch et al., 1998; Nwauzoma et al., 2002). Future disease management programs will continue to
be directed on an integrated pest management (IPM) program that embraces early detection of the
pathogens that cause the Sigatoka diseases and encompasses eradication as well as cultural, chemi-
cal, and improved quarantine management strategies.
6.3 Fusarium Wilt Disease in Banana

6.3.1 Introduction
Fusarium wilt, commonly referred to as Panama disease, is regarded as one of the most devastating
diseases of banana and ranked as one of the top six important plant diseases in the world. Although
Fusarium wilt probably originated in Southeast Asia (Ploetz and Pegg, 1997), the disease was first
discovered in Eagle Farm, Brisbane, Queensland, Australia, in 1876 in banana var. Sugar (Silk
AAB) (Bancroft, 1876). In 1940, Snyder and Hansen proposed the name Fusarium oxysporum
Schlecht. f. sp. cubense (E. F. Smith). Presently, Fusarium wilt has been reported in all the banana-
growing regions of the world (Asia, Africa, Australia, and tropical America) except some islands in
the South Pacific, the Mediterranean, Melanesia, and Somalia (Ploetz and Pegg, 2000).
Race 1 of this disease completely wiped out the commercially important ‘Gros Michel’ cul-
tivar in Central America in the 1950s (Stover, 1962). The Cavendish clones, which are resis-
tant to race 1, were identified as a substitute for ‘Gros Michel’ for the export industry. Of late,
these Cavendish cultivars also succumb to Fusarium wilt in many banana-producing countries
such as Taiwan (Stover and Malo, 1972), Vietnam, Malaysia, Indonesia, Cambodia, China, the
Philippines, South Africa, and Australia (Ploetz, 1990). The disease poses a serious threat to the
multibillion-dollar export industry and also to the livelihoods of millions of small-scale banana
growers (Ploetz, 2005).
6.3.2 General Symptoms
The fungus infects the roots of banana plants, colonizing the vascular system of the rhizome and
pseudostem, and inducing characteristic wilting symptoms usually 5–6 months after planting, and
the symptoms are expressed both externally and internally (Stover, 1962). The external symptom
initially appears as yellowing of the leaf margins of older leaves, which gradually spreads across
the entire leaf surface. Generally yellowing progresses from the oldest to the youngest leaves, but
in some cases, the yellowing may be seen in the middle position (third or fourth leaves). The leaf
petiole turns brown and leaves gradually collapse at the petiole and hang down to form a “skirt”
of dead leaves around the pseudostem (Figure 6.1). The youngest leaves often stand erect, giving
the plant a “spiky” appearance. Newly emerging leaves are paler in appearance, are reduced in
size, and exhibit wrinkling and a distorted appearance (Moore et al., 1995). In the pseudostem,
longitudinal splits may develop near the soil level. The plant also produces many suckers before
it dies.
The internal symptoms are characterized by vascular discoloration beginning with yellowing of
the vascular tissues in the roots and corm/rhizome (Figure 6.2), which progress to form yellow, red,
or brown discolored vascular strands in the pseudostem and sometimes in the stalk. The disease
also spreads to suckers and the internal symptoms can be seen even after 1 or 2 months of emer-
gence of suckers (Moore et al., 1995).
Figure 6.1 Fusarium wilt-infected plant with yellowing of leaves in banana cv ‘Cavendish.’
Figure 6.2 Vascular discoloration due to Fusarium wilt in banana.
6.3.3 Causal Organism
The disease is caused by the soil-borne hyphomycete Fusarium oxysporum Schlecht. f. sp. cubense
(Foc). On potato dextrose agar (PDA), fungal colonies produce white aerial mycelia that may turn
purple in the center. Sporodochia are cream to orange on carnation leaves on carnation leaf agar
(CLA), and sclerotia are blue and submerged. Three types of asexual spores—macroconidia, micro-
conidia, and chlamydospores—are formed. Micro- and macroconidia are produced on branched
and unbranched monophialides, and the size of these spores range from 27 to 55 µm × 3.3 to 5.5
µm, and from 5 to 16 µm × 2.4 to 3.5 µm, respectively. Microconidia are produced on false heads,
are one- or two-celled, and oval- to kidney-shaped. Macroconidia are abundant, four to eight celled,
thin walled and delicate, slightly sickle shaped with an attenuated apical cell and a foot-shaped
basal cell. Chlamydospores are thick walled, terminal, and intercalary, usually globose, 7–11 µm in
diameter, and are formed singly or in pairs in hyphae or conidia but may also be found in clusters
or short chains. These spores are formed during the latter stages of disease in dead host-plant tissue
and in the soil. They are capable of surviving in the soil for several years. No perfect stage (teleo-
morph) of F. oxysporum is known (Su et al., 1986; Jones, 2000a; Ploetz, 2000).
6.3.4 Infection Processes of Foc

The fungus survives as chlamydospores in soil and on plant debris and invades the host through
the root hairs, root tips, and natural wounds along the lateral root base and also through the
wounds caused by farm implements, insect pests, and parasitic nematodes (Rishbeth and
Naylor, 1957; Lee et al., 2009). Once Foc enters the root, it colonizes the cortex and enters the
xylem in the vascular system, rhizome, and pseudostem (Rishbeth, 1955). After colonization,
the pathogen blocks the plant’s vascular system, which will lead to wilting and finally to plant
mortality (Ploetz and Pegg, 2000). After plant death, the fungus grows out of the xylem into
the surrounding tissues, forming many chlamydospores, which are returned to the soil after the
plant decays.
6.3.5 Survival and Spread

The fungus survives primarily in the soil and on plant debris as chlamydospores for more than 30
years (Moore et al., 1995; Ploetz, 2005). The pathogen is also able to infect and persist in the roots
of alternative hosts such as Paspalum, Panicum, Ixophorus, Commelina, and Chloris inflata, which
have been identified as nonsymptomatic hosts (Waite and Dunlap, 1953; Sun, 1977). The pathogen
populations tend to be higher and survive longer in light-textured soils than in heavy alkaline soils.
In the suppressive soil, which contains more microbial population, the pathogen development is
suppressed and this soil type has been reported in Central America, the Canary Islands, Australia,
and South Africa (Moore et al., 1995).
Spread of the pathogen locally, nationally, and internationally is through infected rhizomes or
suckers, and also through soil attached to infested planting material or implements/vehicles (Ploetz,
1994). Surface water is also responsible for the short- and long-distance dispersal of the disease. The
spread between the plants within the field is also affected by roots (Moore et al., 1995). Wind that
carries infested dust and trash is also involved in the spread of the fungus. There is no evidence that
the disease is disseminated in true seed (Ploetz and Pegg, 1997).
6.3.6 Classification
Foc can be classified into many races based on their ability to infect certain banana cultivars and
also vegetative compatibility groups (VCG) (Ploetz and Pegg, 1997). Four major races have been
reported (Moore et al., 1995) and their characteristics are:
1. Race 1: It occurs throughout the world and cultivars like ‘Gros Michel’ (AAA), ‘Silk’
(AAB), ‘Pome’ (AAB), abacá, ‘Maqueño’ (AAB), ‘Pisang Awak’ (ABB), and I.C.2 (AAAA)
(Ploetz et al., 1990; Bentley et al., 1995) are affected. Recent studies conducted by National
Research for Banana (NRCB) in India has indicated that some VCGs of race 1 also attack
race 2 suscepts and vice versa.
2. Race 2: This race attacks mainly ‘Bluggoe’ and ‘Monthan’ (ABB) and other closely related
cooking bananas. It also affects tetraploid ‘Bodles Altafort’ (hybrid between ‘Gros Michel’
and ‘Pisang lilin’) and enset (Ensete ventricosum—an important food crop in Ethiopia).
3. Race 3: Waite (1963) first identified the wilt disease in several species of Heliconia in
Central and South America in the mid 1900s and named the causal strain as race 3 of F.
oxysporum f. sp. cubense. This was recorded in Australia, Honduras, and Costa Rica. This
is nonpathogenic or weakly pathogenic on Musa spp. (Ploetz, 1990).
4. Race 4: It occurs in most of the banana-growing regions and is most destructive since it
affects race 1 and race 2 susceptible clones as well as the Cavendish cultivars and ‘Pisang
Mas’ (AA). This race is separated into two groups: subtropical race 4 (SR4) (VCGs, 0120,
0121, 0129, 01211 found in Australia, 0120 in Canaries and South Africa, and 0122 in
the Philippines) and tropical race 4 (TR4) (VCG-01213–01216 complex). SR4 affects
Cavendish plants that have been predisposed to disease by cold temperatures in the sub-
tropics, whereas TR4 attacks Cavendish more aggressively under tropical conditions in the
absence of any predisposing factors. There are no reports on occurrence of Foc TR4 in the
major banana-growing countries in Asia, including India (Thangavelu et al., 2001).
6.3.7 Disease Management
Effective management of this disease is possible only with integration of different strategies avail-
able, since no single method is able to contain the disease effectively. This approach not only
reduces the initial inoculum but also increases the plant resistance, delays the disease development,
and reduces the effectiveness of initial inoculum (Agrios, 1997). Quarantine and sanitation mainly
involve restricting the movement of corm, suckers, plant or plant parts, soil, and so forth from
infected areas to uninfected/clean areas. Trenches may be dug to separate the diseased areas to
prevent the movement of fungal spores through surface runoff water (Moore et al., 1999).
Creation of anaerobic condition by flooding is successful in managing the disease at least for the
first cycle of the crop (Wardlaw, 1961; Stover, 1962). In India, crop rotation with paddy and flooding
for 3–4 months before planting banana is effective (Sun and Huang, 1985; Thangavelu et al., 2001);
interplanting with cassava lowered the inoculum (Buddenhagen, 2009a). In Central America, cer-
tain fungicides such as Vapam, mylone, allyl alcohol, or formaldehyde controlled the disease very
effectively for 2–3 years (Stover, 1962).
Soil application of calcium compounds and phosphate salts, such as Ca(OH) 2, Ca(NO3)2 4H2O,
CaCO3, CaSO4, K2HPO4, and NaH2PO4 2H2O, strongly inhibited chlamydospore germination and
promoted lysis of germ tubes of Foc in soil (Sun and Huang 1985; Huang et al., 1989). However,
application of ammonia nitrogen increased the disease severity, whereas the nitrate nitrogen forms
decreased it (Huber and Watson, 1974).
Of late, biological control of Fusarium wilt disease is becoming more popular because it is
environmentally friendly and also offers a potential alternative to the use of resistant banana vari-
eties (Weller et al., 2002; Fravel et al., 2003). Biological control agents (BCAs) like Trichoderma,
Pseudomonas, Streptomyces, and nonpathogenic Fusarium (npFo) of both rhizospheric and endo-
phytic origin are effective against Fusarium wilt diseases (Lemanceau and Alabouvette, 1991;
Alabouvette et al., 1993; Larkin and Fravel, 1998; Weller et al., 2002; Thangavelu et al., 2003). The
suggested modes of action involved in the control of the diseases are: mycoparasitism, antibiotic
production, production of cell-wall-degrading enzymes such as chitinases and glucanases, competi-
tion for space and nutrients, and induced resistance. Pseudomonas fluorescens strain WCS 417 (Nel
et al., 2006) and endophytic isolates of nonpathogenic F. oxysporum (npFo) (Gerlach et al., 1999;
Nowak, 1998; Lian, 2009) are effective.
The use of fungicides is part of an integrated approach for Fusarium wilt management (Stover,
1962). Davis et al. (1994) demonstrated that the phosphonate fungicides were effective in con-
trolling Fusarium wilt disease. Several studies have indicated that chemical R&H–3888 (nitrile)
was most effective, followed by EP-161 (methyl isothiocyanate), Vapam (sodium n-methyl dithio-
carbamate), allyl alcohol, and mylone (3, 5-dimethyl tetrahydro-1, 3, 5 2H-thiadiazine-2-thione)
(Corden and Young, 1959, 1960). In India, stem injection with carbendazim decreased the disease
incidence in cultivar ‘Rasthali’ (Silk), but this treatment proved to be ineffective in South Africa
(Lakshmanan et al., 1987; Herbert and Marx, 1990). The fungicide benomyl and demethyla-
tion inhibitors (DMI) fungicides prochloraz, propiconazole, and cyproconazole/propiconazole
significantly reduced the incidence of Foc when applied as root-dip and soil-drench treatments
1 week after planting (Nel et al., 2007). Root dipping of young plants with propiconazole or the
biological Sonata also gave some effect in protecting the young seedlings (Buddenhagen, 2009b).
In South Africa, the spread of early detected Foc was stopped by treatment of the infested sites
with methyl bromide, which was effective only for 3 years. In the Philippines, heat treatment of
soil was used to control the spread of the pathogen, but effective only for a few years (Herbert
and Marx, 1990; Ploetz, 2000).
6.3.8 Host Resistance
This is the most economical and practical long-term option and must be part of an integrated dis-
ease management program for the small-scale farmers in many developing countries (Daniells,
2009). The Taiwan Banana Research Institute (TBRI) had released race 4 Foc-resistant Cavendish
clones viz. ‘Tai Chiao no. 1,’ ‘Tai Chiao no. 3,’ ‘TC3-1035,’ ‘5,’ ‘GCTCV-218’ (Farmosana),
‘GCTCV-119,’ and ‘GCTCV-215-1,’ which were resistant to Foc TR4 (Hwang and Ko, 2004).
The FHIA (Fundación Hondureña de Investigación Agrícola) breeding program has produced
several resistant hybrids to both Foc race 1 and race 4, such as ‘FHIA-01’ (Goldfinger, AAAB)
(Moore et al., 1995; Jones, 2000a) and ‘SH-3640/10’ (High Noon) (De Beer, 1997). EMBRAPA
(Empresa Brasileira de Pesquisa Agropecuaria) in Brazil has developed ‘Prata,’ ‘Maca,’ and
‘Prata Ana’ tetraploids, which showed resistance against Foc (de Matos et al., 1999). Recently,
seven diploid (AA) and 11 tetraploid (AAAB) banana hybrids resistant to VCGs 0124 and 0125
have been developed (de Matos et al. 2009). In India, nine hybrids resistant to Foc race 1 have been
developed. Among these, three were diploids (‘H-02-09,’ ‘H-02-10,’ and ‘H-02-15’), two were
triploids (‘H-02-08’ and ‘NPH-02-01’), and four were tetraploids (‘H-02-19,’ ‘H-02-23,’ ‘H-02-
26,’ and ‘H-02-4’) (Kumar et al., 2009). The Tropical Research and Education Center (TREC) of
the University of Florida has identified four dessert clones (‘Pisang Ceylon,’ ‘FHIA-01’ [AAAB],
‘FIAH-02’ [AAAA], and ‘FHIA-17’ [AAAA]) and three cooking clones (‘Kumunamba’ [AAB],
‘Kandrian’ [ABB], and ‘Saba’ [ABB]) resistant to VCG 01210, 0124, and 1210 of Foc (Ploetz
et al., 1990). Gamma irradiation of ’Dwarf Parfitt,’ an extra-dwarf Cavendish banana cultivar
with poor agronomic characters and resistant to subtropical race 4, resulted in the selection of
‘DPM 25,’ with improved productivity and also tolerance to subtropical race 4 of Foc (Smith et
al., 2006).
6.3.9 Conclusion
Fusarium wilt disease caused by Fusarium oxsporum f. sp. cubense is considered as a highly dev-
astating disease of banana. The disease affects almost all the important cultivars grown in different
parts of the world. The pathogen of the disease can survive as chlamydospores for more than 30
years, which is very dangerous for the perennial system of cultivation. As the fungus Fusarium
is highly variable in nature, wide diversity among the Fusarium wilt pathogen has been reported,
which is again making difficulties in identifying the strains as well as development of suitable
effective management practices including quarantine. Among the four races of Foc, tropical race 4,
which is infecting commercially important Cavendish cultivars, is spreading very fast and posing a
dangerous situation for the sustainable banana production throughout the world. Although several
management practices such as cultural, chemical, and biocontrol methods have been evolved, no
single method has been found effective so far. Hence all the available management strategies have
to be integrated to contain the disease effectively.
6.4 Banana Bacterial Wilt Diseases

6.4.1 Introduction
Such was the speed with which banana Xanthomonas wilt (BXW) has come to prominence as a
major disease of banana in Africa that it received little or no attention in textbooks or monographs
produced before the turn of the last century (Jeger et al., 1995; Thwaites et al., 1999; Ploetz et al.,
2003). Following spread of the disease to central Uganda and the Democratic Republic of Congo
(DRC), there has been a dramatic increase in investigations, which are documented in several com-
prehensive reviews (Biruma et al., 2007; Smith et al., 2008; Tripathi et al., 2009). These studies
have brought into focus some remarkable similarities and also some important differences between
diseases caused by a taxonomically diverse group of bacterial vascular pathogens of banana.
Implications for disease management are considered here.
6.4.2 The Diseases and Their Distribution

The main groups of vascular pathogens of banana are summarized in Table 6.1. Xanthomonas
campestris pv. musacearum (Xcm) was originally reported from Ethiopia (Yirgou and Bradbury,
1968) as causing an endemic disease of enset (Ensete ventricosum) and was subsequently observed
in cultivated bananas (Yirgou and Bradbury, 1974). Although a potential threat to the region was
noted, the disease remained confined to Ethiopia until around 2000, when isolated outbreaks were
identified in central Uganda (Tushemereirwe et al., 2003) and eastern DRC (Ndungo et al., 2004).
It has since spread rapidly and now affects at least six countries in the Great Lakes region of East
Africa. Although enset is generally cultivated only in southern and central Ethiopia, it grows in a
wild state in northeastern DRC, where natural infections have been noted (Ndungo et al., 2008).
Table 6.1
Bacterial Wilts of Banana
Common Name Causal Agent Distribution and Natural Hosts
Banana Xanthomonas wilt, BXW Xanthomonas campestris Ethiopia, Uganda, D. R. Congo, Rwanda,
(enset wilt, banana bacterial wilt pathovar musacearum (Xcm) Tanzania, Kenya. Reports from Burundi are
BBW). Also known as kiwatoka as yet unconfirmed (Enset and all cultivated
(Uganda) and Unyanjano wa banana types; M. balbisiana less
migomba (Tanzania) susceptible). Cultivars with persistent bracts
and flowers may escape the disease
Moko diseases (including Bugtok) Ralstonia solanacearum Americas: (Mexico, Central and South
biovar 1, race 2 America, southern Caribbean, Jamaica): all
cultivated types and wild Heliconia
Philippines: recognized as Moko on AAA
types; inflorescence infection of ABB types
incompletely systemic and recognized as
Bugtok/Tibaglon/Tapurok disease
Blood disease “Blood disease bacterium” Indonesia: all cultivated and some wild Musa
(Ralstonia sp.) species
Bacterial wilt of solanaceous crops Ralstonia solanacearum Central/South America, may be introduced
(brown rot, Granville wilt) biovar 1, race 1 elsewhere: occasionally or experimentally on
AA diploids, only
Recent genomic studies (Aritua et al., 2008) support a probable eastern African origin of Xcm
and indicate that it may share common ancestry with pathogens of graminaceous hosts (sorghum,
sugarcane, maize) from Ethiopia, now classified as X. vasicola. However, a change of name awaits
formal taxonomic revision.
The origins and recent history of BXW in eastern Africa share striking similarities to those of
banana blood disease in Indonesia. Blood disease was originally reported when intensive banana
cultivation was introduced to two small offshore islands in the Salayar archipelago, to the southeast
of the Indonesian island of Sulawesi (formerly Celebes; Rijks, 1916). In subsequent investigations,
Gäumann (1921, 1923) found the disease to be widespread in cultivated bananas and also in wild
(forest) Musa spp. in southern Sulawesi. At some locations, farmers reported historical epidemics
(“waves” of disease) going back for many years, and had coined the local name “penyakit darah” on
account of the reddish “blood-like” bacterial ooze that emerges from cut vascular tissues. The dis-
ease apparently remained confined to Sulawesi until the late 1980s when an outbreak was identified
in west Java (Eden-Green and Sastraatmadja, 1990). Since then, it has spread across the Indonesian
archipelago, from Aceh in the west to Irian Jaya in the east, a distance of over 4000 km. Gäumann
(1921) showed the causal agent was a Gram-negative bacterium that he named Pseudomonas cele-
bensis. Confusingly, this species was briefly reclassified as Xanthomonas, along with other nonfluo-
rescent pseudomonads (Dowson, 1943). The epithet “celebensis” was then used for another organism
and the specific binomial is no longer valid. On the basis of DNA homologies and fatty acid pro-
files, recent isolates have been shown to be a homogeneous group belonging to phylotype IV of the
Ralstonia solanacearum complex, together with isolates from other hosts in Indonesia (Fegan and
Prior, 2005, 2006). However, phenotypic properties, notably pathogenicity for banana but not for
any solanaceous host, differ from those generally accepted for R. solanacearum and, pending for-
mal taxonomic description, the causal agent is referred to as the blood disease bacterium (BDB).
Epidemics of Moko were first reported over 100 years ago in the ‘Moko’ (= ‘Bluggoe’) cultivar and
came to prominence in the 1950s as a threat to commercial plantations of dessert bananas in Central
America (Sequeira, 1998). Strains of Ralstonia (formerly Pseudomonas) solanacearum pathogenic
to banana, collectively referred to as race 2, are thought to have evolved into specialized niches in
Central and South America. Banana (B) strains, persisting in soils and spread mainly through culti-
vation and planting practices, originated in plantations developed from land previously growing wild
Heliconia species, from which isolates pathogenic to banana were obtained (Buddenhagen, 1960).
Some isolates from Heliconia were indistinguishable from B strains on Kelman’s (1954) tetrazolium
(TZC) medium but caused stunting, distortion, and slow wilting of young banana plants (D strains),
whereas others produced colonies with dense red formazan pigment from TZC and were pathogenic
to Heliconia and plantain (AAB) but not dessert bananas (H strains) or mildly pathogenic only to
Heliconia (R strains; French, 1986). The pathogenicity of D, but not R, strains increased after serial
passage through banana (Sequeira and Averre, 1961; French and Sequeira, 1970), supporting the
view that pathogenicity to banana evolved from naturally infected Heliconia.
Isolates showing a small fluidal round (SFR) colony morphology on TZC were first reported
from an unusual outbreak of Moko disease on ‘Bluggoe’ bananas in Honduras in the early 1960s
(Buddenhagen and Elsasser, 1962), apparently originating in planting materials introduced from
Venezuela (Sequeira, 1998). In contrast to B strains, these produced copious amounts of bacterial
exudates (ooze) from infected inflorescences, a feature considered an adaptation to insect transmis-
sion. SFR and other colony variants (SFR-C and SFR-A strains) were associated with other insect-
transmitted epidemics originating in Colombia and spreading in ‘Chato’ (‘Bluggoe’) bananas in the
Amazon basin into Peru and Brazil (French and Sequeira, 1970). Strains with SFR characteristics
are now widely distributed in South and Central America and the southern Caribbean (Lehmann-
Danziger, 1987; Phelps, 1987).
The polyphyletic origins of banana pathogens within the R. solanacearum group are supported
by studies on the molecular phylogeny and taxonomy. From analyses of 16s-23s rDNA interspa-
tial region and endoglucanase sequences, Prior and Fegan (2005) and Fegan and Prior (2006)
showed that Moko and Bugtok strains clustered into four sequevar subgroups within a single divi-
sion (Phylotype II). These findings are consistent with earlier multilocation genotype (MLG) clus-
ters proposed by Cook et al. (1991) from whole-genome restriction fragment length polymorphism
(RFLP) analyses. Banana (B) and Heliconia (H) strains clustered with Bugtok isolates in sequevar
3 (MLG24), whereas insect-transmitted SFR and Amazon (A) strains grouped into two different
and more distantly related clusters (sequevar 4/MLG25 and sequevar 6/MLG28), to which two
new isolates from Brazil were related. In contrast, BDB isolates, previously classified as a novel
MLG (Cook et al., 1991; Cook and Sequeira, 1994), clustered in a more distantly related division
(Phylotype IV), together with other isolates of R. solanacearum and R. syzygii from Indonesia.
These results suggest that capabilities for insect transmission in banana pathogens have evolved
from several independent origins.
6.4.3 Symptoms and Disease Cycle

Symptoms of banana bacterial wilts are very much related to the mode of infection, the host cultivar,
and, in the case of Moko, the strain of the pathogen. Where infection takes place via mature leaves,
roots, or rhizome, systemic invasion proceeds via the corm into the pseudostem of the affected
plant, and frequently into daughter suckers. In this “bottom-up” mode of infection, early symptoms
are a progressive transient yellowing and flaccidity starting with the oldest leaves, which become
necrotic and collapse at the base of the petiole. Pale green or whitish/yellowish panels develop in
one or more of the youngest leaves, which become flaccid and necrotic. The development of fruit
bunches is arrested and some of the fingers may ripen prematurely or split. Young suckers or follow-
ers may show a generalized wilting with little foliar discoloration. Internally, discolored vascular
bundles, initially cream or yellow but later becoming brown or black, may be seen throughout the
plant, eventually extending into the daughter suckers. In fruit-bearing plants, discoloration tends
to be concentrated in the fruit stem and the younger, central leaf bases. Within a few minutes of
cutting, vascular tissues of plants affected by Moko or blood disease exude viscous bacterial ooze
ranging in color from cream to reddish brown or black. A distinguishing feature of BXW is that the
ooze tends to be yellow and extremely copious, often filling airspaces within the leaf bases as well
as vascular tissues.
“Top-down” infection via the inflorescence, whether by insect transmission to male flower
buds or via contaminated cutting knives used in debudding, typically gives rise to a sequence of
symptoms that are very specific to bacterial wilts and serve as useful distinguishing features from
Fusarium wilt. The male bud shows blackening, shriveling, or distortion of one or more bracts and
associated male flowers, progressing to the whole bud and into the peduncle. Within a few days
of infection, droplets of cream-colored or pale yellow bacterial ooze (Figure 6.3) may emerge on
recently exposed scars of bracts and peduncle cushions. The ooze is said to be attractive to sting-
less bees (Trigona spp.) and other generalist feeders (Buddenhagen and Elsasser, 1962). Depending
on the cultivar and stage of fruit development at the time of infection, necrosis may extend into
the younger fruit bunches. Often fruit clusters at first appear outwardly normal but subsequently
exhibit premature ripening. By this stage, all fruits typically show internal discoloration and rot-
ting, which can vary in color (red, brown, or black) and texture (firm or gelatinous). As infection
progresses, internal vascular discoloration can be traced from the male bud into the peduncle,
down the fruit stem, and eventually into the corm and other parts of the plant. In this case, the
youngest leaves, which are subtended from the flower stem rather than the corm, may be the first
to show symptoms, and experienced farmers or field staff may recognize this as the first tell-tale
sign of infection.
Following inflorescence infection in ABB cultivars, and occasionally other types, bacterial inva-
sion can be incompletely systemic and may remain confined to the affected pseudostem or fail to
spread into all of the daughter suckers. This is typified by Bugtok disease of ‘Saba,’ ‘Cardaba,’ and
similar cultivars in the Philippines (Soguilon et al., 1994) but has also been described in ‘Bluggoe’
Figure 6.3 Bacterial ooze from banana infected with Xanthomonas wilt.
infected by SFR strains in Central America (Black and Delbeke, 1991). Suckers that become infected
will rapidly die, but those that do not will survive to produce flower buds that can be infected by
insects. In this way, infected mats may persist indefinitely and act as sources of inoculum for further
spread of the disease.
6.4.4 Disease Cycle and Implications for Control

Opportunities for control interventions relate to the ecology and epidemiology of different strains of
the pathogen, to the host cultivar, and to cultivation practices. In commercial plantations of dessert
(AAA) cultivars developed in Central America in the 1950s, pruning, desuckering, and debudding
were routinely practiced for agronomic reasons. There were thus ample opportunities for spread of
B strains on cultivation tools and contaminated planting materials and, although occasional insect
transmission of B strains probably occurred, routine elimination of male flower buds apparently
prevented the development of insect-transmitted epidemics. Once these factors were understood,
Moko was successfully brought under control by the early 1960s. It generally remains of minor
concern under plantation conditions although continued vigilance is necessary.
In contrast, ABB cultivars such as ‘Bluggoe’ are confined largely to smallholder and domestic
plantings, where they are rarely debudded in time to prevent inflorescence infection (if at all) or with
precautions to prevent spread on cutting tools. Most ABB types have dehiscent male flowers and
bracts, and probably other characteristics that render them particularly susceptible to inflorescence
infection—features that strains such as SFR appear to have evolved to exploit. These factors have
contributed to epidemics of Moko that, like those of BXW and blood disease, have proven much
more difficult to control. The two pathosystems reach their apotheosis in the Philippines, where
Bugtok in smallholder plantings of ABB/BBB cooking bananas such as ‘Saba’ and ‘Cardaba,’ and
Moko in ‘Dwarf Cavendish’ plantations, behave as two different diseases but are caused by the same
strain of R. solanacearum (Fegan, 2005; Fegan and Prior, 2006).
6.4.4.1 Infection via Soil and Roots

During the development of commercial plantations on newly cleared land in Central America, the
incidence of Moko was closely related to the distribution of infected wild species of Heliconia
(Sequeira and Averre, 1961). Although passive spread via the soil was slow, areas abandoned to bac-
terial wilt slowly encroached on healthy ones (Buddenhagen, 1960). Fallowing or rotation regimes
were successfully developed to reduce the risk of infection (Sequeira, 1962). “Buffer zones” around
infected plants, free of banana and other potential hosts of the pathogen, were also advocated to
reduce the risk of reinfection (Stover, 1972), although there are conflicting reports on the role of
alternative weed hosts and specific recommendations have varied (see discussion in Jeger et al.,
1995). The persistence of bacteria in soil following previous disease infection was also recognized
for blood disease (Gäumann, 1923) but experimental data are still lacking.
These findings contributed to early recommendations for control of BXW by uprooting and
burying diseased plants. However, there is an increasing body of data to suggest that Xcm survives
poorly in soil or plant residues (Mwebaze et al., 2006) and that, following rigorous uprooting of
infected mats/removal of diseased plants, replanting can safely take place after a 6-month fallow
period (Turyagyenda et al., 2007). Planting practices such as avoiding topsoil and allowing the
cut surfaces of corms to heal for a few days before planting have been advocated to further reduce
the risks of infection during replanting (Mwangi et al., 2007). However, a study carried out in
Ethiopia by Shehabu et al. (2010) found that paring and air-drying of banana suckers before plant-
ing increased soil-mediated Xanthomonas wilt infections. Likewise, the practice of using discarded
leaves, flower stalks, buds, or other infected plant residues as mulch presents risks for reintroducing
soil-borne inoculum from external sources (Mwangi and Nakato, 2009).
Race 2 of R. solanacearum can reportedly infect roots without mechanical injury (Kelman and
Sequeira, 1965), but this has not been systematically investigated and it is likely that for all diseases
most soil-borne infection probably occurs through mechanical injury of roots and corms during
planting or cultivation (Tumushabe et al., 2006).
6.4.4.2 Spread on Cutting and Cultivation Tools

The early recognition that the majority of Moko infections occurred via cutting tools contaminated
during pruning and harvesting (Buddenhagen and Sequeira, 1958; Sequeira, 1958) led to standard
adoption and rigid enforcement of field sanitation practices that remain successful elements of
Moko control under plantation conditions (Stover, 1972). Similar practices have been recommended
for BXW but are difficult for smallholders to adopt. Field observations on the spread of BXW in
East Africa provide strong circumstantial evidence of the importance of mechanical transmission
both within and between plantings, especially in areas where cultivation practices such as leaf
pruning and debudding are practiced intensively (Mwangi and Nakato, 2009). In Ethiopia, Addis et
al. (2008) confirmed the high susceptibility of leaves, floral rachis, and pseudostems but to a much
lesser extent of roots to infection via contaminated cutting knives. Buregyeya et al. (2008) showed
that cutting knives transmitted disease up to 19 days (stainless knife) or 6 days (steel panga) after
artificial contamination with sap from plants infected with Xcm—quite long enough for the disease
to be spread by farmers, or by itinerant traders who frequently undertake harvesting operations
themselves, using knives that may have been contaminated from distant fields. Similar consider-
ations apply to blood disease in Indonesia, where interisland spread has been attributed to traders
using their own harvesting knives (Supriadi, 2005). However, in practice the cutting of leaves for
packing or padding, rather than harvesting mature fruit bunches, may be more important for spread
of infection. Pseudostems bearing mature fruit bunches are no longer in active growth and observa-
tions on Moko (SFR strain) reported by Hunt (1992) indicated that harvesting mature bunches with
contaminated knives did not result in disease transmission to the mat.
The standard treatment originally recommended for disinfecting knives against Moko was form-
aldehyde (Stover, 1972), but now that its toxicity is better appreciated other treatments need to be
found. Quaternary ammonium compounds (quats), which are now widely used in human hygiene,
food, and agriculture, showed promise in early trials with Moko and one compound was considered
cheaper and only marginally less effective than formaldehyde (Buddenhagen and Sequeira, 1958).
In East Africa, domestic bleach (sodium hypochlorite, widely known by the trade name of Jik) at
dilutions of up to 20% is currently recommended for cleaning tools in fields affected by BXW.
However, hypochlorite is not very stable and neither this, nor the alternative recommendation of
heating over a fire, is very practical for smallholders. Other strategies include avoidance of pruning
where there are risks that BXW is present, strict avoidance of harvesting fruits or leaves using
knives provided by traders, and of course use of forked sticks rather than knives to twist and break
off male buds (see Section 6.4.4.3).
6.4.4.3 Inflorescence Infection

The probable role of inflorescence infection in blood disease epidemics was postulated by Gäumann
(1923) who observed natural infections and showed that bacteria applied to female flowers could
infect the fruits. However, these infections were not attributed to insect vectors at the time, and the
significance of these observations was overlooked until investigation by Buddenhagen and Elsasser
(1962) of an unusual outbreak of Moko disease on ‘Bluggoe’ bananas in Honduras. The authors
observed up to 100 bees and wasps per hour frequenting a single male inflorescence, and recovered
bacteria exhibiting SFR characteristics from 5% of stingless bees (Trigona corvina) and wasps
collected from a patch of diseased plants over a 20-day period. Inflorescence symptoms developed
rapidly: bacteria exuded from pedicels and bract scars after 15–25 days of infection and attracted
insects including Trigona spp., wasps (Polyoba spp.), and fruit flies (Drosophila spp.; Buddenhagen
and Kelman, 1964). Healthy plants were infected when bacteria were carried by insects to moist,
newly exposed pedicels (“cushions”) within about 2 days of abscission of the male flowers. Bagging
whole inflorescences to exclude insects and breaking off the male buds before the first male flower
cushions became exposed prevented infections, which otherwise spread rapidly to neighboring
plants. Transmissions between ‘Bluggoe’ plantings more than 1 mile (1.6 km) apart were apparently
rare, but in isolated cases plants up to 5 miles (8 km) apart became infected and acted as new foci
of infection.
Similar groups of insects have been implicated as vectors of blood disease in Indonesia
(Leiwkabessy, 1999, cited by Supriadi, 2005; Subandiya et al., 2005) and BXW in Uganda (Tinzaara
et al., 2006), DRC (Fiaboe, Beed, et al., 2008), and Ethiopia (Shimelash et al., 2008).
However, although the causal bacteria have frequently been recovered from insects, very few
experimental transmissions have been demonstrated, and the possible role of stingless bees in the
spread of BXW has not been confirmed (Namu, 2008). Symptoms of inflorescence infection appear
to be uncommon at altitudes exceeding 1700 m above sea level in the Great Lakes region of East
Africa, presumably reflecting reduced vector activity at cooler temperatures (Addis et al., 2004;
Mwangi et al., 2007; Ndungo et al., 2008). Bats and birds have also been implicated in spreading
BXW over longer distances. Buregyeya et al. (2008) recovered Xcm from certain birds and bats for
up to 5 days after they had visited diseased banana flowers, but further work is needed to confirm
their epidemiological significance.
Removal of male flower buds after fruit clusters have formed has long been standard agronomic
practice in commercial dessert banana plantations and also under intensive East African Highland
(EAH) production in East Africa. This undoubtedly reduces the risk of transmission by insects but
may make matters worse unless strict precautions are taken to avoid spread on contaminated knives.
For dwarf cultivars such as ‘Dwarf Cavendish,’ buds can be broken off by twisting the peduncle
by hand, and a forked stick can be used for taller plants. Experiments under on-farm conditions in
Uganda have shown that removal of male flower buds in this manner is highly effective in prevent-
ing BXW infection (Blomme et al., 2009), provided this is done immediately after formation of the
last female flower cluster. This practice has been particularly successful in preventing infection of
‘Pisang Awak’ (ABB, ‘Kayinja’) and farmers who have adopted it have come back into full produc-
tion. The effectiveness of debudding suggests that, in practice, infection takes place only via the
male flower parts: in dehiscent cultivars, male flowers and bracts are shed approximately daily as
new ones emerge, so there is continuous availability of infection courts over a period of several
weeks. However, a preliminary report suggests that infection can also occur via the female part of
the inflorescence, and at a high frequency, when bananas are grown in isolation and there are high
levels of inoculum in adjacent plants (Fiaboe, Kubiriba, et al., 2009). It is not yet clear whether these
infections took place via female bract scars or the flowers themselves. In other studies (Tinzaara et
al., 2006), Xcm has been recovered from nectar of male flowers and while male flowers may be too
short lived to allow time for bacteria to multiply and invade the peduncle, this would not apply in
female flowers or in cultivars with persistent male flowers.
6.4.4.4 Planting Materials
Daughter suckers are readily invaded if bacteria reach the corm, but this may not always occur
depending on cultivar and route of infection. Thus contaminated planting materials present signifi-
cant risk of infection, even if the remainder of the mat appears healthy. Spread of Moko between
plantation operations in Central America is well documented (Buddenhagen, 1961) and is thought to
be the means that the disease reached the Philippines as early as the 1940s (Rillo, 1979; Hayward,
2006). Similar mechanisms are likely to have contributed to the spread of blood disease in Indonesia
and BXW in East Africa. The use of tissue-cultured plantlets eliminates the risks for commercial
producers, but access to disease-free planting materials remains a challenge for smallholders (www.
c3project.iita.org, accessed 18 May 2010).
6.4.4.5 Other Modes of Infection

Some strains of R. solanacearum are known to be spread in rivers, and flooding or irrigation has
been suggested as means of dispersal of Moko (Sequeira, 1998). Circumstantial observations have
suggested that BXW may be dispersed via wind-blown rain, on the feet or hooves of man and ani-
mals, or by browsing animals, but experimental evidence is lacking. Trade may play an important
role in the long-distance spread of BXW, both from cutting by contaminated knives and from move-
ment and subsequent discard of infected plant parts. An outbreak in Muleba district, Tanzania, is
thought to have originated from infected male buds used as stoppers for containers of banana beer
imported from Uganda (Mwangi and Nakato, 2009).
6.4.5 Screening and Breeding for Resistance

Several authors have reported differential responses of cultivated and noncultivated Musa to Moko
(Stover, 1972), blood disease (Gäumann, 1921; Supriadi, 2005), and BXW (Ssekiwoko et al., 2006;
Welde Michael et al., 2006; Tripathi et al., 2008) following either natural infection or experimen-
tal inoculation. Although no cultivated type has been reported to be truly resistant to any of these
diseases, differences in speed and severity of symptom development have been noted following
mechanical inoculation, and some clones have shown partial recovery with production of new suck-
ers. However, such plants are likely to be tolerant rather than resistant, and symptomless plants
can serve as hidden sources of inoculum. Stover (1972) reported that 34 of 345 accessions tested
in Honduras showed some resistance to a Moko SFR strain injected into the pseudostem. These
included the ABB clone ‘Pelipita,’ the ‘Manang’ accession of M. acuminata spp. banksii, and
M. balbisiana. The latter has been reported to survive mechanical inoculation with B strains of
Moko and also Xcm (Ssekiwoko et al., 2006; Tripathi et al., 2008).
In the absence of cell-mediated resistance, a more promising breeding strategy may be to exploit
differences in susceptibility to inflorescence infection. The ABB clone ‘Pelipita’ has been suggested
as a replacement for ‘Bluggoe’ because it has indehiscent male bracts and flowers, and is much less
susceptible to insect transmission (Stover and Richardson, 1968). Cavendish types, which tend to
retain male flowers and bracts, are less susceptible to inflorescence infection to Moko than the ‘Gros
Michel’ plants affected by Moko in Central America. ‘Dwarf Cavendish’ is not widely grown in
East Africa but observations in Ethiopia suggest that it is also less susceptible to BXW (Addis et al.,
2004). Some EAH (AAA) and plantain (AAB) cultivars do not shed male flowers and bracts and
hence exhibit apparent resistance, or escape, from BXW (Mwangi and Nakato, 2009). Others have
dehiscent flower parts but appear to be less susceptible to infection either because the flowers are less
attractive to insects or the flower scars are less conducive to penetration or survival of Xcm (Mwangi
et al., 2006). Recently, Buddenhagen (2009b) has drawn attention to clones from Indonesia that do
not produce male buds at all. One of these, known locally as ‘Pisang Puju,’ has fruit characteristics
very similar to ‘Pisang Kepok’ (Saba) and is proposed as a replacement. However, although able to
escape insect transmission, it is likely that these clones will be susceptible to infection of roots or
leaves damaged by contaminated cultivation tools.
These features underline the importance of considering different routes of infection when screen-
ing for resistance that may prove useful in the field. Mechanical inoculation by injection of bacte-
ria into pseudostem or leaf bases is a convenient technique for screening large numbers of plants
(Tripathi, Odipio, et al., 2008) but may be unnaturally severe, especially if high doses of inoculum
are used. Welde Michael et al. (2006) found all of 40 banana cultivars were susceptible following
inoculation of a high dose of Xcm into leaf bases, but acknowledged that the inoculation method
could have masked differences in susceptibility to infection via inflorescences.
Given the lack of resistance and difficulties in conventional breeding of bananas, transgenic
approaches merit attention. Tripathi et al. (2008) report promising progress in generating several
East African Highland banana (EAHB) and ‘Pisang Awak’ lines with enhanced defense mecha-
nisms induced by introduction of ferredoxin-like amphiphatic protein (pflp) from sweet pepper.
These are currently being evaluated for resistance to Xcm under containment conditions.
6.4.6 Impact and Future Outlook

Once main avenues of infection were recognized and appropriate phytosanitary measures devised,
Moko was quickly brought under control in commercial plantations of dessert bananas, although
at some considerable costs (Stover, 1972; Sequeira, 1998). It has remained a serious and intractable
problem for smaller farmers since epidemics were first described in South and Central America in
the 19th and early 20th centuries. Within the past 50 years, well-documented pandemics driven by
insect transmission in highly susceptible ABB cultivars have continued to limit or destroy banana
cultivations in both the Americas and the Philippines (French and Sequeira, 1970; Lehmann-
Danziger, 1987; Sequeira, 1998; Molina, 2006). A similar pandemic has occurred in Indonesia,
following spread of blood disease from Sulawesi to west Java in the late 1980s (Davis et al., 2001;
Supriadi, 2005; Buddenhagen, 2009b) and now threatens the Southeast Asian mainland (Promed,
2009). In East and Central Africa, high densities of ‘Kayinja’ (‘Pisang Awak’) and similar suscep-
tible cultivars have driven the insect-transmitted spread of BXW in some regions (Eden-Green,
2004), but the situation is more complex. Under intensive cultivation of AAA-EAHB types, where
banana tends to have greatest value for income and food security, insect transmission is probably
rare, but cultivation practices favor mechanical transmission.
Much has been written about the social, economic, cultural, and environmental impacts of
banana bacterial wilts on affected smallholder communities but these aspects are difficult to quan-
tify. Kalyebara et al. (2006) estimated that, in purely economic terms, cumulative losses to BXW in
Uganda could reach US$5.6 billion over 10–15 years, representing an annual loss of about US$200
of food and income per farming household. Adoption of control measures could reduce losses by
40% in matooke but less in brewing types. Similar conclusions were reached independently from
surveys carried out in 2005 by Karamura (2006) and co-workers, who predicted that, unchecked,
the disease would spread throughout Uganda, resulting in a cumulative loss of over $4 billion by
2010, significant price increases, and reduction in production. However, in the short term, moderate
production losses could produce shortages and higher prices that would benefit producers (Abele
and Pillay, 2007). Increased market prices have acted as incentives to farmers prepared to adopt
debudding to control BXW in ‘Kayinja’ juice bananas (Blomme and Karamura, 2006). Perhaps the
greatest concerns arise in areas destabilized through conflict and civil unrest, where annual crops
become disrupted or neglected and bananas assume increased importance for food security (Abele
et al., 2007).
In purely technical terms, banana bacterial wilts are not difficult to control. Avoidance and
sanitation measures are known and easy to implement, although complete eradication is more
difficult to achieve. The problem for smallholder systems lies both in communicating knowl-
edge and in gaining ownership and adoption of control measures that, to be fully effective, need
to be applied on a community scale. Considerable efforts have been made in Uganda to gain
community adoption through carefully structured interactions such as working groups and task
forces, involving technical staff, local government, community leaders, and farmers, a process
that has been termed participatory development communication (PDC; Nankinga and Okaasai,
2006; Odoi, 2006). These measures have met with considerable success in reducing the incidence
and spread of BXW in smallholder communities (Kubiriba, 2009), but dealing with resurgence
of infection remains a problem.
6.5 Banana Viruses

6.5.1 Introduction
Viral diseases of banana constitute a very large group of important infective agents that reduce
banana and plantain production in different parts of the world. Since banana is a vegetatively propa-
gated crop, the viruses simply multiply in them and are spread during planting. Tissue culture can
also help in spreading banana viruses if infected plants are multiplied in culture. For this reason,
proper and careful indexing of planting materials for viral pathogens using molecular tools is neces-
sary. The following viral pathogens have been reported from different banana growing areas of the
world and they include: banana streak virus (BSV), banana bunchy top virus (BBTV), banana bract
mosaic virus (BBMV), and banana mosaic or infectious chlorosis (also called cucumber mosaic
virus, CMV).
6.5.2 Banana Streak Virus

6.5.2.1 Distribution
Viral leaf streak was first reported on Musa (AAA group Cavendish subgroup) ‘Poyo’ from Côte
d’Ivoire (Lassoudiére, 1974). The causal agent was identified in Morocco in 1986 and called banana
streak badnavirus (BSV) (Lockhart, 1986) and the disease was called banana streak virus. BSV
is a para-retrovirus (Bouhida et al., 1993). BSV has been identified in different banana-growing
areas of the world, including Benin, Côte d’Ivoire, Ghana, Malawi, Madagascar, Mauritius, Nigeria,
Rwanda, South Africa, Tanzania, Australia, Brazil, the Canary Islands, and Trinidad (Lockhart and
Olszewski, 1993). The disease has also been found in India where it is most severe on ‘Mysore,’
an important commercial AAB group, and in Latin America and Australia (Jones and Lockhart,
1993; Diekmann and Putter, 1996; Riechel et al., 1996). Although BSV disease occurs in all banana-
producing areas, the amount of field infection may greatly vary.
6.5.2.2 Structure and Composition

Badnaviruses are a group of plant viruses with non-enveloped virions and average particle size of
120–150 nm × 30 nm and double-stranded DNA (dsDNA) genome of 7.4–7.8 kbp (Lockhart, 1990).
Particle sizes of 60–900 nm in length have been observed in purified preparations. Badnaviruses
differ from other plant viruses in particle morphology, genome size, vector relationships, and histo-
pathology. Under the microscope, particles of CMV and BSV are distinctly different; whereas CMV
is isometric (28–30 nm), BSV has a bacilliform shape (30 × 130–150 nm). Badnavirus particles con-
tain a single species of dsDNA. Under nondenaturing conditions, the DNA migrates in agarose as
three or more bands, corresponding to linear, circular, and twisted configurations. The virus codes
for three proteins from three well-defined open reading frames (ORFs). ORF 111 polyprotein is the
most characterized part of the virus.
6.5.2.3 Host Range and Viral Transmission

Most badnaviruses are present in clonally propagated tropical crops and spread in nature by
vegetative propagation. The individual viruses typically have a restricted natural host range,
frequently limited to a few species within a single plant genus. BSV is serologically related to
the sugarcane bacilliform virus (ScBV; Lockhart and Autrey, 1988), which also produces streak
symptoms in experimentally inoculated plants (Lockhart and Autrey, 1991). However, prelimi-
nary host-range studies with BSV isolates indicate differences in the ability to infect species
within the Musaceae as well as sugarcane (Lockhart and Autrey, 1991). ScBV is transmitted
from sugarcane to banana by the pink sugarcane mealy bug Saccharicaccus sacharri and from
banana to banana by Planococcus citri in a semipersistent manner. Spread within short distances
is accomplished when the vector (mealy bug) makes contact with foliage within the crop canopy
from infected to healthy plant. The spread of badnaviruses occurs primarily within a single culti-
vated species. There is no evidence that mechanical contact, cutting tools, or cultural operations
can spread BSV.
In most tropics, sugarcane is grown in close proximity to banana, and transmission from the for-
mer to the latter undoubtedly occurs. In India where sugarcane is grown in rotation with rice, BSV is
very common, especially in ‘Mysore’ (AAB). Almost all the clones of this cultivar carry BSV, sug-
gesting that the disease symptoms were of genetic origin. It is now known that BSV can be transmit-
ted to new hybrids through the activation of integrants present in the parent B genomes, and tissue
culture triggers symptom expression. The mode of replication in badnaviruses encourages a high
level of mutation, and a number of serologically distinct, naturally occurring isolates of BSV have
been identified. Hence, it would be useful to know if all strains are vector transmitted, and whether
there is any vector specificity, to explain why BSV spreads in some areas and not in others.
6.5.2.4 Symptom Expression
Jones and Lockhart (1993) have reviewed symptoms associated with BSV and found that they are
similar to cucumber mosaic virus (CMV), especially in early stages. However, necrotic streaks and
periodicity of symptom expression are characteristic features of BSV. Plants may not show streak
symptoms on all leaves, and for several months, emerging leaves may not exhibit streak symptoms
at all or show only light symptoms. This explains why plants in quarantine need to be grown for
some time and examined regularly for symptoms. For this reason, Musa pathologists may have
mistaken BSV for CMV. A few photographs reputed to show symptoms of CMV in some publica-
tions look identical to those of BSV (Wardlaw, 1961; Stover, 1972). Stover (1972) and Simmonds
(1987) showed that incidence of CMV in Horn plantain (AAB) in Honduras was extremely high,
with greater symptoms being expressed in cooler conditions. In Morocco, BSV-infected banana
(Musa spp. [AAA group] cv ‘Dwarf Cavendish’) had broken or continuous streaks and/or sparse
or concentrated spindle-shaped lesions on the leaf lamina that often turned necrotic upon aging
(Lockhart, 1986).
The following symptoms are associated with BSV-infected Musa germplasm in Nigeria: discrete
whitish/yellow short streak on the leaf lamina turning darker yellow or orange with age, distortion
of leaf lamina, internal necrosis of the pseudostem, length-wise cracking of outer leaf sheaths,
and cigar leaf death (die-back) followed by plant death and distorted bunch or fingers (Gauhl and
Pasberg-Gauhl, 1995). Others are abnormal emergence of flowers and bunches, shortened and dis-
torted peduncle, and bunch bursting out from the side of the pseudostem (Gauhl and Pasberg-Gauhl,
1994b). Diseased plants have reduced growth and vigor, resulting in poor yield due to small or
absence of bunches (Ortiz, 1996). These symptoms are more severe in the rainy season than in the
dry season (Dahal et al., 1998). Pronounced streak symptoms occur sporadically throughout the
year when inflorescence initiation coincides with an episode of increased replication.
6.5.2.5 Diagnosis and Detection

The successful control of most diseases depends on accurate diagnosis. It is known that BSV can
persist in the host for a long time without producing any symptoms. The integration of more than a
full-length genome of the virus in the host cell is a challenge to quarantine authorities and has seri-
ous implications on the exchange of planting materials across international boundaries. Previously,
diagnosis of BSV was based on the recognition of symptoms. Unfortunately, the inherent difficul-
ties in the use of symptoms as the only diagnostic technique resulted in confusion in differentiating
BSV from CMV. In addition, the detection and diagnosis of badnaviruses is hindered by their low
concentrations in host plants and the presence of secondary products such as tannins, mucilage, and
other compounds (PBIP, 1997). Detection by visual observations is not reliable because of period-
icity of symptoms, and the use of indicator plants is not applicable because BSV has a restricted
host range. Detection by electrophoresis of virus-specific dsRNAs in plants has been found to be
a highly sensitive method (Dodds et al., 1984). However, this technique is only applicable to plant
viruses whose genomes consist of single-stranded RNA (ssRNA). But BSV and other badnaviruses
that have dsDNA genomes do not have dsRNA replicative intermediates and therefore cannot be
detected using this method. Serological methods, using immunoprecipitation, enzyme immunoas-
say (EIA), and immunoelectron microscopy (IEM) have been advocated (Lockhart, 1986). Both
EIA and IEM provide rapid, sensitive, and reliable methods for the detection of badnaviruses when
a viral antigen occurs in plant tissue. The application of these techniques is, however, restricted by
the occurrence of serologically distinct badnavirus populations within a given crop. This serological
heterogeneity among isolates has been illustrated in BSV using EIA and IEM (Lockhart, 1990).
To overcome these difficulties, an alternative approach based on the polymerase chain reaction
(PCR) amplification of the conserved badnavirus genomic sequences was introduced (Sidi et al.,
1988). This method has the potential to detect any variant of BSV. Thus, an isolate of BSV can be
purified and the genome of this isolate is cloned and sequenced, allowing for the synthesis of BSV-
specific primers. These primers are used to screen a diverse range of Musa germplasm to confirm
their ability to detect BSV isolate from different sources. Medberry et al. (1990) employed this
method to identify and analyze the nucleotide sequences of three badnaviruses (CoYMV, RTBV,
and KTSV) and identified three conserved regions in the ORF 111, which include the tRNA Met bind-
ing domain, ribonuclease H (RNAse H), and reverse transcriptase (RT) regions. The PCR-based
amplification method therefore appears to represent a workable solution to the problem of serologi-
cal and genomic heterogeneity among BSV and other badnavirus triple-antibody sandwich enzyme-
linked immunosorbent assay (TAS-ELISA) has been used to detect the virus (Thottapilly et al.,
1997). Immunocapture PCR (IC-PCR) has also been used to differentiate the episomal and retrovi-
ral sequences in banana. Real-time PCR has also been used for quick detection of virus genomes,
but the technique is expensive and may not be affordable by most tissue-culture laboratories.
6.5.2.6 Economic Importance
BSV has been reported to cause losses of 7–90% in Côte d’Ivoire (Lassoudière, 1979). BSV infec-
tion can reduce bunch size and distort fruit shape in banana. It has been reported that yield loss var-
ies from cultivar to cultivar and with climatic conditions. In India, BSV severely infects the Mysore
group of banana, namely ‘Poovan,’ ‘Red Banana,’ ‘Robusta,’ ‘Nendran,’ ‘Cheni Champa,’ and also
the Cavendish group (‘Basrai’ and ‘Grand Nain’). It is likely that BSV disease occurs in all banana-
producing areas, although the amount of field infection may vary greatly.
6.5.2.7 Control and Elimination

The use of virus-free planting materials remains the best way to control badnaviruses. A combi-
nation of meristem culture and thermotherapy is another reliable method (Quak, 1977). However,
this does not guarantee “virus-free” materials (George and Sherrington, 1984; Drew et al., 1989;
Nwauzoma et al., 2004). This was further confirmed in thermotherapy studies with the closely
related sugarcane bacilliform virus. As the ambient temperature was increased, the replication rate
of the sugarcane virus also increased. Virions of badnaviruses are thought to be stable at room tem-
perature for several weeks but infectivity is lost on exposure to 53–55°C for 10 minutes.
It has been demonstrated that the BSV genome is integrated into the Musa genome (Ndowora et
al., 1999; INIBAP, 1997). Ortiz and Vuylsteke (1998) suggested the use of virus-tolerant genotypes
as the most appropriate short-term control measure. In vitro culture in the presence of “anti-viral”
compounds can be used to eliminate viruses from germplasms. One of the most effective and com-
monly used compounds is Ribavirin (1-D-ribofuranosil-1-H-1, 2,4-triazole-3-carboxamide). The
actual mode of action of Ribavirin is still unclear; it appears that it has RNA-specific activity but
it has been demonstrated that Ribavirin does not inhibit RNA polymerases (Lerch, 1987; Senula et
al., 2000). Since there are no reports of its activity against dsDNA viruses of plants, it cannot be
regarded as a potential therapeutic agent for BSV elimination.
6.5.3 Banana Bunchy Top Virus (BBTV)

6.5.3.1 Distribution
Bunchy top virus disease (BBTV) is a major production constraint of banana in most tropical
countries, especially in Asia. The disease was first reported in Fiji and subsequently in Taiwan at
about 1900 and Egypt in 1901. It is believed that the disease spread to Australia and Sri Lanka in
1913 through infected suckers from Fiji. It was introduced to India from Sri Lanka in 1943 and is
now predominant in the following states: Andhra Pradesh, Tamil Nadu, Orissa, Maharastra, Bihar,
Karnataka, West Bengal, Assam, and Uttar Pradesh (Selvarajan et al., 2007). Some important local
cultivars grown by small-scale growers in several Asian countries are susceptible. Bunchy top is
present in some parts of Africa and the South Pacific, but has not been recorded in South and
Central America or the Caribbean.

BBTV was formally classified as a luteovirus due to the following characteristics: non-sap-transmis-
sible nature but persistently transmitted by aphids, and presence of yellowish symptoms restricted
to the phloem tissues. An analysis of infected plants (Dale et al., 1986) and electrophoretic patterns
of dsRNA from infected plants (Iskra et al., 1989) supported the earlier classification as a luteovi-
rus. However, Harding et al. (1991) and Thomas and Dietzgen (1991) used a modified method of
Wu and Su (1990) to purify 18–20 nm isometric virus-like particles (VLPs) from infected plants.
These particles had ssDNA of 1 kb with a molecular weight of about 20.000. Furthermore, Harding
et al. (1993) reported that the sequence of the first component had circular ssDNAs of 1.111 kb,
with other structures that resembled the geminiviruses. Burns et al. (1995) showed that BBTV has
a multicomponent genome with at least six ssDNA components, namely components 1–6, and the
circular genomic DNA of each component varies in size from 1.018 kb to 1.111 kb. All the compo-
nents had a major common region (CR-M) and the stem loop common region (CR-S). Five out of
the six components had single large ORFs in the virion sense. Xie and Hu (1995) showed that the
sequence of three BBTV genomic components from a Hawaiian BBTV isolate had components 1, 3,
and 4 that were almost identical to components 1, 2, and 5 of Harding et al. (1993) and Burns et al.
(1995). Wu et al. (1994) sequenced two more BBTV components from Taiwan and found that both
isolates encoded for replicase-associated proteins. Certainly, BBTV is not a luteovirus but is more
similar to the geminivirus, but differs from the latter in that its irons are isometric. Probably, BBTV
is a member of a new plant virus group, very much likely a babuvirus of the family Nanoviridae. It
is isometric and measures 20 kDa and has a multicomponent genome. The virus is highly concen-
trated in the phloem of infected plants.
6.5.3.3 Transmission and Host Range

The disease is transmitted by an aphid, Pentalonia nigronervosa, especially at the nymphal stage
through suckers and divided corms of infected plants. It can also be transmitted through infected
micropropagated plantlets obtained from infected plants (Drew et al., 1989). It spreads very rapidly
in summer when the vector population is very high and active. Drew et al. (1989) also showed that
tissue-culture plants did not show characteristic symptoms until they were transferred to the green-
house, where a majority expressed symptoms. The virus affects all the species of the genus Musa
(M. acuminata, M. balbisiana, M. sinensis, and M. paradisica with their hybrids and Fe’i bananas).
All commercial and widely grown cultivars of banana and plantain are susceptible. There are no
reports of infection on wild diploid Musa species.
6.5.3.4 Disease Symptoms
Symptoms of the presence of BBTW include intermittent, dark green dots and streaks of different
length on the leaf sheath, petiole, veins, and midrib. Typical streaks may not be present in some
cultivars. Emerging leaves are shorter, brittle in texture, and narrow (Figure 6.4). Infected plants
do not produce flowers, and when infection occurs late, bunches are formed but the fingers do not
develop to maturity. In some cultivars, there is marginal chlorosis of the leaf lamina and vein fleck-
ing. Sometimes, the tip of the bract of the male bud turns green and when infection occurs very late
in the season, the plant shows dark green streaks on the tip of the bracts.
6.5.3.5 Detection and Control

BBTV can be detected through symptoms, the presence of the aphid vector, serology, and nucleic
acid assay. Advances in research in tissue culture and immunoassay diagnostic tools have enabled
the production and use of healthy planting materials as a component to manage banana viruses.
Transgenic virus resistance (TVR) has been widely used as an effective strategy to control plant
viruses. This strategy could be of great relevance to bananas since there has been no immunity to
BBTV, and conventional breeding programs are yet to prove effective. Genetic transformation of the
cultivars ‘Grand Nain’ using Agrobacterium and ‘Bluggoe’ using microprojectile bombardment for
BBTV resistance has been reported (Selvarajan et al., 2007).
Figure 6.4 Symptoms of banana bunchy top disease.

6.5.4 Banana Bract Mosaic Virus (BBrMV)

Banana bract mosaic virus is a potyvirus of the family Potyviridae. BBrMV was prominently
reported first from the island of Mindanao, Philippines (Magnaye and Espino, 1990). Thomas and
Magnaye (1996) confirmed the presence of the causal agent of the disease in the widely cultivated
local cultivar ‘Embul’ (AAB group, Mysore) in Sri Lanka in 1996. BBrMV is also present in Ghana,
Nigeria, and Malawi (Selvarajan et al., 2007). In India, BBrMV is found in the southern states of
Tamil Nadu and Kerala, where it is referred to as “Kookan.”

The genome of the virus is a positive single-stranded RNA (ssRNA) of approximately 10 kb particle
size. The particles are flexuous rod shaped and measure 750 × 15 nm in size. Under the electron
microscope, flexuous rod-shaped particles from the leaf sheath and bracts of infected ‘Nendran’
and ‘Robusta’ cultivars have been observed (Selvarajan et al., 2007). The virus titer is greater in
bracts and the midrib than in the leaf sheath. Cytopathological observations reveal the presence
of pinwheel inclusions and flexuous rod-shaped particles, with typical features of potyvirus. The
coat protein appears as one major polypeptide with a molecular weight of 38 kDa and three minor
polypeptides of 63, 53, and 22 kDa (Selvarajan et al., 2007). The open reading frame (ORF) of the
virus amino acid sequence was found to be similar to the C-terminal half of the maize dwarf mosaic
potyvirus coat protein.
6.5.4.2 Disease Symptoms
Characteristic symptoms include the presence of spindle-shaped, pinkish to reddish streaks on the
pseudostem, midrib, and peduncle. Typically spindle shaped, mild mosaic streaks on bracts, pedun-
cle, and fingers have also been observed. In the cultivar ‘Nendran,’ the leaf orientation changes, giv-
ing it the appearance of “Traveler’s Palm” (Ravenala madagascariensis), with spindle-shaped mosaic
patterns on both upper and lower leaf surfaces. Infected plants show a very long or extremely short
peduncle, production of immature bunches, and raised corky growth on the peduncle. In ‘Red Banana’
and ‘Robusta,’ presence of strips on leaves and shriveled, pencil-like fingers have been observed.
6.5.4.3 Transmission and Detection

BBrMV is transmitted in a nonpersistent manner by three banana aphids (Rhopalosihum maidi,
Aphis gossypii, and Pentalonia nigronervosa) and by the cowpea aphid Aphis craccivora. According
to Sreenivasalu et al. (2006) quoted by Selvarajan et al. (2007), these vectors occur in banana and
plantain in the semiarid tropics. Reverse transcriptase PCR (RT-PCR) was found to be more sen-
sitive than ELISA and immunocapture PCR (IC-PCR) to detect the virus (Rodoni et al., 1999).
Selvarajan et al. (2006) also found RT-PCR more sensitive than DAC-ELISA and dot immunobin-
ding assay (DIBA) to detect viral isolates. RT-PCR amplification of viral RNA from crude plant
sap has been reported (Selvarajan et al., 2007). Other detection techniques are nucleic acid probe
specific to BBrMV (Thomas et al., 1997) and digoxigenin-labeled virus-specific probes.
6.5.4.4 Economic Importance and Control

In India, yield losses of 52.5% and 70% have been reported in the cultivar ‘Nendran’ and ‘Robusta,’
respectively (Cherian et al., 2002). Use of virus-free planting materials is the best way to control the
spread of the virus.
6.5.5 Banana Mosaic or Cucumber Mosaic Virus (CMV)

Banana mosaic or infectious chlorosis caused by cucumber mosaic virus is a disease of relatively
minor importance to banana, infecting more than 1,000 species of plants as hosts in the tropics,
subtropics, and temperate regions of the world, including Australia and parts of Asia. CMV is
distributed worldwide and has perhaps the widest host range of any plant pathogenic virus. First
described in New South Wales (NSW) Australia in 1930, one of the isolates, IB, is restricted to Asia,
while IA and II subgroups are worldwide (Selvarajan et al., 2007). In India, the disease occurred
in the early 1940s in the state of Maharashtra and later reported in Tamil Nadu and other parts of
the country. CMV isolates can be divided into two major serological subgroups defined as DTL
and ToRS, and this provides valuable information on the geographical location of and symptoms
produced by CMV isolates (Haase et al., 1989).

CMV consists of a spherical particle of 28–30 nm in diameter containing ssRNA and is naturally
transmitted by aphid vectors or by seed (Lockhart and Jones, 2000), with viral particles of about
29 nm and composed of 180 subunits. The particles have a sediment (S) value of 98. The virions
are made of 18% RNA, are highly labile, and rely on RNA-protein interactions for integrity. The
genomic RNAs are SS in addition to sense RNAs with 5´ cap structures and 3´ conserved regions
that can be folded into tRNA-like structures. The virus encodes five proteins, distributed on three
genomic RNAs. RNA 1 is 3.3 kb and is the only monocistronic RNA that encodes the 1a protein
that is required for viral replication. RNA 2 with 3 kb encodes the 2a protein, the viral polymerase,
and the 2b protein, expressed from a low-abundance subgenomic RNA and RNA 4A. The 2b ORF
is overlapped on the carbolic acid (COOH) terminal portion of the 2a ORF, which inhibits host
post-transcriptional gene silencing (PTGS). RNA 3 has a particle size of 2.2 kb and is packaged
in individual particles as a subgenomic RNA. RNA 4 (1 kb) encodes the movement protein (MP)
from the 5 ORF and the coat protein (CP). Both 3a protein and the CP are necessary for intercel-
lular movement of the virus through plasmodesmata, while 2b ORF is important in long-distance
movement of CMV. Also, Hu et al. (1995) identified banana isolates that belong to the subgroup I,
DTL serotype.
6.5.5.2 Disease Symptoms and Host Range

CMV has an incredible host range, infecting over 1,000 hosts comprising of 85 plant families and
approximately 365 genera in both monocots and dicots. Symptoms, which depend on the strain of the
virus pathogen and growth temperature, include yellow streaks or flecks on leaves in a mosaic pat-
tern, leaf yellowing, and leaf mosaic that appears on all the leaves of infected plants. Rotting of cigar
leaf, internal necrosis of pseudostem, and death of the entire plant are associated with CMV infection.
Chlorotic banding and a rosette appearance of leaf arrangement are also symptoms of CMV (Selvarajan
et al., 2007).
6.5.5.3 Viral Transmission

The disease is acquired through a wide range of host plants that grow in proximity with banana
from where it is transmitted by aphid vectors in a nonpersistent manner: cotton aphid, Aphis gos-
sypii, and corn aphid, Rhopalosipum maidis. Transmission has also been recorded by Myzus per-
sicae, Macrosiphum pisi, and Rhopalosipum prunifolae. It can also be transmitted by the parasitic
plant dodder (Cuscuta sp.), in which it replicates, and through seeds. The virus is most frequently
transmitted mechanically and through sap of purified virions and viral RNA.
6.5.5.4 Disease Management
One of the control measures is the use of pathogen-free planting material and control of alternate
hosts (weeds, legumes, cucurbits, and members of the Solanaceae, such as tomato). Meristem cul-
ture has also been developed to eradicate viruses of various vegetative propagating plants, including
CMV. It was found that CMV particles could invade meristems and that a gradient of increasing
virus concentration from the dome to the successive primordial exists (Walkey and Webb, 1968).
This means that the probability of obtaining virus-free plants is inversely related to the size of the
meristem excised. Thermo- and chemotherapy coupled with meristem culture can be used when
meristem culture alone fails (Kartha, 1986). Berg and Bustamante (1974) observed that heat treat-
ment (35–43°C) for 100 days performed on rhizomes with meristem culture was inefficient for
cleaning commercial banana cultivars.
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7 Integrated Pest
Management of Banana
Thomas Dubois and Daniel L. Coyne
Contents
7.1 Introduction........................................................................................................................... 122
7.2 Plant-Parasitic Nematodes..................................................................................................... 122
7.2.1 An Overview of Nematode Species and Their Distribution...................................... 122
7.2.2 Nematode Damage.................................................................................................... 124
7.2.3 Nematode Management............................................................................................. 124
7.3 Insect and Mite Pests............................................................................................................. 125
7.3.1 Plant-Boring Pests..................................................................................................... 125
7.3.1.1 The Banana Weevil..................................................................................... 125
7.3.1.2 Stem Borers................................................................................................. 126
7.3.2 Fruit and Flower Pests............................................................................................... 126
7.3.2.1 Banana Moths............................................................................................. 126
7.3.2.2 Thrips.......................................................................................................... 127
7.3.2.3 Peel-Scarring Beetles.................................................................................. 128
7.3.2.4 Fruit Flies.................................................................................................... 128
7.3.2.5 The Sugarcane Bud Moth Caterpillar......................................................... 128
7.3.3 Sucking Insects and Associated Arthropoda............................................................ 128
7.3.3.1 The Banana Aphid...................................................................................... 128
7.3.3.2 Whiteflies.................................................................................................... 129
7.3.3.3 Scales and Mealybugs................................................................................. 129
7.3.3.4 Mites........................................................................................................... 130
7.3.4 Foliage Feeders.......................................................................................................... 130
7.3.4.1 The Banana Skipper.................................................................................... 130
7.3.4.2 The Chinese Rose Beetle............................................................................ 130
7.3.4.3 Other Foliage Feeders................................................................................. 130
7.4 Pest Interactions..................................................................................................................... 131
7.4.1 Ants............................................................................................................................ 131
7.4.2 Natural Enemies........................................................................................................ 131
7.4.3 Weeds......................................................................................................................... 132
7.5 Integrated Pest Management................................................................................................. 132
7.5.1 History and Context of IPM...................................................................................... 132
7.5.2 Principles of IPM....................................................................................................... 132
7.5.2.1 Integrated.................................................................................................... 132
7.5.2.2 Pest.............................................................................................................. 133
7.5.2.3 Management................................................................................................ 133
7.5.3 Practical IPM as a Continuum................................................................................... 133
7.6 IPM in Banana....................................................................................................................... 134
7.6.1 IPM in Commercial Plantations................................................................................ 134
121
7.6.2 IPM in Smallholder Systems..................................................................................... 135

7.6.2.1 Problems..................................................................................................... 135
7.6.2.2 Focus on Cultural Management.................................................................. 135
7.6.2.3 Technology Transfer................................................................................... 136
7.7 Advances in Seed-Based Microbial Management................................................................. 136
7.8 Conclusion............................................................................................................................. 138
References....................................................................................................................................... 138
7.1 Introduction
Bananas (Musa spp.) are plagued by a variety of nonmicrobial pests. Most attention has focused on
the banana weevil Cosmopolites sordidus Germar and a complex of plant-parasitic nematodes, of
which the burrowing nematode Radopholus similis Cobb Thorne has received the most attention.
However, banana is grown widely across tropical and subtropical regions, attracting a wide range
of associated pests. These can vary greatly according to geography and clone, while changes in
cropping practices and the introduction of new or unfamiliar cultivars can introduce new pest spe-
cies. In addition, banana serves varying purposes, ranging from the genetically diverse production
systems of subsistence foods to commercially managed plantations of genetically uniform dessert
bananas for export markets. For example, flower and fruit pests that cause cosmetic damage are of
limited importance to subsistence cooking bananas but can result in refusal of export shipments
when detected in even low numbers. Often, the management of pests is discussed in the context of
integrated pest management (IPM). IPM, however, is often misused, referring instead to a plethora
of ill-linked management options that can at times still be at the research stage. Within this chap-
ter, therefore, the full spectrum of banana production systems will be taken into account when
discussing the vast diversity of banana pests, while providing an important assessment on how IPM
principles can be applied to manage them.
7.2 Plant-Parasitic Nematodes
7.2.1 An Overview of Nematode Species and Their Distribution
Plant-parasitic nematodes are the most detrimental soil-borne pests of banana (Gowen et al., 2005).
On a global basis, the key pest species are Helicotylenchus multicinctus (Cobb) Golden, root knot
nematodes Meloidogyne spp. (Figure 7.1), the root-lesion nematode Pratylenchus coffeae Sher
and Allen, Pratylenchus goodeyi Sher and Allen, and R. similis (Coyne, 2009). Other species not
generally viewed as key pests may, however, be of local significance such as the reniform nema-
tode Rotylenchulus reniformis Linford and Oliveira or Hoplolaimus pararobustus Schuurmans
Stekhoven and Teunissen Sher. Virtually without exception, species occur in mixed communities.
Radopholus similis has been considered as the most damaging nematode affecting bananas
worldwide, especially in lowland tropical areas (Sarah, 2000). However, this perception has essen-
tially stemmed from the nuisance R. similis poses to commercial dessert banana plantations, where
it has wreaked havoc and resulted in the substantial application of carbamate- and organophos-
phate-based pesticides (Cianco and Mukerji, 2009). Consequently, R. similis has traditionally been
the main focus in breeding programs. In subsistence farming systems, though, the situation is less
clearly defined. The nematode is thermophobic and in the tropics does not occur at high, cool alti-
tudes, above 1400 m in the East African highlands (Price, 2006) where a substantial proportion of
Africa’s banana production is concentrated, nor does it occur at high latitudes, such as Taiwan and
the Canary Islands (Jones, 2009). R. similis was previously the key nematode pest species in West
Africa (Speijer and Fogain, 1999), but recent surveys show P. coffeae is often the most damaging
species (Coyne, 2009). Since P. coffeae is also prevalent across the Pacific and Southeast Asia, it is
Integrated Pest Management of Banana 123
Figure 7.1 Root knot nematodes in banana (Courtesy of D. Coyne.)
of concern for banana and requires a greater attention in respect to pest management and resistance
breeding.
Pratylenchus goodeyi, on the other hand, is viewed as thermophilic, and in the East African high-
lands, for example, replaces R. similis as the dominant species above 1400 m altitude (Speijer and
Fogain, 1999). Its status as a pest of banana, however, is unclear. It can occur in extremely high densi-
ties, such as on banana in Tanzania (Speijer and Bosch, 1996) and enset in Ethiopia (Peregrine and
Bridge, 1992), where it undoubtedly causes some damage. In Rwanda and Uganda, however, no cor-
relation could be established between P. goodeyi and cooking banana losses (Gaidoshova et al., 2009;
D. Coyne, unpublished). It is also interesting that P. goodeyi represents a major pest in commercial
banana plantations in the Canary Islands (De Guiran and Vilardebo, 1962) and in Australia (Pattison
et al., 2002) where prevailing temperatures tend to be higher than is optimal for this species. Recently,
P. goodeyi was identified from bananas in Kenya. Further examination of P. goodeyi from toppled
bananas on the Kenyan coast and the Canary Islands using molecular techniques demonstrated distinct
molecular differences of these nematodes compared with P. goodeyi from the highlands of Uganda
(Coyne and Waeyenberge, 2008). Results indicated that the “tropical” (Kenyan) P. goodeyi were more
closely linked to P. crenatus Loof, P. penetrans Cobb, and P. neglectus Rensch than P. goodeyi, even
though they physically resembled P. goodeyi. Within-species variability is a well-known phenomenon,
which can explain differences in virulence and host range of some species (Starr et al., 2002). There
are good reasons to separate certain strains into separate species, such as for the P. coffeae complex,
which hitherto was a single species based on morphology (Duncan et al., 1999). For R. similis, studies
have demonstrated that a series of different strains exist, with the Sri Lanka strain responsible for the
severe damage to Ugandan bananas, amongst the most aggressive (Price, 2006), and able to over-
come the resistance present in cv ‘Yangambi Km5’ (Plowright, 2000; Dochez, 2004). Such variability
and diagnostic difficulties have significant implications to the development of management programs,
especially for the use of resistance through breeding programs. Knowledge of the key pests and access
to sources of resistance against these species and their variable strains are essential to make progress
in managing these pests.
Helicotylenchus multicinctus is regularly associated with losses to banana, but almost exclu-
sively in combination with other nematode species, especially R. similis and Meloidogyne spp.
(Gowen et al., 2005). Its status has been subject to speculation, as determining the contribution of
the individual nematode species to damage and losses is often difficult. Accumulating evidence,
however, demonstrates that H. multicinctus is indeed responsible for large proportions of damage to
banana production, even when other species are present (Ssango et al., 2004).
Meloidogyne spp. are amongst the most abundant nematode pests across all crops, with a global
distribution. Their importance on bananas has been underestimated as they also regularly occur in
combination with other damaging species (Gowen et al., 2005). However, in some instances they
dominate the nematode populations and contribute significantly to production losses.
7.2.2 Nematode Damage
Nematodes generally cause damage through the destruction of root and rhizome tissue. Damaged
tissue becomes necrotic and dies, reducing nutrient and water uptake, reducing bunch weights, and
retarding harvest. Severe damage underscores plant anchorage, which can result in plant toppling
(Sarah, 2000; Jones, 2009), while reduced plant turgidity can result in snapping of plant stems dur-
ing periods of low water availability (D. Coyne, unpublished). Fruit on fallen plants generally have
no value, resulting in extreme yield losses where infection levels and plant losses are high (Gowen
et al., 2005). Common symptoms of severe nematode infection include stunting, poor plant growth,
narrow and weak stems, foliar chlorosis, root rotting and galling, and plant toppling. Determining
infestation levels can be difficult, especially to the untrained, as nematodes exist below ground
and remain out of sight, until severe damage symptoms are observed. Nematodes almost always
occur as species combinations that may be complex. Establishing the specific species contributions
to damage is difficult, resulting in complications for developing management options that may be
species specific.
7.2.3 Nematode Management
The discrepancy between management options for smallholders and commercial growers is vast.
Nevertheless, nematodes remain a difficult group to manage effectively. However, because new
infestations are primarily perpetuated through infected planting material, the use of clean, healthy,
nematode-free planting material cannot be overemphasized for either system. Hot water treatment
of suckers after removal of infected roots is a simple and effective technique for sanitizing mate-
rial. For smallholder systems, this technique has been further adapted using a short 30 s exposure
period in boiling water, which is less time and energy consuming, and more appealing to resource-
poor farmers (Viaenne et al., 2006). For commercial systems, such as in Australia and Hawaii,
hot water treatment is used to provide nematode-free material (Colbran, 1967). However, sterile
plants produced using tissue culture and certified pest- and disease-free are ideal. Such material is
now routinely used in commercial banana production but has yet to gain wider use by smallholder
farmers (Dubois, Coyne, et al., 2006). The lack of virus indexing, suboptimal weaning procedures,
accidental cultivar mixing during production, inappropriate farmer handling, and subsidization by
governmental and nongovernmental organizations remain some of the major hurdles to overcome
before tissue-culture technology can be widely rolled out among smallholder farmers.
In commercial plantations, postplanting nematicide applications continue to provide the most
universal method of nematode management, primarily against R. similis, administered through
granular applications or drip irrigation (Sarah, 2000; Jones, 2009). Soil sanitation can be achieved
through a cleansing system based on glyphosate injection into banana plants before uprooting
(Risède et al., 2009). However, many nematicides are being progressively removed from the market
(Zum Felde et al., 2009). In the French West Indies, management of R. similis is based primarily
upon the repeated application of carbamate or organophosphate nematicides; however, with increas-
ing restrictions on their use, the search for alternative and environmentally responsible options has
intensified. An environmentally sound scheme supported by three key pillars is being devised: use
of tissue culture, fallow, and intercropping with nonhosts (Risède et al., 2009). In Hawaii’s IPM
scheme, incorporation of crop residue and fallowing fields for 6–8 months is common. Emphasis
is also being increasingly placed on efforts to identify suitable biologically based solutions, such as
mycorrhizae, endophytes, and biopesticides (Meyer and Roberts, 2002; Viaene et al., 2006; Sikora
et al., 2008).
The use of healthy planting material is not only critical, but for smallholder farmers it also
often is their only realistic option for nematode management. However, the use of locally grown,
nematode-resistant cultivars, in combination with healthy planting material, is highly desirable
(Coyne, 2009). Establishing nematode resistance is also a key target in banana breeding programs
(Tenkouano and Swennen, 2004; Pillay and Tripathi, 2007; Lorenzen et al., 2009). Commercial
dessert bananas are characterized by few landraces with an extremely narrow genetic base (Ortiz et
al., 1995), while sources of resistance to nematode species are limited (De Waele and Elsen, 2002).
To date no widely grown clone of export banana is known to be resistant to the important nematode
species (Gowen et al., 2005). There are confirmed sources of resistance against R. similis but not
necessarily against Pratylenchus spp. (De Waele and Elsen, 2002). Resistance to R. similis from
cv ‘Pisang Jari Buaya’ has been incorporated into the widely used diploid parent cv SH-3142 of the
Fundación Hondureña de Investigación Agrícola (FHIA) (Pinochet and Rowe, 1979). Recent suc-
cesses have also been achieved in the breeding programs at the International Institute of Tropical
Agriculture (IITA) (Pillay and Tripathi, 2007; Lorenzen et al., 2009) and the Centre de Coopération
Internationale en Recherche Agronomique pour le Développement (CIRAD). At IITA, the diploid
banana hybrids TMB2×5105-1 and TMB2×9128-3 have good combining ability and are resistant to
R. similis (Tenkouano et al., 2003). At CIRAD, partial resistance to both R. similis and P. coffeae
is reported within synthetic hybrids of M. acuminata (Quénéhervé et al., 2009). Meanwhile, the
genetic modification of existing cultivars is also becoming a realistic option for nematode manage-
ment (Roderick et al., 2009; Tripathi, 2009). Research efforts for biologically based solutions are
equally being sought for smallholder farmers in Africa and India to compensate for the unsuitability
and removal from use of nematicides.
7.3 Insect and Mite Pests

Bananas can be attacked by a wide range of insect and mite pests. Rather than a taxonomic over-
view, it is best to group these into functional groups, as members from widely different taxa often
pose similar problems and require similar management options.
7.3.1 Plant-Boring Pests

7.3.1.1 The Banana Weevil
The biology, distribution, and damage caused by Cosmopolites sordidus is comprehensively
reviewed by Gold et al. (2001). Banana weevils feed only on bananas. Adults are most commonly
found between leaf sheaths, in the soil at the base of the mat, or associated with crop residues. The
banana weevil is nocturnally active and particularly susceptible to desiccation. As adults tend to
have limited movement between mats and rarely fly, dissemination is primarily through infested
planting material. The banana weevil is a typical k-selected insect with long life span and low fecun-
dity. Adults normally survive for longer than 1 year, and oviposition has been estimated at 1 egg/
week. Oviposition occurs in the leaf sheaths and rhizome surface, especially in flowered plants and
in crop residues. Crop damage is inflicted by the larvae. The emerging larvae preferentially feed in
the rhizome but will also attack the true stem and occasionally the pseudostem. Larval developmen-
tal rates are temperature dependent. Under tropical conditions, egg to adult development takes 5–7
weeks. Egg development does not occur below 12°C, restricting its distribution to lower altitudes.
Adult banana weevils are attracted by volatiles emanating from host plants, explaining why cut rhi-
zomes of fresh suckers for planting material are especially susceptible to attack (Gold et al., 2001).
The banana weevil has a cosmopolitan distribution, occurring in smallholder banana systems
to commercial plantations. It is present in all banana and plantain production regions in the tropics
and subtropics, and is generally considered the most important insect pest of banana (Jones, 2009).
Beer, roasting, and cooking bananas are most susceptible, and therefore banana weevil problems
appear to be most severe in smallholder systems and less in commercial cv Cavendish plantations
(Gold and Messiaen, 2000).
Banana weevil attack has been reported to interfere with root initiation, kill existing roots, limit
nutrient uptake, reduce plant vigor, delay flowering, and increase susceptibility to other pests and
diseases. Yield reductions stem from both plant loss (plant death, rhizome snapping) and reduced
bunch weights. Losses of more than 40% have been recorded (Gold and Messiaen, 2000; Gold et al.,
2001). Young banana plants are most at risk because tunneling by the banana weevil can be fatal at
this stage (Constantinides and McHugh, 2003).
As with nematodes, banana weevils are dispersed through contaminated planting material,
emphasizing the importance of clean planting material as an essential prophylactic management
measure. Rigorous field sanitation measures also take advantage of the adult’s dependency on resi-
dues, lack of movement, and need for moisture. Despite numerous surveys, no known effective para-
sitoids of the banana weevil have been identified. In commercial systems, insecticides are applied.
For resource-poor farmers, cultural management is the only means currently available to reduce
banana weevil populations (Gold and Messiaen, 2000).
7.3.1.2 Stem Borers
Stem borers, such as the giant banana stem borer Castniomera humboldti Boisduval and the banana
stem weevil Odoiporus longicollis Olivier, tunnel through the banana pseudostem (Jones, 2009).
Castniomera humboldti occurs in Central and South America where it is a relatively minor pest,
whereas O. longicollis can be a serious pest in Asia. The latter is among the main insect pests of
quarantine importance for Australia (Pinese, 1999) and considered the most important insect pest in
India. Eggs are laid inside air chambers through incisions made on the leaf sheath. In the advanced
stage of infestation, severely affected plants break. Banana stem weevils often inflict total crop
failures in susceptible cultivars (Jayanthi and Verghese, 1999). The pest survives in pseudostem
stumps, which often remain as trash in the field after harvest. In India, the distribution of O. longi-
collis is aggravated when farmers cut the pseudostems at up to 1 m high from the ground level and
allow them to decompose slowly until the establishment of the succeeding ratoon crop, which they
believe transfers nutrition to subsequent ratoons (Padmanaban and Kandasamy, 2003).
7.3.2 Fruit and Flower Pests

Fruit and flower pests are especially important on exported banana. For example, in Hawaii, pres-
ence of the banana moth Opogona sacchari Bojer on fruit for export will result in their rejection
(Constantinides, 2003). A small number of larvae of the banana scab moth Nacoleia octasema
Meyrick may lead to the destruction of an entire bunch otherwise destined for export. The mere
presence of Bactrocera spp. fruit flies, an insignificant pest of bananas, on shipments from Australia
to New Zealand requires destruction of the fruit (Pinese, 1999; Ministry of Agriculture and Forestry,
2006). Most often, fruit and flower pests are managed using insecticide-treated bags that enclose the
flower. Chlorpyrifos-treated bags are especially effective and used abundantly in commercial plan-
tations, but environmentally safer alternatives, such as bifethrin, are increasingly sought (Chiquita
Brands International, 2001).
7.3.2.1 Banana Moths

Opogona sacchari is endemic to Africa, where it is an insignificant pest. The pest has a wide host
range and has been considered a serious pest of banana in the Canary Islands since the 1920s. In the
1970s, it was introduced into Brazil, where it has since become an important banana pest. The insect
has also started to appear in a number of European countries on various tropical and subtropical
glasshouse crops, and is now considered a serious quarantine pest (Smith et al., 1996; OEPP/EPPO,
2006). The banana moth oviposits on senescing flowers, decaying leaves, pseudostems, and fruits
on which the larvae feed, although they will also feed on healthy adjacent tissue. Preventive man-
agement measures, such as the removal of plant debris and flowers, in addition to the application
of insecticides to bunches prior to bagging, greatly reduces damage (Peña et al., 2002). Recently,
a pheromone was discovered that attracts this pest, which may additionally aid the development of
more efficient monitoring schemes (Wageningen University, 2009).
The banana fruit-piercing moth Eudocima fullonia Clercq attacks many fruits and vegetable
crops, and can pose a serious banana risk. Unlike most moth and butterfly pests, the caterpillar stage
is not the damaging stage. Instead, the adult moth punctures and feeds on ripening fruit, not only
administering direct damage but also indirectly facilitating fungal and bacterial infections. High
moth populations can result in premature ripening and fruit drop (CAPS online). Interestingly, in
some endemic areas, such as Papua New Guinea, the pest is effectively managed below threshold
levels by egg parasitoids (Sands and Liebregts, 2005).
The banana scab moth Nacoleia octasema Meyrick is one of the most serious pests in Malaysia,
the southwest Pacific, and Queensland, Australia. Females lay eggs on flower bracts as the inflores-
cence emerges. Larvae feed on the surface of young fingers. They enter the flower and feed on the
developing fruits within, gradually progressing down the maturing bunch. This causes brown scars,
scabs, and severe cracking on the developing fruits. Cultural and biological control methods are not
particularly effective due to the cryptic nature of the feeding larvae, and their management is based
largely on injection of insecticides (Paine, 1964; Morton, 1987; Stover and Simmonds, 1987; Botha
et al., 2000; Nelson et al., 2006).
7.3.2.2 Thrips
Numerous species of thrips of the family Thripidae feed on banana (CABI, 2005). Most thrips
prefer sunny and dry areas, have a broad host range, and feed on flowers, fruits, or other young
tissues, with both larvae and adults causing damage (Parker et al., 1995). Thrips cause superficial
skin blemishes on immature banana fruits. Although severe infestations can cause peel splitting, the
damage they cause is primarily cosmetic, and therefore only commercial banana systems require
prophylactic management measures to meet stringent export requirements (Peña et al., 2002).
Banana is affected by several members of Chaetanaphothrips: the orchid thrips
Chaetanaphothrips orchidii Moulton, the banana rust thrips Chaetanaphothrips signipennis
Bagnall, and Chaetanaphothrips leeuweni Karny. These species are cosmopolitan pests, with most
damage resulting from larval feeding. Chaetanaphothrips signipennis is a problem in Australia,
while C. orchidii induces similar damage in Central and South America (Peña et al., 2002). Feeding
on leaf sheaths results in damage on the outer surface of leaf petioles and is characterized by dark,
V-shaped marks, while damage to the fruit initially presents a water-soaked appearance that later
turns bronze- or rust-colored. The pest can split the fruit peel, exposing the flesh. It also feeds on the
area where adjacent fingers touch, resulting in a reddish discoloration (Williams et al., 1990; CABI,
2005; Jones, 2009). The life cycle can be completed in 28 days. The insect is managed by spraying
banana fruits with insecticide at bunch emergence and covering them with polyethylene bags prior
to harvest (Morton, 1987; Hara et al., 2002; CABI, 2005).
The Hawaiian flower thrips, Thrips hawaiiensis Morgan and T. florum Schmutz, are similar,
often confused with each other, and as a cosmopolitan species complex, feed on a wide variety
of tropical flowers (Hollingsworth, 2003). The insect enters the developing fruit while the bracts
remain present and oviposits on the young fruit. Feeding results in corky scabbing of the peel,
flecked, spotted, or deformed flowers, and sometimes cracked fruits, especially during hot and dry
weather. Infestations are lessened by removal of the terminal male bud, which tends to harbor the
pest (Morton, 1987; CABI, 2005; Jones, 2009; Peña et al., 2002). Unlike most flower thrips, this
species complex prefers wet and shady areas (Sakimura and Krauss, 1944).
The banded greenhouse thrips Hercinothrips femoralis Reuter is a cosmopolitan thrips species
with a wide host range and has been recorded on bananas in various parts of the world. The closely
related rind thrips H. bicinctus Bagnall, which is equally cosmopolitan, is considered a more impor-
tant banana pest (Roditakis et al., 2006). Feeding by this insect causes unsightly silver and bronze fruit
scars, reducing their marketability (Hawaiian Banana Industry, 2010a). The silvering usually occurs
with small infestations. With larger infestations, especially in combination with the two-spotted spi-
der mite Tetranychus urticae Koch, the fruits turn smoky-red in color, occasionally leading to skin
cracks, further reducing the market value of the fruit (Lewis, 1997). Hercinothrips spp. are closely
related to the rind thrips Elixothrips brevisetis Bagnall. Elixothrips brevitis is also a polyphagous foli-
age feeder and a common pest in commercial banana stands, and feeds on leaves, flowers, or stems.
In Martinique, E. brevisetis has replaced H. femoralis as the predominant thrips pest (Rey, 2002).
Flowers, buds, and the undersides of leaves become spotted with small black fecal specks. Injured tis-
sue develops a silvery appearance and eventually turns dark brown, affecting banana marketing (Rey,
2002). Elixothrips brevitis also feeds on leaf tips, resulting in wilting and curling. When affected, buds
may fail to open (Constantinides and McHugh, 2003; Hawaiian Banana Industry, 2010a).
Banana is also damaged by Frankliniella spp. The banana flower thrips Frankliniella par-
vula Hood pupates in the soil and only emerges during daylight hours to oviposit in the epidermis
of young banana fruits. The host range of this thrips species seems restricted to banana plants
(Harrison, 1963; Peña et al., 2002). The blossom thrips Frankliniella insularis Franklin mainly
occurs in Central America (Mound and Marullo, 1996).
7.3.2.3 Peel-Scarring Beetles
Several species of Colaspis spp. are reported as banana pests, especially in Central and South
America (Ostmark, 1975; Jones, 2009). Colaspis hypochlora Lefèvre in particular appears a trouble-
some pest in Venezuela, Guyana, and Mexico, where it invades young fruit on developing bunches,
although this species is often confounded with other members of the genus. Severe outbreaks of
this pest have been documented in Panama and Colombia (Ostmark, 1975; Morton, 1987). In the
Philippines, several peel-scarring beetles belonging to Philicoptus spp. have also been reported as
pests (Stephens, 1984).
7.3.2.4 Fruit Flies

Fruit flies only attack ripe banana fruits. Although minor pests, fruit flies, particularly of the genus
Batrocera spp., can be highly significant quarantine pests (Nelson et al., 2006), disrupting interna-
tional banana shipments. In October 2009, Mexico, for instance, halted all imports of fresh banana
from regions where the banana fruit fly Bactrocera musae Tryon or the oriental fruit fly B. dorsa-
lis Hendel occur (USDA, 2009). Bactrocera musae is considered among the most serious banana
pests of Papua New Guinea (Kambuou, 2003). In Sri Lanka, severe outbreaks occurred of several
Bactrocera spp. in the late 1990s (Ekanayake et al., 2002).
7.3.2.5 The Sugarcane Bud Moth Caterpillar

The sugarcane bud moth caterpillar Decadarchis flavistriata Walsingham is a localized pest in the
Pacific, especially Hawaii. Caterpillars feed on decaying flowers, which can cause fruit scarring.
Removing flowers prior to bagging reduces damage from this pest (Nelson et al., 2006).
7.3.3 Sucking Insects and Associated Arthropoda

7.3.3.1 The Banana Aphid
Colonies of the banana aphid Pentalonia nigronervosa Coquerel can occur anywhere on the plant
but are most often found at the base (Robson et al., 2007). Young suckers are typically most heavily
infested. The banana aphid is a phloem feeder, which causes plants to become deformed; the leaves
become curled and shriveled, and in some cases galls form on the leaves (Metcalf, 1962). Direct
damage from the banana aphid is normally negligible. More important is the role of P. nigronervosa
as a virus vector. The alate form of the banana aphid is the sole vector of banana bunchy top virus
(BBTV) disease, among the most serious of banana viruses in Asia, Africa, and the Pacific (Hu et
al., 1996; Dale and Harding, 1998). With the exception of vector transmission, use of infected plant-
ing material is the only other mode of transmission of BBTV (Robson et al., 2006). Consequently
BBTV management is highly dependent upon prophylactic vector management. Once BBTV con-
tamination occurs, eradication is both difficult and costly, with vector management merely tending
to reduce the rate of spread to healthy plants. In commercial plantations, pesticide applications are
applied regularly, with newly released products, such as imidacloprid, being investigated (Robson
et al., 2007). Utilization of disease-free planting material, windbreaks, horticultural oils, and deter-
gents provide alternatives to pesticide treatments. Destruction of diseased plants immediately upon
detection will also slow the spread. In Hawaii, eradication efforts continue to be conducted on an
island-to-island basis. In Australia, a zero-tolerance policy and strict quarantine measures are estab-
lished against the pest (Magee, 1967; Hawaii Banana Industry Association, 2010a, 2010b; Robinson,
1996; Robson et al., 2006).
7.3.3.2 Whiteflies
The spiraling whitefly Aleurodicus dispersus Russell is native to Central America but now has a
cosmopolitan distribution. It is a polyphagous pest, including on banana. Aleurodicus dispersus is
a sap-sucking insect that damages and discolors plant leaves during its nymphal stages but does not
damage banana fruits directly (Waterhouse and Norris, 1989; Nelson et al., 2006). Whiteflies excrete
honeydew, which serves as a substrate for mold fungi. Sooty mold blackens the leaves and decreases
photosynthetic activity. During severe infestations in Costa Rica and Hawaii, high levels of sooty
mold cause premature leaf drop and reduced yields (Botha et al., 2000; Nelson et al., 2006). The
spiraling whitefly is not considered a principal threat to banana, as populations are usually main-
tained below economic thresholds by natural enemies, especially in the regions where it is endemic
(Ramani et al., 2002), although in some countries it is considered a quarantine pest (Pinese, 1999).
Other whiteflies have been reported as major pests locally, such as Lecanoideus floccissimus Martin
in commercial banana greenhouses in the Canary Islands (Hernández-Suárez et al., 2006).
7.3.3.3 Scales and Mealybugs

The coconut scale Aspidiotus destructor Signoret is a common polyphagous pest of banana
(Williams and Watson, 1988). The coconut scale usually occurs on the underside of leaves but
can also affect petioles, peduncles, and fruits. The pest usually occurs in densely massed colonies
on the lower leaf surface, except in extremely heavy infestations where it may be present on both
sides. Mature scales are found on the older leaves. Their piercing and sucking mouthparts extract
plant juices and inject toxic saliva, leading to discoloration and yellowing of plant tissue (Beardsley,
1970; Waterhouse and Norris, 1987; Wright and Diez, 2005). When attached to fruits, they pose a
significant phytosanitary issue because of their quarantine status in many regions. The presence
even of a single live coconut scale can effect the complete rejection of a California-bound banana
shipment, causing substantial loss for Hawaii’s growers and exports (Ming-yi, 2003; Nelson et al.,
2006). Other species of scales have equally been reported as banana pests, such as the hemispheri-
cal scale Saissetia coffeae (Walker) and the green scale Coccus viridis De Lotto in the South Pacific
(Ben-Dov, 1994; Nafus, 2000a, 2000b). Numerous species of mealybugs, which are closely related
to scales, can be detrimental to bananas, such as the gray pineapple mealybug Dysmicoccus neo-
brevipes Beardsley in the Philippines, while severe outbreaks of D. alazon Williams, Aspidiotus
elaeidis Marchal, and A. hederae (Vallot) are reported from organic banana plantations in Tenerife
(Ben-Dov, 1994; Alvarez et al., 2001; Nafus 2000a, 2000b). The mango mealybug Rastrococcus
invadens Williams, occasionally affecting bananas, has been identified as the primary exotic insect
quarantine pest to banana production in Australia (Pinese, 1999). Other pests include members of
Planococcus spp., Pseudococcus spp., and Ferrisia spp. Although not a significant pest of banana
in most locations, mealybugs have also been associated with transfer of banana streak virus (BSV)
(Nelson et al., 2006).
7.3.3.4 Mites
Mites are generally considered a minor but frequent pest of bananas. However, several mites of the
genus Tetranychus can cause significant damage to banana, such as T. urticae and especially the
banana spider mite T. lambi Pritchard and Baker (Morton, 1987; Pinese and Piper, 1994). In West
Bengal, India, Oligonychus oryzae Hirst was found to be the more damaging mite species (Karmakar
and Dey, 2006). Mite activity and damage are mainly confined to localized, dry conditions, such as
the underside of old leaves. In severe infestations, whole leaves turn brown-gray and wilt, resulting
in sunburned bunches and a reduction in plant growth. However, in warm weather and during severe
outbreaks, mites may migrate to the bunches and damage fruits. Dry and warm conditions under
plastic bunch covers are particularly favorable for the buildup of banana spider mites. Fruit damage
is characterized by a silver-gray discoloration of the fruit tip, and fruits may dry out and crack when
serious infestations occur (Morton, 1987; Pinese and Piper, 1994). Mites are also implicated in fruit
speckling, a disease with unknown etiology that, particularly during the rainy season, has caused up
to 70% rejection of export banana in Central America (Pasberg-Gauhl, 2002).
7.3.4 Foliage Feeders
A large group of foliage-feeding insects, originating from several taxa, can cause damage to
banana. The economic damage they cause is usually limited, with populations remaining below
economic injury levels through natural predation and parasitism. However, serious crop losses
can occur.
7.3.4.1 The Banana Skipper

The banana skipper Erionota thrax L. is considered to be the main insect pest in Papua New Guinea
(Kambuou, 2003). In Australia, it is a quarantine pest (Pinese, 1999), where it is sometimes referred
to as the banana leaf roller due to its habit of rolling leaves to make shelters. Caterpillars secrete a
protective, white, waxy covering inside the rolled leaves. The feeding and rolling destroys the leaves
and significantly reduces the plant’s leaf area. Leaf defoliation can occur quickly with only three
caterpillars per leaf (Queensland Horticulture Institute, 2000). In Asia, from where the banana skip-
per originates, the parasitic wasp Cotesia erionotae Wilkinson effectively manages banana skipper
infestations, which were previously a serious problem. In Malaysia, populations are kept in check by
at least five primary endoparasitoids (Queensland Horticulture Institute, 2000; Okolle et al., 2006;
Jones, 2009), while in Papua New Guinea, parasitoids have been introduced to manage outbreaks of
leaf rollers in areas of the country (Kambuou 2003).
7.3.4.2 The Chinese Rose Beetle

The Chinese rose beetle Adoretus sinicus Burmeister is a minor but common pest on all major
banana-producing islands in Hawaii and in the Pacific. The larvae reside in the soil and litter, with
damage caused only by adult feeding. The adult is nocturnal and feeds primarily on leaf and inter-
venal tissue. It most commonly attacks young plants (Nelson et al., 2006).
7.3.4.3 Other Foliage Feeders

Caterpillars from the genera Antichoris, Caligo, Opsiphanes, and Sibene have been reported to
partially defoliate banana plants in Central and South America (Jones, 2009). The larval stages of
Opsiphanes tamarindi Felder can consume large areas of leaf, making it a potentially serious pest
(Uquillas, 2002). Especially the tamarind owlet Opsiphanes tamarindis Felder, the owl butterfly
Caligo mennon Felder, and Antichloris viridis Druce are considered economically important pests
in countries such as Venezuela (Ramirez et al., 1999). Bagworm (Oiketicus kirbyi Guilding) can be a
problem in Central America, such as in Costa Rica. Females live for only a maximum of 14 days but
can produce over 6,500 eggs during their adult life span. However, natural parasites usually limit
outbreaks (Stover and Simmonds, 1987).
7.4 Pest Interactions
Of the wide range of pests observed on banana, the level of damage inflicted depends on numerous
factors. Banana pests are often highly interactive, occurring within a complex ecosystem that ulti-
mately influences the damage they cause. As such, there is need to avoid pest management solutions
that tend to focus on a single pest without considering its relation and interactions to other factors.
It is necessary that pest management options be holistic in their approach.
7.4.1 Ants
Ants have at times been heralded as natural enemies for biological control in conservation pro-
grams. For example, encouraging colonies of ants has been suggested as a means to manage C.
signipennis (CABI, 2005). Myrmicine ants such as Tetramorium guinense F. and the big-headed
ant Pheidole megacephala F. have reportedly contributed to the successful management of banana
weevils in plantain in Cuba and are even encouraged to nest in pseudostem sections that can then be
used for their dissemination (Gold and Messiaen, 2000).
However, whereas ants are antagonistic to most other insect taxa, they can be highly protective
of some honeydew-producing pest species, such as scales, whiteflies, and aphids. Ants will seek
out honeydew sources to protect the supply, effectively farming the source, which may include
their aggressive defense of the honeydew-producing insects. For example, honeydew produced by
A. dispersus attracts ants, which, in turn, offer protection to the whiteflies, aggravating its dam-
age and indirectly contributing to quarantine problems for export fruits (Waterhouse and Norris,
1989; Nelson et al., 2006).
Of particular concern is the intimate relationship of ants with the banana aphid, which pro-
duces honeydew. Aphid populations prosper in the presence of ant colonies, and thus ants indirectly
aggravate BBTV incidence, increasing the probability of BBTV spread by the aphids. In Hawaii,
P. megacephala and, more recently, the long-legged ant Anoplolepis longipes Jerdon are primarily
associated with the banana aphid. By moving round aphids feeding on banana plants, they contrib-
ute to the spread of BBTV. Even directly, A. longipes feeds on the surface of the banana fruit, caus-
ing scarring of the fruit surface and reducing marketability (Brooks, 2003).
7.4.2 Natural Enemies
Several banana pests that require significant population densities before damage occurs are main-
tained below damage thresholds by natural enemies. Particularly good examples of this are demon-
strated with the spiraling whitefly and the banana skipper (Ramani et al., 2002; Okolle et al., 2006).
However, broad-spectrum insecticide applications, when relied upon for management of many pests
simultaneously, may cause secondary outbreaks of otherwise minor pests, especially following the
use of aerial or cover sprays (Pinese and Piper, 1994). Historical Lepidopteran outbreaks in banana
have been associated with pesticide-induced disturbance of their natural enemies, such as the local-
ized outbreaks of the banana skipper in Malaysia (Okolle et al., 2006). In a related study in Costa
Rica, Hymenopteran parasitoid abundance and species richness were inversely related to applica-
tion rates of nematicide and insecticide (Matlock and De La Cruz, 2002).
7.4.3 Weeds
Weeds not only compete with the banana crop for water and nutrients but also provide important
pest havens, both by providing shelter and, more importantly, by serving as alternative hosts, espe-
cially for polyphagous thrips, banana moths, whiteflies, and mites. Consequently, weed manage-
ment is an important component in many banana production areas for the indirect management of
pests. In Hawaiian banana orchards, weed management strategies involve the prevention of weed
seed formation and using pre-emergence herbicides, with emphasis on weed management prior to
canopy closure (Hawaii Banana Industry Association, 2010b).
7.5 Integrated Pest Management

7.5.1 History and Context of IPM
IPM is a term widely used but often misused in reference to banana. In relation to the literature,
researchers and practitioners tend to equate IPM with a list of control options for a particular pest
(often still at the research phase and biased towards biological control), which is not IPM. The term
IPM was first coined in 1972 following a speech by President Nixon to the U.S. Congress, and origi-
nally defined in 1975 by the Food and Agricultural Organization (FAO, 1975). Since then, IPM has
become a frequently used and misused term, often without the needful consideration of the subtle-
ties and implications of its true meaning or impact on modern agriculture (Kogan, 1998). IPM was
originally envisaged as a concept to counter the excessive applications of pesticides, particularly
insecticides. Although pesticides can, and have, greatly increased crop productivity, their use has
led to unintended adverse effects on human health and the environment. Furthermore, pesticide
resistance among the target pests can result in secondary pest outbreaks through their ill effects on
natural enemies (Stephenson, 2001). Originally, IPM was entomocentric, and only much later were
weed science and plant pathology included in IPM principles (Kogan, 1998). Numerous definitions
of IPM abound, with the concept of decision-making central to most (Bajwa and Kogan, 2002).
Based on an analysis of the various definitions spanning the preceding 35 years, Kogan (1998) pro-
posed a consensual definition of current thought: “IPM is a decision support system for the selection
and use of pest control tactics, singly or harmoniously coordinated into a management strategy,
based on cost/benefit analyses that take into account the interests of and impacts on producers,
society and the environment.”
7.5.2 Principles of IPM

The principles of IPM can be best explained by examining the terms of the acronym: integrated,
pest, and management.
7.5.2.1 Integrated
“Integrated” refers to the harmonious use of multiple management methods to control single
pests, as well as the impacts of these methods on multiple pests (Kogan, 1998). Management
methods are traditionally categorized as chemical (for example, pesticides), cultural (such as
intercropping), biological (for example, parasitoids), host plant resistance-based (such as breed-
ing genetically modified organisms), and genetic (sterile insect release, for example). A mere list
of categorized control options, however, does not necessarily enable the farmer to practice IPM.
It is important to distinguish between preventive/prophylactic and curative management options.
As IPM is founded on a decision-making process—namely, before economic damage levels
are incurred—IPM implicitly relies on prophylactic management options (Bajwa and Kogan,
2002). The successful and harmonious integration of management options is a difficult, if not a
near-impossible, task. Integration can be viewed as either vertical (that is, within a pest taxon,
sometimes referred to as first level) or horizontal (that is, among pest taxa, sometimes referred
to as second level). For example, an insecticide that affects both the target pest and its natural
enemies represents a lack of vertical integration; similarly, application of a fungicide that is det-
rimental to the natural enemies of pests provides a lack of horizontal integration. Historically, the
lack of such integration has been a major impediment to the implementation of IPM in agriculture
(Ehler, 2006).
7.5.2.2 Pest
“Pest” refers to any organism causing crop damage, including invertebrate and vertebrate ani-
mals, pathogens, and weeds (Kogan, 1998). Pest is an anthropocentric term, and highly relative
and dynamic. Any insect living in or on banana plants can, at some stage and in some locations,
become a pest or cease to be a pest. As such, sampling and monitoring schemes are of paramount
importance and are necessary components before IPM can be conducted. Even with a pest incur-
ring identical levels of injury in different locations, the economic damage acceptance level can
differ between production systems. This implies that sampling and monitoring schemes need to be
adapted to be location, crop, crop system, and season specific (Stephenson, 2001).
7.5.2.3 Management
“Management,” the most important term, refers to a series of decision rules based on ecological and
economic considerations, and equipped with sound and specific information related to the pest and
its management options. The key principle for this decision-making process is often the economic
injury level (EIL) concept (Stern et al., 1959). EIL is based on economics: the study of the rela-
tionships between pest densities, host responses to injury, and resultant economic losses. EIL is a
theoretical value that, if actually attained by a pest population, will result in economic damage. The
EIL formula [C/(V × I × D × K)] is determined using five primary variables: cost of the management
tactic per production unit (C), market value per production unit (V), injury units per pest (I), damage
per injury unit (D), and the proportional reduction in pest attack (K). From the EIL, the economic
threshold (ET) is calculated. The ET differs from the EIL in that it is a practical or operational rule,
rather than a theoretical one. The ET is defined as the population density at which control action
should be determined (initiated) to prevent an increasing pest population (injury) from reaching the
economic injury level. The ET is effectively an action threshold and is more complex to calculate
than the EIL. Besides information on the EIL, several other parameters need to be known to cal-
culate the ET, such as pest and host phenology, population growth and injury rates, and time delays
associated with the IPM tactics utilized. These parameters are also location, crop, crop system, and
season specific, and require extensive research before their implementation.
7.5.3 Practical IPM as a Continuum

Decision making, based on pest populations, is the most critical element in any IPM program.
Without the critical components of ET and EIL, there will be no decision making and hence no
IPM. However, because ETs and EILs are difficult to calculate, the practical implementation of IPM
has become less strict and is often applied as a continuum. For example, the U.S. Department of
Agriculture (USDA) uses a four-tier approach. As a first line of defense, prophylactic cultural meth-
ods (rotation, resistant cultivars, and pest-free planting material) are encouraged. Once monitoring,
identification, and action thresholds indicate that pest management is required and preventive meth-
ods are no longer effective, the least risky curative pest-management options are initially employed,
including highly targeted pesticides, such as pheromones to disrupt pest mating, or mechanical
control, such as trapping or weeding. If further monitoring, identification, and action thresholds
indicate that less risky controls are not sufficiently effective, additional pest-management methods
would be needed, such as targeted application of pesticides. Broadcast spraying of nonspecific pes-
ticides is a last resort (Stephenson, 2001).
Practically, efficient and operational IPM programs exist for specific crops in specific locations.
These programs take on a variety of formats: protocols, checklists, standards, and definitions. Many
of these assign point values to each practice, facilitating use as a performance assessment tool
(Green and Petzoldt, 2009).
7.6 IPM in Banana

7.6.1 IPM in Commercial Plantations
Pest management in commercial banana plantations is primarily chemical based, using nemati-
cides with insecticidal activity and applying specific insecticides to the plant base or on bunches.
Management of R. similis to date has essentially been achieved through the application of carbam-
ates (aldicarb, carbofuran, and oxamyl) and organophosphates (fenamiphos, ethoprop, and terbufos)
(Berg, 1991). Cyclodiene insecticides, once widely used but eventually abandoned following the
development of pest resistance and the emergence of environmental concerns, are now replaced by
less persistent organophosphates. However, pests such as the banana weevil have demonstrated the
ability to develop resistance to most pesticide classes (Gold and Messiaen, 2000). In Hawaii, to avoid
pesticide resistance, the industry is actively developing a pesticide resistance program. The organo-
phosphate diazinon, the primary pesticide used for thrips management, is being replaced with low-
risk pesticides such as imidacloprid and spinosad (Hawaii Banana Industry Association, 2010b).
Compared to smallholder systems, much focus is directed towards managing fruit and flower pests
in commercial banana plantations, because there tends to be a zero-tolerance policy on damage or
even presence for export markets (Jones, 2009), leading to much lower EILs and ETs.
In commercial plantations, IPM is especially sought to substitute for the excessive use of nem-
aticides particularly of late, following the imminent removal from use of many nematicides (http://
www.pesticideinfo.org/) and the increasingly strict regulations on the maximum permitted residue
levels of imported fruit, vegetable, and cereal products (European Commission, 2007). Furthermore,
most systemic nematicides are short lived (2–5 weeks) (Zum Felde et al., 2009), with rapid microbe-
enhanced biodegradation greatly reducing their effect, following their repeated and consistent use
(Moens et al., 2004), leading to an ever increasing but untenable number of applications.
Examples of true banana IPM schemes are rare but can be found in Hawaii. Their banana
IPM protocol uses a combination of guidelines and point values to determine the level of IPM
being utilized on a particular farm, which is constantly subject to change with new IPM devel-
opments. Pest management practices are grouped according to five categories (cultural, physi-
cal, mechanical, biological, and chemical) and each category is assigned a point value. Those
practices that require more active management decisions or present reduced environmental risks
receive higher point values. In practicality, points are low for pesticide-dependent practices
while high for biologically dependent ones. A grower is certified as an IPM practitioner if he
or she enrolls in the program and provides documentation that at least 70% of the total possible
points is achieved. Although not applied for all pests, binomial pest sampling plans have been
developed to allow for informed decision making regarding pesticide applications (Robson et
al., 2006).
Currently, the public demand for quality products grown with environmentally responsible meth-
ods is strongly encouraging organic banana farming. Nonpesticide pest-management methods are
paramount for the promotion and adoption of such cropping systems (Roditakis et al., 2006). There
is the erroneous tendency to associate the production of organic banana with smallholder farmers,
possibly because of the perception that they can least afford the pesticides used on conventional
crops and are more likely to use naturally occurring inputs. Current price premiums for organic
produce, only enjoyed in niche Western markets and required to offset increased production costs,
are also decreasing, perhaps limiting organic banana production in the long run (Reid, 2000; Abele
et al., 2007).
7.6.2 IPM in Smallholder Systems

7.6.2.1 Problems
Compared to commercial systems, smallholder banana systems are intrinsically different, making
it virtually impossible to implement IPM, while adoption of IPM has remained low in the developed
world, contrary to original expectations (Vereijken, 1989; World Bank, 2005; Ehler, 2006). Pest
problem recognition, and more specifically species identification, is required as a basis for IPM.
However, in resource-poor situations, the obstacles to pest problem recognition and species iden-
tification are often much greater than for commercial situations. For example, in Kenya, only 15%
of the farmers had knowledge on the damage caused by banana weevils and none for nematodes or
their damage symptoms, mostly mistaking their damage for that of banana weevils. Importantly,
and of greater concern, was that both farmers and extension officers made this mistake (Seshu
Reddy et al., 1999). In addition, pest profiles and their management cannot be simply transferred
from developed countries, as banana clones are highly variable and differ from the commercial cv
Cavendish types. For example, in East Africa, based on a comprehensive farm baseline study, sev-
eral banana pests, such as banana weevils and plant-parasitic nematodes, may not be as important
as traditionally assumed (CIALCA, 2009).
Government support and resources for IPM implementation at the national level in less-devel-
oped countries are often scarce or absent. Through the Cooperative Global Program, sponsored by
the Food and Agricultural Organization (FAO) and the United Nations Environmental Program
(UNEP), IPM strategies were targeted at cropping systems and regions, which included implemen-
tation, research, training, and education, and which have led to some success stories (such as the
reduction of pesticide use against the brown plant hopper Nilaparvata lugens Stål on rice (Oryza
sativa L.) (FAO, 1995). Since the 1990s, the Consultative Group for International Agricultural
Research (CGIAR) has increased its attention towards crop protection in general and IPM in par-
ticular, away from host-plant-resistance breeding (Kogan, 1998).
Smallholder banana systems are typically low input/low output, or resource poor, meaning that
pest-management practices are often limited at best. In Cameroon, for example, insecticide applica-
tions among smallholder banana farmers are negligible, because the returns are too low to allow
meaningful investment into pest-management measures. Only management options that offer less
capital investment, therefore, offer long-term sustainability (Tomekpe and Sadom, 2008).
7.6.2.2 Focus on Cultural Management

Mainly because of limited resources and availability of other options, pest management in small-
holder systems is heavily dependent on cultural options. However, cultural control options against
insect pests in smallholder systems differ markedly from those of commercial systems. For exam-
ple, one of the characteristics of smallholder banana systems is the perennial nature of banana, cre-
ating difficulties for short-term rotation options, while annual or single-cycle cropping characterize
some commercial systems. In addition, a combination of banana cultivars with varying levels of
resistance is often present in the same field in smallholder systems (Seshu Reddy et al., 1999).
Most of the cultural management options available to smallholder farmers are simple and
rely on good crop husbandry and habitat management—practices that encourage vigorous crop
growth, which leads to less damage. These practices include deep planting, weeding, mulching,
and the application of organic manure. For example, systematic trapping with pseudostem or
rhizome pieces, as well as removal of crop residues, can also be effective in reducing popula-
tions of adult banana weevils (Masanza et al., 2006). Using crop residues as mulch is effective at
decreasing nematode populations, especially when applied to low fertility systems (McIntyre et
al., 2000). One of the most critical management options for borers and nematodes is the use of
clean, healthy planting material. Tissue culture plantlets are widely used in commercial banana
plantations for pest and disease prevention but less available or understood in smallholder sys-
tems. Where tissue culture is not available, removal of roots and emersion of cleaned (pared)
suckers in hot water treatments can be highly effective against banana weevils and nematodes
(Speijer et al., 1999; Gold and Messiaen, 2000), while adapting the system to a simpler system
of using 30 s periods of immersion in boiling water can be equally effective against nematodes
(Tenkouano et al., 2006; Viaenne et al., 2006).
Besides cultural management options, much research currently focuses on biologically based
management options. How feasible or economical these will be to smallholder farmers remains to be
seen. However, based on interviews, Mugisha-Kamatenesi (2008) observed that subsistence farmers
around the Lake Victoria basin in East Africa commonly use botanical pesticides. Botanical com-
pounds especially are seen as substitutes for costly pesticides. Applications of neem (Azadirachta
indica A. Juss) in the field, such as neem oil for treating planting material or pseudostem traps,
protect bananas from weevil and nematode attack, inhibiting weevil larvae development by up to
14 days (ICIPE, 1997).
7.6.2.3 Technology Transfer
As a consequence of the intrinsic differences between smallholder and commercial systems, the
focus for pest management for smallholder farmers is on transfer of the basic technologies, mostly
cultural based, to the farming communities and extension personnel. For example, in Zanzibar, pest
management includes formation of farmers’ groups, training of trainers, establishment of plots used
for participatory action, and farmer field schools, which are used for demonstrating basic technolo-
gies, such as good crop husbandry (Rajab and Fundi, 1999). In Kenya, mobile training workshops
were initiated on a trial basis, which proved very effective in information dissemination (Seshu
Reddy et al., 1998). Global Plant Clinics is a recent initiative to link smallholder farmers with pest
information. The initiative aims to improve access to effective plant health services by adopting
similar approaches used in human health, through regular advisory services made available in local
communities (Boa, 2007).
7.7 Advances in Seed-Based Microbial Management

Endophytes are organisms that, at some time during their life cycle, live within plant tissues yet cause
no disease symptoms to their host (Petrini, 1986). Endophytes are natural and integral components
of all plants. The relationship can be mutualistic: Endophytes protect the host plant against pests
and diseases, and increase plant growth and vigor. Endophytes occupy a niche with relatively low
competition from other microorganisms, provided they gain access initially. As such, endophytes
have received increasing attention as biological control organisms in vegetatively multiplied crops,
such as banana (Sikora et al., 2008). Tissue-culture banana plants are becoming increasingly used in
banana production, even in smallholder systems, because of the advantages offered by these plants.
Tissue-culture plants are free from pests and pathogens, simple and quick to multiply in larger num-
bers, and exhibit faster and more uniform growth in the field than sucker-planted fields (Vuylsteke,
1989; Mateille et al., 1994; Robinson, 1996; Dubois, Coyne, et al., 2006; Pocasangre, 2006).
However, because tissue-culture plants are propagated under sterile conditions, they are void
of all beneficial organisms, including endophytes (Pocasangre, 2006). By selecting the best per-
forming endophytic strains and reintroducing them early into tissue-culture plantlets, the natural
equilibrium is somewhat restored, extending the benefits of clean planting material. Research into
enhancing banana with endophytes started in the early 1970s (Sikora and Schlosser, 1973; Sikora
and Schönbeck, 1975).
The use of endophyte-enhanced tissue-culture plants is a unique form of microbial pest con-
trol, mainly because it is seed based and thus circumvents many of the obstacles normally associ-
ated with augmentative biological control. Thus tissue-culture plantlets can be supplied to farmers
already fortified with endophytes, eliminating the need for farmers to apply the biopesticides. Costs
and know-how associated with formulation, distribution, application, and storage of endophytes can
be transferred to commercial tissue-culture laboratories (Dubois and Coyne, 2006). As endophytes
exist in planta, they offer great potential to manage cryptic pests and diseases. Furthermore, endo-
phytes escape the rhizosphere community, where they would otherwise compete with the native
flora and where they would be exposed to environmental factors that may adversely affect their
efficacy. Avoiding this competition and exposure allows for low initial inoculation levels, improving
consistency of endophyte performance and substantially reducing costs.
Endophyte-enhanced tissue-culture technology is sought after both in smallholder as well as
commercial systems. In smallholder systems, the early stages of plant growth can be challeng-
ing because banana tissue-culture plantlets need higher levels of care and attention than conven-
tional planting material. Where soils are depleted and pests and diseases abundant, tissue culture
is only superior to conventional planting material if accompanied by significant field maintenance.
Especially in the smallholder banana production systems, where high-input field maintenance
routines are largely absent, endophyte enhancement creates more robust tissue-culture plants. In
commercial systems, endophytes are also being investigated as replacements for nematicides (Zum
Felde et al., 2009).
How endophytes protect bananas is only just beginning to be understood. The primary mode
of protection of the endophyte Fusarium oxysporum Schlecht.: Fries against R. similis appears to
involve a number of mechanisms, including induced resistance (Athman, 2006). Induced resistance
is the activation of defense mechanisms in plants after contact with biotic initiators, such as endo-
phytes. The endophyte triggers pathways that induce physiological changes in the plant, enabling a
susceptible cultivar to express similar properties as a resistant cultivar (Dubois, Gold, et al., 2006).
This mode of action is economically interesting because it may transfer host resistance across a
broad range of pest groups. Also, endophytic inoculum can be further reduced and may not neces-
sarily need to persist for long periods, as long as the resistance remains triggered. Furthermore,
F. oxysporum seems to prime the banana plant against pests and diseases rather than inducing a
constitutive response. The priming of plants in this way thus avoids waste and helps optimize the
use of resources, a prerequisite for the implementation of the plant-enhancement technology (Heil
et al., 2000).
Several groups of endophytes are currently under investigation worldwide. A first group is com-
prised of mostly hyphomyceteous fungi that are nonobligate endophytes, which have a saprophytic
stage in the rhizosphere. Fusarium oxysporum is the most predominant endophytic taxon in banana
(Hallman and Sikora, 1996; Dubois, Gold, et al., 2006) and offers great commercial potential,
mainly due to the relative ease of inoculum production (Dubois, Coyne, et al., 2006). Endophytic
protection of tissue-culture banana plants has been demonstrated in the field under both commercial
and smallholder settings. In Panama, inoculation with Trichoderma atroviride P. Karst. protected
tissue-culture banana plants from R. similis better than two applications of ethoprop and temephos
nematicides, reducing R. similis population levels by 30–50% (Pocasangre et al., 2006; Pocasangre
et al., 2007). In Kenya, inoculation with F. oxysporum reduced nematode population densities by >
45% and damage by > 20% over one growth cycle (JKUAT, 2008; Waithira, 2009). Mass multiplica-
tion mechanisms of promising strains are currently being researched, in coordination with private
industry in East Africa and Central America.
A second group is the arbuscular mycorrhizal fungi (AMF), obligate symbionts of almost all
higher plants, including most cultivated plant species (Abbott and Robson, 1984; Sikora et al., 2003).
AMF not only antagonize banana pests but also improve plant growth and survival through water and
nutrient uptake. Tissue-culture plants enhanced with Glomus fasciculatum Thaxter and G. mosseae
Thaxter have demonstrated suppression of nematodes, such as R. similis (Umesh et al., 1988) and P.
goodeyi (Jaizme-Vega and Pinochet, 1997). However, to obtain the inoculum needed for application,
AMF need to be produced on living plants (Sikora et al., 2003), creating difficulties and expense in
their production and application. Until recently, the use of AMF was not viewed to be commercially
viable, although products are now beginning to appear on the market. As intercropping is such a
common aspect of subsistence farming, some intercrops that favor AMF inoculum buildup, such as
sorghum (Sorghum spp.), could be promoted (Elsen et al., 2003; Elsen et al., 2009).
Several entomopathogenic fungal products based on Metarrhizium spp. and Beauveria spp.
are commercially available for use as insect biopesticides. Despite near 100% efficacy in vitro
against pests such as the banana weevil (Kaaya et al., 1993), their efficacy in the field tends to be
slow, erratic, and ultimately an expensive option. The development of efficient and cost-effective
field delivery systems currently hampers their use in smallholder and commercial banana systems.
Recently, it was demonstrated that such fungi can be applied as artificial endophytes in banana
plants, reducing banana weevil populations and damage (Akello et al., 2007; Akello, Dubois, et al.,
2008a, 2008b; Akello, Coyne, et al., 2008).
7.8 Conclusion
This chapter provides an overview of nonmicrobial pests of bananas. Pest profiles are highly vari-
able and depend on region, clone, and crop system. Of particular importance is that recommen-
dations for their management, and research leading to these, vary greatly between smallholder
systems and commercially managed plantations. Whereas IPM is necessary and can lead to reduced
pesticide reliance, especially nematicides, in commercial systems, the situation contrasts mark-
edly with smallholder systems. For smallholder systems, ill-linked management options, some that
are often unsuitable, should be avoided for use in IPM, and a focus on key basic issues, such as
pest identification, cultural management options, and deployment of basic training for farmers and
extension workers should prevail.
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8 Reproductive Biology
Jeanie Anne Fortescue and David William Turner
Contents
8.1 Floral Biology........................................................................................................................ 146
8.1.1 Development of the Inflorescence............................................................................. 146
8.1.2 Development of the Female Flower........................................................................... 147
8.1.2.1 Pre-Anthesis................................................................................................ 147
8.1.2.2 Anthesis (Opening of the Female Flowers)................................................ 148
8.1.2.3 Post-Anthesis............................................................................................... 151
8.1.2.4 Ovule Growth in Cross Section.................................................................. 152
8.1.2.5 Embryology of Musa.................................................................................. 152
8.1.3 Development of Male Organs and Gametes.............................................................. 154
8.1.3.1 Microsporogenesis...................................................................................... 154
8.1.3.2 Pollination................................................................................................... 155
8.1.4 Floral Factors and Breeding Systems........................................................................ 155
8.1.4.1 Outbreeding or Inbreeding?........................................................................ 155
8.1.4.2 Reproductive Abnormalities....................................................................... 156
8.1.4.3 Female Sterility........................................................................................... 157
8.1.4.4 Female–Male Interaction............................................................................ 157
8.1.4.5 Pollen–Pistil Interaction............................................................................. 157
8.1.4.6 Self-Incompatibility.................................................................................... 158
8.2 Pollen Production, Viability, and Germination..................................................................... 158
8.2.1 Pollen Production....................................................................................................... 159
8.2.2 Polyspory................................................................................................................... 161
8.2.3 Pollen Germination.................................................................................................... 162
8.3 Seed Production..................................................................................................................... 163
8.3.1 Seed Morphology and Anatomy................................................................................ 163
8.3.2 The Seed Coat............................................................................................................ 164
8.3.3 Pollinators.................................................................................................................. 165
8.3.4 Factors Affecting Seed Set........................................................................................ 166
8.3.4.1 Maximum Seed Set..................................................................................... 166
8.3.4.2 Bunch and Fruit Factors.............................................................................. 166
8.3.4.3 Environmental Factors................................................................................ 168
8.3.5 Seed Growth.............................................................................................................. 169
8.3.6 Seed Storage.............................................................................................................. 170
8.4 Seed Germination.................................................................................................................. 170
8.4.1 Dormancy.................................................................................................................. 170
8.4.2 Viability..................................................................................................................... 174
8.4.3 Germination............................................................................................................... 174
8.5 Conclusion............................................................................................................................. 175
Acknowledgments........................................................................................................................... 176
References....................................................................................................................................... 176
145
8.1 Floral Biology

Bananas and plantains (Musaceae) are a unique group of plants within the Zingiberales. They con-
tribute to the nourishment and culture of millions of people, especially in tropical regions, and are
exported to temperate regions. This chapter discusses the reproductive biology of the Musa spp.
because it is central to working with the breeding system and the relative success of banana breed-
ing schemes.
In recent years knowledge of the sexual reproduction of plants has progressed considerably,
but few studies have been made on members of the Musaceae, despite their economic importance
and scientific interest. Many of the key references on floral biology of Musa spp. were published
between 1930 and 1980, especially those involving ovules and the female gametophyte.
8.1.1 Development of the Inflorescence

The vegetative stage of each shoot ends when the apical meristem produces an inflorescence that
undergoes much of its early development inside the pseudostem. Extension of the internodes below
it thrusts the inflorescence to the top of the plant and clear of the surrounding leaf sheaths. Until its
emergence, the changes from a vegetative to floral apex and even the development of the inflores-
cence is unmarked by any external sign. The only indication is leaf number and size. Approximately
26 to 50 laminate leaves are produced, increasing in size until the last two, which are much reduced
in size, just before the inflorescence emerges (Simmonds, 1962; Stover and Simmonds, 1987). The
vegetative apical meristem of Musa is a flattened dome. The main meristem lies deep under the
center of the dome. As in other monocotyledons, vegetative growth is delegated to lateral organs;
leaves are differentiated in regular succession from the outer flanks of the corpus. The transition
to flowering is accompanied by a broadening of the apex by both cell division and cell expansion.
After floral initiation, the meristem becomes convex and rises above the surrounding leaf bases.
Flower bracts appear instead of leaves.
Initiation of the inflorescence is accompanied by a great increase in mitotic activity deep in the
corpus and thickening of the tunica. The products of all this activity and growth are a stem with
elongated internodes, non-encircling bracts in place of encircling sheaths, and a regular system of
axillary lateral branches—the flowers (Simmonds, 1966; Stover and Simmonds, 1987). In quantita-
tive terms, before floral initiation, the meristem produces a leaf and a lateral bud (phytomer) every
10 days, but after floral initiation it produces a bract and up to 20 flower initials every 1 to 2 days.
At the earliest stages of development, the floral parts are undifferentiated mounds of tissues.
The leaf and following bracts are homologous organs and have the same morphology during early
floral initiation. The major difference between the two is that the bracts have flower primordia in
their axils. The early floral meristem produces secondary meristems that then initiate the individual
flowers along the central axis. Swellings soon appear at the base of the flower bracts that enlarge
and differentiate into female flowers. Subsequent bracts subtend male flowers (White, 1928). In tree
crops the determinate floral meristems are axillary and the terminal apex remains vegetative; this
situation is typical in tropical crops such as avocado, a dicotyledon (Sedgley and Griffin, 1989). In
Musa it is not known whether the apex truly changes from vegetative to reproductive or whether the
lateral activity around it becomes reproductive (White, 1928).
The inflorescence axis is the distal part of the aerial stem, which is terminal on the corm. It is a
raceme or spike of cymose clusters of flowers at nodes covered by colored bracts. The female flow-
ers are within the basal (proximal) bracts and the male flowers in the apical (distal) bracts; the inter-
mediary clusters or neuters are of transitional structure. Musa flowers are predominantly unisexual
and the plant monoecious. However, bananas grow in a clump with several generations connected
to the underground corm and geitonogamous pollination may occur between inflorescences on the
one clump or mat. Within an inflorescence, transition from nodes with female flowers to nodes
with male flowers is marked in the juvenile inflorescence by a sudden decline in ovary length from
Reproductive Biology 147
one node to another. This is the first sign of gross sex differentiation. The underlying biochemical
processes must take place much earlier in the sequence of floral differentiation. The major differ-
ence between the male and female ovary is the latter are larger, have a massive style that exceeds
the perianth in length, and the stamens are reduced to staminodes. In the male flowers the ovary is
small and in many cultivars and species they develop an abscission zone at their base and are shed a
few days after anthesis. The female flowers develop no such abscission zone; however, the style and
staminodes may abscise, leaving a calloused scar at the top of the ovary (Simmonds, 1966; Stover
and Simmonds, 1987).
The inflorescence bears 1 to 30 nodes (or hands) of pistillate female flowers depending on geno-
type, environment, and edaphic conditions, followed by 0 to 4 hands of neuter flowers or pseudo-
hermaphrodite hands. The remainder of the inflorescence contains staminate flowers, of which
there are from 150 to 300 hands. Spikes of cv ‘Gros Michel’ (AAA) can contain over 100 male
hands representing 2,500 male flowers compared with fewer than 200 female flowers (White, 1928).
The apex may continue to produce male flowers long after the female fruits have rotted. In some
clones, especially among plantains, the apex is short lived so that growth ceases soon after the
bunch emerges from the top of the pseudostem; Horn plantains are characterized by the absence of
male flowers at maturity (Simmonds, 1966; Stover and Simmonds, 1987; Swennen et al., 1995; De
Langhe et al., 2005).
The basal nodes of the banana inflorescence bear the female flowers, and the upper nodes the
male flowers. When the ovaries of the female flowers are developing into fruit, the axis continues to
grow. As the stem grows, the bracts open to expose the male flowers and then both bract and male
flowers usually abscise after a day or two. The distribution of the female and male flowers and the
continued growth of the distal portion of the axis confer on the mature inflorescence a characteristic
appearance. At the basal end is a mass of fruit, then a length of axis to which the male flowers and
their subtending bracts may adhere, or abscise, and a “bell” of new male bracts and flowers at the
distal end. The whole inflorescence may be 1 to 3 m long.
8.1.2 Development of the Female Flower

Musa has an inferior trilocular ovary with axile placentation. The ovules, of which there may be 300
to 1,500 per ovary, spring as relatively late outgrowths from the placenta and are two or four rowed
in each locule. This character is expressed differently by the acuminata (A) and balbisiana (B)
genomes and is diagnostic for determining the genomic origins of the edible bananas (Simmonds
and Shepherd, 1955; Simmonds, 1966; Stover and Simmonds, 1987). The development of the female
gametophyte of Musa is typical of angiosperms.
8.1.2.1 Pre-Anthesis
The ovary has septal nectaries with copious nectar and a tough outer skin that splits open longi-
tudinally. The stigma is trilobate with a constantly wet and papillate surface that is sticky when
receptive. It has two to four rows of ovules in each locule. The ovules are numerous, embedded in a
strip of mucilage, and axile to the placenta. Development from floral initiation to ovule primordium
to megaspore mother cell occurs while the inflorescence is inside the pseudostem (White, 1928;
Juliano and Alcala, 1933; Dodds, 1945; Bouharmont, 1963; Dahlgren et al., 1985; Krishnamoorthy
et al., 2004). As the inflorescence moves upward, the flowers arise in each node as a double row of
closely grouped protuberances numbering 12 to 20 in ‘Gros Michel’ (White, 1928). When the length
of the bract that envelops the inflorescence is twice its width, the fascicular primordium, which pro-
duces the flowers, appears as a mamillate hump at the axis. Development of the floral organs of the
three kinds of flowers is acropetal. They arise in the following sequence: outer perianth lobes, inner
perianth lobes, stamen, and pistil (Juliano and Alcala, 1933).
When it is midway up the pseudostem, the inflorescence of cv ‘Gros Michel’ (AAA) is about
120 mm long and the staminate and pistillate flowers are distinguishable. The ovary of the female
Placental
wall
Funiculus
Ovule
Placental
hairs
Figure 8.1 Musa acuminata ssp. (AA), an undescribed seeded banana similar to microcarpa but with lon-
ger pedicels. Also in Figures 8.3, 8.4, and 8.8. Bar = 50 µm. The ovule is small and orthotropous in orientation.
It is only a fingerlike projection from the placenta. The outer and inner integuments, nucellus, and nucellar cap
have not differentiated. L, locule. (Reprinted from Sci. Hort. 104, J.A. Fortescue, and D.W. Turner, Growth
and development of ovules of banana, plantain and ensat (Musaceae), 463–478, Copyright 2005, with permis-
sion from Elsevier.)
flowers is about 10 mm long and 2.5 mm in diameter. The megasporangia are differentiating and
appear as a rounded protuberance growing at right angles from the placental wall. Initially the ovule
primordia are slender, fingerlike projections not more than five cells in width and two to four times
longer (Figure 8.1) (White, 1928; Bouharmont, 1963). The ovule is at first atropous and by differen-
tial growth becomes anatropous. The micropyle points towards the placental wall.
The inner integument has already formed when the archesporium arises from any subepi-
dermal cell near the summit of the nucellus. The differentiation of the archesporia takes place
before the differentiation of the outer integument and just at the time the megasporange is half
anatropous. It is easily distinguished from the surrounding cells by its relatively large size and
great affinity for stains (Dodds, 1945; Bouharmont, 1963). The ovules attain almost maximum
size when the megaspore mother cell begins to divide. When the inflorescence emerges from
the top of the pseudostem, the embryo sac has differentiated and the nuclei are in their respec-
tive positions.
The stage when the ovule is half anatropous is critical in its development because changes are
rapid and profound. The outer and inner integuments have differentiated but not the nucellar cap;
however, the integuments are not large enough to form a micropyle. A large cell is visible in the
nucellus and is most probably the megaspore mother cell (Figure 8.2). Within a few days the ovule
is circular and the inner and outer integuments have enlarged to encircle the nucellus and form
the micropyle. A nucellar cap is evident and an elongated and vacuolated megaspore has formed
(Figure 8.3). Environmental conditions at the stage between archesporia and embryo sac are critical,
because a cold snap can produce malformations only inside the ovule and not the fruit (Fortescue
and Turner, 2005c).
8.1.2.2 Anthesis (Opening of the Female Flowers)

In Musa spp. the ovule is anatropous, bitegmic, and crassinuclear. The external integument is thick,
distinct from the internal integument, and the funicle is partly united to the external integument.
Outer
integument
Inner Megaspore
integument mother cell
F
Outer
integument
Ph
Figure 8.2 Musa spp. Cavendish subgroup, (AAA), sterile edible cultivar, also in Figures 8.5, 8.6, and 8.7.
Bar = 50 µm. The ovule is now anatropous, the outer and inner integuments have formed but not the nucellar
cap. The megaspore mother cell has formed with a central vacuole. F, funiculus; N, nucellus; Ph, placental
hairs. (Reprinted from Sci. Hort. 104, J.A. Fortescue and D.W. Turner, The anatomy of ovule development of
banana, plantain and ensat (Musaceae), 479–492, Copyright 2005, with permission from Elsevier.)
Embryo
sac
N
Nucellar
cap
Oi
Ii Micropyle
F
Figure 8.3 Musa acuminata ssp. (AA), bar = 100 µm. All the components of the ovule have now differ-
entiated. F, funiculus; Ii, inner integument; N, nucellus; Oi, outer integument. (Reprinted from Sci. Hort. 104,
J.A. Fortescue and D.W. Turner, The anatomy of ovule development of banana, plantain and ensat (Musaceae),
479–492, Copyright 2005, with permission from Elsevier.)
Antipodal
Polar nuclei
Egg Nuclei
apparatus
Nc
Ii
Oi
Figure 8.4 Musa acuminata ssp. (AA), bar = 25 µm. The diploid embryo sac at anthesis. It is very large,
round, and lies flush against the nucellar cap. All the constituent embryo sac components are present. Two
polar nuclei are visible over the antipodal pit where one antipodal is in view. A nucleus is visible in the egg
apparatus. Ii, inner integument; Nc, nucellar cap, Oi, outer integument. (Reprinted from Sci. Hort. 104, J.A.
Fortescue and D.W. Turner, The anatomy of ovule development of banana, plantain and ensat (Musaceae),
479–492, Copyright 2005, with permission from Elsevier.)
The chalazal mass is a transitional zone between the integuments, the funicle, and the base of the
nucellus, which is massive. The nucellus consists of two parts; on the outside there are parenchy-
matous cells with very small nuclei, and towards the center there are large cells with voluminous
and very colored nuclei. The micropyle is formed by both integuments and points in all directions
towards the placental wall. At anthesis, the ovule of seeded Musa ssp. is 0.6 mm wide and 0.7 mm
long. The embryo sac is large and located next to the nucellar cap; it is eight nucleate and has a
characteristic bell shape (Figure 8.4). Its broad base is embedded in the nucellus and it has two large
unfused polar nuclei, usually immediately over the antipodal pit in which lie the three antipodals.
Its tapered end is flush with the columnar cells of the micropylar cap and contains the egg apparatus
and two large and active synergids (White, 1928; Dodds, 1945; Maheshwari, 1950; Bouharmont,
1963; Fortescue and Turner, 2005a, 2005b). Figure 8.4 shows a median cross-section of an embryo
sac of Musa sp. at anthesis; in view are two antipodals, two polar nuclei, and the egg apparatus with
a nucleus.
The primary vascular bundles are found in the placental wall and outer integuments, in which
they appear to be in a functioning state at anthesis and probably during the maturation of the seed.
The chalazal mass consists of two doughnut-shaped discs positioned one above the other. Before
anthesis, the chalaza is not very apparent but as anthesis approaches it grows larger and becomes very
obvious after fertilization. In the unfertilized ovule, the chalaza remains thin and lightly stained.
The developmental progression of the embryo sac of the edible triploids is very similar to
that of the seeded diploids; examples can be found in all stages of development from tetrad
to embryo sac. At anthesis the majority of the embryo sacs are not found flush with the nucel-
lar cap and their contents appear in disarray, particularly in the AAA genotypes but also in
the ABB genotypes. They are often oval shaped and vacuolated. However, many embryo sacs
still showed internal order with one or more nuclei present (Figure 8.5) (Fortescue and Turner,
2005a, 2005b).
Nuclei
Second
chamber
Remote from
nucellar cap
Nc
Ii
Figure 8.5 Musa spp. Cavendish subgroup, (AAA), bar = 25 µm. At anthesis the triploid embryo sac is
present, usually with its contents in disarray, but with some order and one or more nuclei present. Note that
this sac is not flush with the nucellar cap. Ii, inner integument; Nc, nucellar cap.
8.1.2.3 Post-Anthesis
A few days after anthesis, the nucellus, lying subadjacent to the integument and extending from the
walls of the embryo sac outward and downward to the chalaza, begins to break down to form an
inverted funnel-shaped cavity in the center of the main mass of the nucellus. This autolytic process
is independent of the development of the embryo sac. Without the division of the endospermic
nucleus to fill the cavity, the unfertilized ovule shrinks, collapsing on itself. After fertilization the
internal part of the nucellus entirely disintegrates; after 25 days none of the inner cells remain
and a large internal cavity has formed. The thickness of the larger parenchymatous cells does not
decrease. By 55 days after fertilization, these parenchymatous cells are almost empty, becoming
the perisperm, which later disintegrates when the endosperm becomes cellular (Bouharmont, 1963;
Fortescue and Turner, 2005a, 2005b).
In fertilized ovules the development of the endosperm and zygote begins, and the endospermic
nucleus commences to divide almost immediately. Three days after fertilization, the endosperm is
20 to 40 nucleate. Divisions proceed rapidly, resulting in a convoluted vesicle of free nuclei devoid
of walls, situated immediately over the antipodal pit. The divisions are not synchronous, forming
isolated vesicles and nuclei that wander the periphery of the embryo sac. The nucellar autolysis
begins at the outer basal regions of the embryo sac and endosperm nuclei pass into the resultant cav-
ity. After 22 days, the endosperm becomes cellular. The cells are large, separate from one another,
uninucleate, and do not divide further. Later they increase in volume a little and crush the perisperm
against the integuments. After 32 to 40 days, the endosperm thickly carpets the embryo sac.
After fertilization the zygote possesses one or two nucleoli and the synergids disappear. It does
not begin to divide until after endosperm formation is well underway. It develops slowly, the multi-
cellular bi- or quadra-cellular proembryo can be observed after 13 days. After 25 days, the proem-
bryo is a small multicellular, undifferentiated, and elongated mass measuring 65 by 35 µm. After
40 days it measures 110 by 40 µm and has partially emerged from the hollow of the micropylar cap.
After 55 days the proembryo measures 120 to 250 by 80 to 170 µm. After 62 days, what is now the
embryo is fixed to the nucellar cap by the suspensor and nearly fills the space inside what is now the
micropylar collar. The embryo continues to grow until it fills the micropylar cavity as a short cylin-
drical body and finally pushes into the main nucellar cavity where it encounters the dense nutritive
Lysing
nucellar
cells
Lysing
embryo
Sac wall
Figure 8.6 Musa spp. Cavendish subgroup, (AAA), bar = 25 µm. The post-anthesis embryo sac and sur-
rounding nucellus tissues begin to lyse. The nucellus cells at the posterior end and to the lateral sides of the
embryo sac have begun to lyse, the cells have become compressed, and their walls are disrupted. The embryo
sac also has begun to lyse, the walls have ruptured, splitting the sac and spilling the contents into the sur-
rounding nucellus tissue. (Reprinted from Sci. Hort. 104, J.A. Fortescue and D.W. Turner, The anatomy of
ovule development of banana, plantain and ensat (Musaceae), 479–492, Copyright 2005, with permission
from Elsevier.)
endosperm. The protruding portion flattens against this to form the fungiform digestive pad char-
acteristic of the embryos of Musa. After 68 days the meristems of the shoot and root appear. The
mature embryo is very small and does not contain a well-defined suspensor. The fertilized ovule
is enormous, and the outer integuments enlarge to close the micropyle and form the operculum.
The nucellar cap cells alter their arrangement to form a large cradle in which the proembyron rests
(Fortescue and Turner, 2005c).
In the edible triploids the embryo sac begins to show deterioration soon after anthesis; the cells
surrounding and in contact with the sac from the posterior end lyse along with the embryo sac
walls and their contents fill the space the sac had occupied (Figure 8.6). The lysing cells continue to
spread laterally to create an internal chamber (Figure 8.7) very similar to the conditions seen in the
ovules of seeded diploid bananas immediately after fertilization (Figure 8.8).
8.1.2.4 Ovule Growth in Cross Section

At anthesis the ovules of Musa ssp. measure 0.49±0.03 mm2 in cross-sectional area. At 47 days after
fertilization, the size increases eightfold to 3.8±0.5 mm2. Unfertilized ovules remained a similar size
of 0.48±0.06 mm2 after 47 days. At anthesis the triploid ovules are approximately twice as large,
in cross-sectional area, as the diploid ovules, averaging 0.82±0.08 mm2. Within Musa, ovule size
increases with ploidy, the ovules of triploids are larger than diploids and those of tetraploids are larger
again. The acuminata/balbisiana content does not appear to significantly affect ovule size or shape.
In Ensete sp., the ovules at anthesis are up to 11 times larger than those of Musa, measuring up to 5.0
mm2 (Fortescue and Turner, 2005b). This difference is reflected later in the size of mature seeds.
8.1.2.5 Embryology of Musa
The megasporogenesis and gametogenesis of Musa is typical of angiosperms with a few minor
exceptions. The megasporocyte undergoes the usual meiotic divisions to form a tetrad. The tetrads
are usually linear, but isobilateral T and inverted T shapes can occur in the same species. The
Antipodal
attachment point Ruptured
embryo sac
Nucellar chamber
Figure 8.7 Musa spp. Cavendish subgroup, (AAA), bar = 50 µm. The lysis has spread laterally to cre-
ate an internal chamber, very similar to the early postfertilized circumstances found in the fertilized dip-
loid. (Reprinted from Sci. Hort. 104, J.A. Fortescue and D.W. Turner, The anatomy of ovule development of
Liquid endosperm
Nuclei
Proembryon
Remnant
pollen tube
Figure 8.8 Musa acuminata ssp. (AA), bar = 25 µm. The fertilized ovule 20 days post-anthesis. The fertil-
ized ovule is large, the nucellus has completely lysed, creating a large central cavity, the micropyle is closed,
and the nucellar cap has formed a large cradle in which sits the proembryo. The endosperm is beginning to
form but is not yet cellular. A nucleus is present inside the proembryo and the remnants of the pollen tube
can be seen—it has pushed apart the nucellar cap cells and pierced the embryo sac with an apparent forked
tip. (Reprinted from Sci. Hort. 104, J.A. Fortescue and D.W. Turner, The anatomy of ovule development of
embryo sac is large, with a characteristic bell shape and located next to the columnar cells of the
micropylar cap. It is eight nucleate, containing the egg apparatus with two large and active synergids,
two large unfused polar nuclei, and three antipodals. The antipodals are ephemeral but sometimes
persistent (White, 1928; Dodds, 1945; Maheshwari, 1950; Bouharmont, 1963; Fortescue and Turner,
2005a, 2005b). The polar nuclei fuse at the time of fertilization. The development of the endosperm
is the nuclear type. In Musa, not only is nuclear division nonsynchronous (which in itself is not
unusual) but some nuclei divide more actively, forming isolated vesicles or nodules that develop into
separate endosperm masses. Usually one large vesicle is formed over the antipodal pit and lesser
free nuclei are dispersed about the sac. Later the endosperm becomes cellular beginning at the
micropylar region. The embryology of Musa has not been studied in sufficient detail. Bouharmont
(1963) describes the embryology as of the Asterad type. According to Johri et al. (1992), Asterad is
the common type of embryology in the order Zingiberales. It is reasonable to assume, from lack of
evidence to the contrary, that the embryology of Musa is also the Asterad type. The mature embryo
is capitate, more or less basal, relatively small, and restricted to the lower part of the seed, distally
expanded and in copious endosperm. The seed is medium to large.
8.1.3 Development of Male Organs and Gametes

In angiosperms the stamen differentiates into an anther and filament. The anther usually develops
into two lobes with two pollen sacs with four groups of archesporial tissue. The archesporial tis-
sue differentiates into a mass of pollen mother cells surrounded by various wall layers. The pollen
mother cells become surrounded by callose, undergo meiosis, and produce four haploid cells. These
haploid cells subsequently develop into pollen grains surrounded by the pollen grain wall. At matu-
rity the size, shape, surface pattern, and stickiness of the pollen grains are highly characteristic of
species (Sedgley and Griffin, 1989). Bananas do not differ from the normal angiosperm example,
except in gross morphological appearance. Five of the six stamens are functional and the anthers are
elongate to linear, basifixed, and tetrasporangiate. The pollen grains are two locular and nonapertu-
rate. The anthers of Musaceae have a glandular-secretory tapetum and the pollen grains have a thin
or virtually nonexistent exine but a thick intine (Dahlgren et al., 1985; Fortescue and Turner, 2004).
The tapetum synthesizes substances that contain fibrogranular proteins and carotenoid lipid drop-
lets that help form the exine (Johri et al., 1992). As in most angiosperms, the anthers dehisce by two
longitudinal slits; each microsporangium opens by a common slit with the other microsporangium.
8.1.3.1 Microsporogenesis
Microsporogenesis and male gametogenesis in Musaceae are not well studied and there is insuf-
ficient data from each of the families of Zingiberales to draw close comparisons. However, from the
literature it does appear to be a uniform order (Dahlgren et al., 1985). The following account relates
to the anther development in Musella lasiocarpa (Musaceae) by Xue et al. (2005). The anthers
are tetrasproangiate. The formation of the anther wall is of the basic type. The mature anther wall
consists of an epidermis, an endothecium, many middle layers, and a two-layered glandular tape-
tum with uninucleate cells. The old anther wall consists of an epidermis with annular and helical
thickening and reduced endothecium. Successive cytokinesis follows meiosis of the microspore
mother cells, forming a T-shaped or isobilateral tetrad of microspores. Pollen grains are two celled.
The generative cell nucleus is clavate in shape (Xue et al., 2005). The archesporial cells are recog-
nized by their dense cytoplasm and conspicuous nuclei. A row of sporogenous cells produced by
the archesporia gives rise to a mass of microspore mother cells by several mitotic divisions. While
changes take place in the anther walls, the primary sporogenous cells undergo mitosis forming sec-
ondary sporogenous cells from which the microsporocytes are derived. Microspores are separated
from the tetrad as uninucleate free microspores. Each microspore has a dense cytoplasm with a
prominent and centrally placed nucleus that moves to a peripheral position as the vacuole develops.
The first mitotic division of the microspore results in the formation of two unequal cells, the large
vegetative and smaller generative cell. The pollen grains are two celled and nonaperturate at the
time of anther dehiscence. The callose surrounding the microsporocytes is thin, and the pollen
grains have a thin or virtually nonexistent exine and a thick intine (Xue et al., 2005).
The tapetal cells are uninucleate throughout their development. At the time of microsporocyte
meiosis, the walls of the tapetal cells become indistinct and the tapetal cells begin to degenerate.
The endothecium reduces and does not develop fibrous thickenings as in most angiosperms. At the
mature pollen grain stage, the tapetal cells have degenerated completely. During maturation the epi-
dermal cells enlarge and thicken. Thus the mature anther wall is composed of the fibrous thickened
epidermis and the reduced endothecium (Xue et al., 2005).
8.1.3.2 Pollination
In terms of pollination, no fewer than about 4,000 pollen grains are needed to cover the stigmatic
surface of a female flower of a Musa diploid (Dodds, 1945). This is approximately 20 to 40 times the
number of ovules in an ovary. About 12 hr after pollination, the pollen tubes transverse the entire
length of the style. The style is approximately 30 mm long, so growth rate is 0.33 mm/hr. There
appears to be no inhibition of the pollen tube growth and more tubes enter the ovary than there are
embryo sacs awaiting fertilization. Pollen tubes enter the ovule only through the micropyle. The
styles abscise about 30 hr after the maturation of their receptive surfaces. Ovules must be fertilized
within 24 hrs of flower opening, after which they begin to disintegrate.
8.1.4 Floral Factors and Breeding Systems

8.1.4.1 Outbreeding or Inbreeding?
Bananas are monoecious: The female and male reproductive organs are separated into different
floral structures on the same inflorescence. This is the dominant condition among gymnosperms,
also frequent in angiosperm monocotyledons and some dicotyledonous timber and crops species
(Sedgley and Griffin, 1989). If the monoecious condition is complete, then self-pollination of an
individual flower is impossible. In the monoecious condition the flowers are functionally unisexual
but still structurally hermaphrodite. Both carpel and stamen primordia are initiated, and female
and male flowers are identical in early stages. Further development results in female flowers with
functional carpel and vestigial sterile staminodes and male flowers with functional stamens but
small and underdeveloped carpels. Most monoecious plants produce considerably more male than
female flowers; the proportions range from 0.1% female flowers in Aesculus sp. to 60% in lychee,
oil palm, and mangoes (Sedgley and Griffin, 1989). In Musa cv ‘Gros Michel’ (AAA), 7% of the
flowers within an inflorescence are female (White, 1928).
Bananas are also dichogamous: There is temporal as well as spatial separation of the sexes
within an inflorescence, and the female organs mature before the male organs. Dichogamy is par-
ticularly common in monoecious plants, especially the gymnosperms. In wild bananas the first
bunch thrown by a new plant would be out-pollinated. But bananas are clump forming; therefore,
later bunches thrown by suckers arising from the parent rhizome could be pollinated by male flow-
ers from the preceding bunches. Therefore selfing cannot be uncommon. Since bananas tend to be
gregarious plants of transient habitats, sib pollination must be frequent. According to Simmonds
(1962) out-pollinations do occur as evidenced by the occasional occurrence of interspecific hybrids
in batches of open pollinated seeds.
Female flowers produce considerable quantities of nectar, and male flowers tend to produce less
or none at all. The female flowers of M. salaccensis, which has a vertical inflorescence, produce dur-
ing the day 17–33 µL/h of nectar that contains 18–19% sugar (Itino et al., 1991). Male flowers pro-
duce less (17–19 µL/h) with similar sugar concentrations (19–20%). Male flowers of M. acuminata
ssp. halabanensis that occur on the pendant section of the inflorescence produce 8 µL/h of nectar
during the night. This nocturnal nectar contains 23–25% sugar (Itino et al., 1991). Similar quantities
of nectar are produced by the flowers of M. paradisiaca ssp. sapientum Kuntze (possibly AAA, cv
‘Pisang Masak Hijau’ or ‘Red-Green Red,’ or AAB, ‘Silk Fig’) but the male flowers produce about
four times more (35 µL/h) nectar than the female flowers (8–9 µL/h) (Fahn and Benouaiche, 1979).
In contrast, the male flowers of Musa spp. AAA Cavendish subgroup ‘Dwarf Cavendish’ do not
produce any nectar because the epithelial nectar cells disintegrate before the nectary matures (Fahn
and Benouaiche, 1979). In M. itinerans, female flowers produce 6 µL/h and male flowers a little less
at 4 µL/h, but the sugar concentration is similar in each flower type (21–22%) (Liu, Li, et al., 2002).
The female flowers of Musella lasiocarpa produce nectar at a rate of 7 µl/h and the male flowers 2
µl/hr, with a sucrose content of 15% (Liu, Kress, et al., 2002). Among species and cultivars there is
considerable variation in the rate of production of nectar from female flowers. The reasons for these
differences among Musa spp. have yet to be determined.
Within the Musaceae, some species have a proportion of functionally hermaphrodite flowers
and presumably would be frequently, if not regularly, self-pollinated. Musa acuminata ssp. banksii,
M. ingens and M. schizocarpa are examples (Simmonds, 1962). In addition, Nur (1976) excluded
pollinators from inflorescences of M. velutina, but flowers set seed because of self-pollination. The
species with hermaphrodite flowers occur at the outer margins of the geographical distribution and
represent a mechanism that favors selfing by isolated plants at the limit of their range (Simmonds,
1962). Bananas are intermediate between the two extremes of inbreeding and outbreeding. If they
were highly outbred, there should be significant loss of vigor on selfing, but this is not so. Bananas
are moderately outbred and can tolerate an occasional generation of close inbreeding without sig-
nificant harm. This strategy is consistent with their status as “jungle weeds” that provide a useful
understory when forest vegetation is damaged by extreme weather events.
8.1.4.2 Reproductive Abnormalities
Abnormalities of the physiological features of the reproductive process can influence the breeding
systems of plants. They cause a breakdown in the sequence of flower and fruit development and
cause marginal or substantial effects on the breeding system. Significant abnormalities can affect
the genetic composition of the subsequent generation. Parthenocarpy is defined as the formation of
fruit without fertilization; the phenomenon reduces the unreliability of fruit set and results in fruit
without seeds that consumers may prefer. There are two types: stimulative parthenocarpy, which
requires pollination before the fruit develops, and vegetative parthenocarpy, which requires no exter-
nal stimulus. Musa has vegetative parthenocarpy (Dodds and Simmonds, 1948; Ortiz and Vuylsteke,
1995a). Parthenocarpy is sometimes inaccurately used to describe fruit without mature seeds.
Ploidy affects the breeding system. It often results in reduced fertility from faulty chromosome
pairing at meiosis. However, polyploids, especially triploids, are very vigorous and produce few
seeds. Triploids are uniformly highly sterile and have an expectation of only 0.05% gametic fertil-
ity. Triploids can still be slightly fertile through chromosome restitution. Tetraploids are moderately
pollen fertile and make effective male parents. In the tetraploid breeding system, the tetraploids
must be very highly female sterile to minimize the risk of seediness in fruit for consumption. Male
sterility is not confined to triploid bananas, as Dumpe and Ortiz (1996) showed that half of their
diploid accessions were male sterile. Structural heterozygosity in the diploids is responsible for
moderate to nearly complete male sterility. Chromosome pairing is generally low and variable with
genotype and environment. The diploids ‘Sucrier’ (AA), ‘Bande’ (AA), and ‘Palembang’ (AA) are
nearly completely male sterile, while ‘Pisang Lilin’ (AA, allied to M. acuminata ssp. malaccensis)
is only 50% sterile.
Female sterility is less often reported as being selected against in cultivation. The implications of
female sterility are more important than that of male sterility since a totally female sterile cultivar
will not grow fruit unless it is parthenocarpic. However, flowers can still contribute to the breeding
system and overall fertility by augmenting the floral display and pollen production. Bananas are
probably the most conspicuously sterile of all cultivated fruits. Female meiosis of bananas shows a
similar behavior range that characterizes male meiosis, and there is no reason for the sexes to differ
in the mechanisms of meiosis. Irregularities notwithstanding, parthenocarpic bananas can produce

a few morphologically normal embryo sacs and fertilized embryo sacs with young endosperm when
suitably pollinated.
8.1.4.3 Female Sterility

Errors in development of seed can occur in the sporophyte, the gametophyte, and female × male
interactions. Sporophytic mutations cause defects in a discrete aspect of ovule development such as
initiation of integuments or development of the nucellus. They can also cause a defect in embryo sac
development by disrupting meiosis. Gametic mutations are determined by the haploid genotype of
the embryo sac and usually affect the number of mitotic divisions, that is, multiple functional egg
cells and embryos within a sac. Gametic mutations are not well understood or documented. The
gametophyte can contain up to five micropylar cells as either eggs or synergids, and seeds have
been obtained with twin, triplet, and quadruplet embryos formed by the fertilization of multiple egg
cells (Haig, 1990; Reiser and Fischer, 1993). Unlike pollen, the sporophyte surrounds the gameto-
phyte and maintains physical contact throughout its development. In Musa none of the sporophyte
problems such as the Arabidopsis sin1, ovm2, ovm3, and be11 mutations have been reported. The
literature does not mention any defects in ovule development. The published works, particularly of
Dodds (1945) and Dodds and Simmonds (1948), focused on problems of female × male interactions.
Dodds (1945), Dodds and Simmonds (1948), Simmonds (1962), and Shepherd (1999) have reported
detailed studies of the cytogenetics of wild and cultivated bananas.
Female infertility can be divided into two basic causes: those that inhibit the development of the
embryo sac and those that prevent its fertilization. In the first case, the two well-defined causes of ste-
rility in Musa spp. are multiple archesporia, in frequencies of 0–45%, and tetrahedral shaped tetrads
(Dodds, 1945; Dodds and Simmonds, 1948; Simmonds, 1962). In the second case, the sporophytic
cause of infertility is failure of the pollen tube to reach the ovule. This is apparently common despite
a great excess of pollen supplied to the stigma and the number of tubes in the style far exceeding that
necessary to fertilize all the ovules (Dodds, 1945; Shepherd, 1954, 1960b, 1999). In female cytol-
ogy, besides structural hybridity and deficiencies in chromosome homology, there are other causes
of sterility, such as abnormal spore development from spindle irregularities and deficient wall forma-
tion, sometimes leading to diploid or tetraploid spores from genetic restitution. Failures in embryo
sac development resulted in morphological errors and errors of polarity and failure of fertilization
and undefined derangement of postfertilization events (Dodds, 1945; Dodds and Simmonds, 1948;
Simmonds, 1962; Shepherd, 1999). Embryo sac failure accounts for most sterility in edible banana
clones. By far the greatest cause is gametogenic, ill-defined irregularities of embryo sac formation
caused by mismatching of chromosomes. Simmonds (1962) considered that all bananas, whether dip-
loid or triploid, suffer one or more of these defects, which may affect as many as 50% of ovules. Early
irregularities notwithstanding, edible diploids can produce a few percent of morphologically normal
embryo sacs whether genetically balanced or not (Fortescue and Turner, 2005b).
8.1.4.4 Female–Male Interaction

The first step in the female–male interaction is pollination, the transfer of pollen from male repro-
ductive structures to receptive stigmas. In the wild-seeded diploid bananas, pollination is essential
for fruit development. The result is mature fruits that contain a mass of hard black or brown seeds
surrounded by a scanty sweetish pulp. The parthenocarpic edible bananas develop as mass of pulp
without pollination and without seeds, and pollination has no detectable effect on the development
of the edible banana fruit (Simmonds, 1966).
8.1.4.5 Pollen–Pistil Interaction
The second step in the female–male interaction, after the arrival of pollen, is the extracellular
secretions of the stigma and pollen grains. The angiosperm stigma is covered by extracellular secre-
tions that may contain carbohydrates, proteins, enzymes, phenolics, and amino acids. The primary
recognition of species occurs at the stigma and its secretions. The pollen tube’s journey from stigma
to embryo sac is long. It grows through the stigma secretions and enters the stigmatic tissue between
the papilla cells. Its route through the stigma, style, and ovary is entirely confined to the extra-
cellular secretions produced by the cells of the transmitting tract (Sedgley and Griffin, 1989). In
angiosperms the embryo sac is generally mature and receptive at the time of anthesis and the pol-
len tube reaches the ovary and fertilizes the egg cell within days of pollination—a relatively short
proportion of the overall time span of the reproductive process. The male gametes are transferred
from the male to female organs via the pollen tubes, a highly complex process consisting of a num-
ber of sequential steps. At all stages of the process, there is potential for acceptance or rejection of
incompatible pollen.
8.1.4.6 Self-Incompatibility
Many plants have prezygotic self-incompatibility (SI), in which the growth of self-pollen tubes is
inhibited and fertilization prevented. Most prezygotic SI is a gametophytic mechanism with pollen
tube growth inhibited in the style. The tubes generally cease growth in the upper portion of the
style and have a characteristic appearance of swollen tips, terminal deposition of callous, and often
discharge their contents into the intercellular matrix. Thus self-pollen tubes show clear signs of
inhibited growth thought to be controlled by the stylar tissue. SI is an out-crossing mechanism that
reduces inbreeding and promotes heterozygosity in natural populations. A disadvantage is that it
reduces the amount of possible crosses available to the plant breeder.
Interspecific and intergeneric hybridizations are important in many plant breeding programs
where a combination of characteristics from different species is required. The ease of these hybrid-
izations varies greatly between species and genera. Research has concentrated on developing meth-
ods to overcome barriers. Methods of overcoming SI involve exploration of the physiology of the
pistil and the utilization of the recognition mechanism between pollen and pistil. Some techniques
employed for overcoming SI at the breeder’s disposal include bud pollination, temperature, and
hormone manipulation. The concentrations of glycoproteins in the style that cause incompatibility
increase immediately before anthesis. Immature flowers and old flowers produced near the end of
the flowering season have weaker SI control. This may be less relevant in Musa spp. where flower-
ing can occur at any time of the year. Temperature can affect SI. Thus, in almond and cherry the
optimum for selfing is lower than that for crossing, 15°C compared with 25°C. In contrast, high
temperatures in apple, 32–60°C, results in seed set following self-pollination presumably due to
denaturing of the glycoprotein. Similar effects are achieved by γ radiation. Applications of auxins,
gibberellins, boric acid, or succinic acid to the base of the pistil before pollination affect SI. The
female–male interaction is still a difficult area of research; the major events occur within the female
structures, making it difficult to locate the organs of interest. In the literature there has been much
work conducted on pollen germination and tube growth, though little specifically on bananas. Most
effort has been placed on the pollen–pistil interactions.
In Musa there are two events that could be interpreted as SI. In the seeded diploid, over half of
normal ovules are unfertilized, despite a great excess of pollen applied to the stigma and the tube
number at the base of the style far in excess of that necessary to fertilize all the ovules. There are fre-
quent failures of fertilization despite normal pollen tube growth. In ‘Gros Michel’ (AAA), erratic pol-
len tube growth contributed to sterility by delayed or slow but otherwise normal growth of the tubes
or abnormal growth manifested by arrest and swelling of pollen tube tip (Dodds, 1945; Shepherd,
1954, 1960a, 1960b). Secondly, in relation to apical bias in fertilization within the fruit, Shepherd
showed that the more fertile the banana fruit and the earlier the pollination, the less the bias.
8.2 Pollen Production, Viability, and Germination

For use as a male parent in conventional Musa breeding, the genotype must produce ample pol-
len that is viable, can germinate, and can fertilize the female gametophyte. A deficiency in any of
these requirements precludes selection, even if the plant possesses the characteristics required in
the progeny, such as resistance to disease. The amount of pollen can be assessed by counting and
expressing the results on a per anther basis (Krishnamoorthy and Kumar, 2005a, 2005b) or by
ranking the amount of pollen using an hedonic scale (0 = none, 6 = extremely high; Ssebuliba et al.,
2008). Pollen viability is often assessed using vital stains; the most common methods are the use of
nuclear and vital dyes such as acetocarmine glycerol jelly (Marks, 1954) and Alexander’s procedure
(Alexander, 1969). However, different stains do not always give the same answer either within or
across species (Rodriguez-Riano and Dafni, 2000) and so results are indicative, depending on the
stains used. In Musa there is not necessarily a significant correlation between viability of pollen
and its capacity to produce seed (r = 0.25, P = 0.36; Ortiz et al., 1998). Germination, expressed as
percent, is usually evaluated in vitro and may equal or more usually be less than the percent viabil-
ity indicated by vital stains (Rodriguez-Riano and Dafni, 2000). This may mean that the stains are
not indicative of the capacity of the pollen to germinate, or it may be because the germination is
expressed on the basis of the whole population rather than the proportion of viable pollen.
8.2.1 Pollen Production

Pollen production is influenced by genetic and environmental factors. In Musa, male meiosis is
essentially regular, and sterility in certain cultivars is caused by chromosome misbehavior at meio-
sis. Structural complexities met at meiosis may be overcome by genetic restitution in mitosis. In
essentially sterile triploids with very low levels of structural hybridity, genetic restitution could
account for pollen present at anthesis.
Musa pollen grains are almost visible to the naked eye and are rather sticky. The amount can
vary from 200 to more than 40,000 pollen grains per anther (Sathiamoorthy, 1994; Krishnamoorthy
and Kumar, 2005a). Sathiamoorthy (1994) presents data on pollen counts for a range of diploid,
triploid, and tetraploid banana genotypes grown in India. All genotypes produced pollen, at least
2,500 per anther, even cv ‘Matti,’ a tall edible diploid (AA) that is male sterile. Different cultivars
contain more viable pollen than others, either within or between genomes or groups. Between ploidy
groups, the diploids contained three times more pollen than the tetraploids and 11 times more than
the triploids. Krishnamoorthy and Kumar (2005b) measured the pollen production of 18 tetraploid
(AABB) banana hybrids in India. The number of pollen grains per anther ranged from 400 to
12,000. Most triploid accessions do not produce enough pollen for use in breeding programs. Low
pollen production in triploids is mostly attributed to meiotic abnormalities, which may be under
genetic control (A or B) and influenced by environment (Simmonds, 1966; Ortiz, 1995; Dumpe and
Ortiz, 1996; Ortiz et al., 1998).
To account for the variation in seed set observed during conventional cross-breeding of Musa,
many researchers have assessed the viability of pollen used in pollination. A reliable method of
detecting pollen viability and germination allows not only for increased efficiency in breeding but
raises the possibility of in vitro pollination of ovules, especially since banana breeding is hampered
by pre- and postzygotic barriers of incompatibility. Here, from the published literature, we sum-
marize data on the pollen viability of species, clones, and cultivars of Musa grown in Australia
(Fortescue and Turner, 2004), India (Sathiamoorthy and Madhava Rao, 1980; Sathiamoorthy, 1994),
tropical America and the Caribbean (Landa et al., 1999; Perea Dallos, 1998; Roman et al., 2004;
Soares et al., 2008), and Africa (Dumpe and Ortiz, 1996; Ortiz et al., 1998; Adeleke et al., 2004;
Nyine and Pillay, 2007; Ssebuliba et al., 2008). The data on pollen viability were sorted into ploidy
and genomic groups (Table 8.1).
Diploids produce pollen with high viability (80–90%), compared with triploids (40–49%), and
these differences are significant at P = 0.05 level of probability (Table 8.1). The pollen viability of
tetraploids was variable but the numbers available were too small to make a comparison with other
ploidy levels. Within the diploids and triploids, there was no significant effect of the proportion
of A or B genome on pollen viability. A feature of pollen viability is the large range within each
Table 8.1
Pollen Viability in Musa spp. in Relation to Ploidy and Genomic Constitution
Genome Viability, %
Ploidy N Mean SE Range
Diploid AA 81 81a 2 28–100
BB 12 90a 5 44–100
Diploid total 93 83A 2
Triploids AAA 39 44b 5 3–92
AAB 15 37b 9 0–91
ABB 23 49b 6 5–90
Triploid total 77 44B 3
Tetraploids AAAA 1 31
AAAB 4 56 16 28–94
AABB 1 14
ABBB 2 33 4 30–37
Tetraploid total 8 42B 10
Note: Mean values of genomes followed by the same lowercase letter are not significantly different by t-test at P = 0.05.
Ploidy means followed by the same uppercase letter are not significantly different (P = 0.05). SE = standard error. The
tetraploids had too few data and were excluded from the t-test to compare genomes within ploidy levels.
Source: Data from Sathiamoorthy, S. and V.N. Madhava Rao, 1980, Pollen production in relation to genome and ploidy in
banana clones, In: National seminar on banana production technology, C.R. Muthukrishnan, and J.B.M.Md. Abdul
Khader, eds., 46–49, Chennai, India, Tamil Nadu Agricultural University; Sathiamoorthy, S., 1994, Musa improve-
ment in India, In: The improvement and testing of Musa: A global partnership, D.R. Jones, ed., 188–200, Montpellier,
France, INIBAP; Dumpe, B.B. and R. Ortiz, 1996, Apparent male fertility in Musa germplasm, HortScience, 31,
1019–1022; Ortiz, R., F. Ulburghs, and J.U. Okoro, 1998, Seasonal variation of apparent male fertility and 2n pol-
len production in plantain and banana, HortScience, 33, 146–148; Landa, R., A. Rayas, T. Ramirez, J. Ventura, J.
Albert, and O. Roca, 1999, Study of the pollen fertility in the INIVIT genetic improvement programme, InfoMusa,
8(1), 27; Perea Dallos, M., 1998, Pollen and anther culture in Musa spp., Acta Hort., 490, 493–497; Adeleke,
M.T.V., M. Pillay, and B.E. Okoli, 2004, Relationship between meiotic irregularities and fertility in diploid and
triploid Musa L., Cytologia, 69, 387–393; Fortescue, J.A. and D.W. Turner, 2004, Pollen fertility in Musa: Viability
in cultivars grown in Southern Australia, Aust. J. Agric. Res., 55, 1085–1091; Roman, M.I., M. Alonso, X. Xiques,
C. Gonzalez, and I. Sanchez, 2004, Estudio del numero cromosomico y la fertilidad del pollen especies y clones
diploids de platano fruta (Musa ssp.), Cultivos Tropicales, 25, 71–73; Nyine, M. and M. Pillay, 2007, Banana nectar
as a medium for testing pollen viability and germination in Musa, Afr. J. Biotechnol., 6, 1175–1180; Soares, T.L.,
S.O. Silva, M.A.P.C. Costa, J.A. Santos-Serejo, A.S. Souza, L.S.M. Lino, et al., 2008, In vitro germination and
viability of pollen grains of banana diploids, Crop Breed. Appl. Biotechnol., 8, 111–118; Ssebuliba, R.N., A.
Tenkouano, and M. Pillay, 2008, Male fertility and occurrence of 2n gametes in East African Highland bananas
(Musa ssp.), Euphytica, 164, 53–62.
genomic group (Table 8.1), suggesting that some genotypes contain high pollen viability irrespec-
tive of their ploidy level. However, viability below 30% is more common among triploid genotypes
than diploids.
This broad picture contains exceptions when applied at the local level. For example, in a range
of genotypes grown across southern Australia, diploids had 88% viable pollen compared with 29%
for tetraploids. Tetraploid cultivars contained three times more viable pollen than the triploids
AAA (9%), ABB (10%), and four times more than AAB cultivars (6%) (Fortescue and Turner,
2004). Similar results were found previously with cultivars grown in India where the diploids had
50 to 66% viable pollen, triploids had 21–29%, and the tetraploids had 28% (Sathiamoorthy and
Madhava Rao, 1980; Sathiamoorthy, 1994). Adeleke et al. (2004) found that the viability of pol-
len in homogenomic triploids (AAA) was 30–48%, while in heterogenomic triploids (AAB, ABB)
it was much less and averaged 8–30%. Musa balbisiana (diploid, section Eumusa) and M. ornata
(section Rhodochlamys) have more viable pollen than M. acuminata (diploid, section Eumusa).
Within triploids the cv ‘Gros Michel’ has more viable pollen than its dwarf mutant cv ‘Highgate’
(AAA) but similar to the Cavendish subgroup (AAA). Shepherd (1960a, 1960b) found that among
M. acuminata, ssp. burmannica had much more viable pollen than ssp. malaccensis.
Flower age, expressed as node position in relation to anthesis, can affect the viability of pol-
len. The day between pre-anthesis and anthesis can see a decrease of 7% per node in viable pollen
(Fortescue and Turner, 2004). Shepherd (1960a) compared pollination in the afternoon of the day
before flower opening with the morning of the day after flower opening. Late pollination always
resulted in lower yields of seed. Ssebuliba et al. (2008) demonstrated a significant relationship
between the node from which the pollen was obtained and variation in pollen viability. All nodes
were at anthesis and all collection was done early in the morning. Pollen viability was highest in
the mid nodes 30 to 39 of the male rachis, especially in the triploids and certain diploids. Therefore,
choice of pollen source should consider the position of the node from which pollen grains are
harvested as well as time of day and day of anthesis. Genotype, node number, and time of day do
not account for all the variation seen by different authors, especially when the same cultivar gives
different results in different locations. It is known that pollen viability is influenced by temperature
and humidity (Ortiz et al., 1998). Variation in pollen viability caused by organ ontogeny, season, or
genotype will influence the practical aspects of pollen storage and use in a breeding scheme.
8.2.2 Polyspory
Polyspory, in this context, is the production of pollen grains of different ploidy levels within the
same anther. This is a feature of plants that have a hybrid origin and is linked to the production of
univalents during pairing of chromosomes in meiosis (Penland, 1923; Griffiths et al., 2000; Adeleke
et al., 2004). The different levels of ploidy in pollen are usually determined by measuring the size
of the pollen grains: Larger grains have a higher ploidy level than smaller grains.
Sathiamoorthy (1994) found that in 8 diploid cultivars, 7 showed polyspory with 39±4% haploid
pollen and 11±3% diploid. Among 17 triploid cultivars, 16 showed polyspory with 16±2% pol-
len being haploid, 15±3% being diploid, and 5±2% being tetraploid. Among 4 tetraploid cultivars,
3 showed polyspory with 7±2% pollen being haploid and 23±4% pollen being diploid. The two
seeded species, M. acuminata and M. balbisiana, produced only haploid pollen (66±4%). In con-
trast, Shepherd (1999) compared pollen samples from 11 plants from the one cross, of which four
produced entirely haploid pollen with 95% viability. Only one uncommonly large grain was seen
and no irregular ones except in two plants where the pollen was extremely variable in size represent-
ing haploid, diploid, and tetraploid pollen.
Gametes with the sporophytic chromosome number are known as 2n gametes. The production of
2n gametes is uncontrolled and fairly rare in diploid clones (Dodds, 1943). However, Sathiamoorthy
(1994) found 11% of 2n gametes in seven diploid banana genotypes grown in India. Ortiz (1997)
examined 2n pollen production in a range of Musa spp. and genotypes in Africa and found that
those genotypes that produced 2n pollen were also parthenocarpic. The formation of 2n pollen has
been described by Simmonds (1962), Ortiz (1995), and Shepherd (1999). Diploid spore formation
results from either the failure of the first meiosis division followed by division restitution mecha-
nisms or the failure of the second division following a successful first division. The usual mode of
2n spore production in bananas is due to second division restitution. Ortiz (1997) found that not all
genotypes can produce 2n pollen and that at least one locus may be involved in the inheritance of
2n pollen production in Musa.
Pollen diameter is a function of genome size since both the nucleus and cytoplasm increase as
chromosome number increases. Diploid or 2n pollen is approximately 25% larger than haploid (n)
pollen, and the diameter of 3n and 4n pollen is considered to be 44 and 59% greater than that of hap-
loid pollen, respectively (Ssebuliba et al., 2008). In Musa, haploid pollen diameter ranges between
Table 8.2
Proportion of Pollen with Different Levels of Ploidy from AA or AAA Cultivars
Diploids, AA East African Highland Bananas, AAA
Pollen Ploidy Mean SE Range Mean SE Range
n 96.0 2.0 86–100 82.0 2.0 68–95
2n 2.9 1.5 0–9 14.0 1.8 4–24
3n 0.8 0.7 0–4 2.5 0.6 0–7
Note: SE = standard error.

Source: Data from Ssebuliba, R.N., A. Tenkouano, and M. Pillay, 2008, Male fertility and occurrence of 2n gametes in East
African Highland bananas (Musa ssp.), Euphytica, 164, 53–62.
78 and 128 µm (Shepherd, 1999; Ortiz et al., 1998; Ssebuliba et al., 2008). In the diploid species M.
balbisiana, the haploid pollen has a diameter of 94±2 µm compared with 105±1 µm for pollen of M.
acuminata. There are also slight differences in mean pollen diameter within ploidy level between
landraces and hybrids: ABB cooking bananas have a slightly smaller pollen than AAB landraces.
Darlington (1937) indicated than pollen with a diameter of 129 µm or less was haploid while the
average diameter of 2n pollen was 148 microns. In Musa, pollen grains with a diameter of 160 µm
are generally considered to be 2n pollen grains.
Triploid genotypes produce less haploid pollen than diploid genotypes (Table 8.2). The range
of pollen that is haploid is large and so there is variation among genotypes within ploidy levels. In
East African Highland bananas, the proportions of haploid (n), diploid (2n), and triploid (3n) pollen
grains differ greatly, the proportion of haploid pollen being significantly higher than that of diploid
and triploid pollen (Ssebuliba et al., 2008). Haploid pollen grains make up 95% of the population in
an anther; diploid pollen constitutes 3–5%, and triploid pollen from 0–1% of the population. This
is consistent with the findings of Ortiz (1997) that the frequency of genotypes that can produce 2n
pollen was 22% in diploid M. acuminata, 56% in AAA, 17% in AAB, and 14% in ABB triploids.
The higher frequency of 2n pollen production in AAA triploids compared with diploids is consistent
with the occurrence of sexual polyploidization in bananas (Ortiz, 1997).
8.2.3 Pollen Germination

There is much information in the broader plant literature on pollen germination and pollen tube
growth; however, there is still little information available for banana. Pollen grains need to be alive
when they reach the stigma (viable), be able to germinate and grow towards the ovule (functional
competence), and be able to complete double fertilization to produce a healthy zygote. The vital
stains used to indicate viability cannot determine which pollen is functionally competent. This is
best observed by pollinating fertile female flowers. Krishnamoorthy and Kumar (2005b) measured
pollen germination, in vitro, of 18 tetraploid (AABB) banana hybrids in India. Germination ranged
from 4 to 17%. There was no relationship between pollen production and germination and so the
amount of germinable pollen produced by a genotype could not be predicted from the amount pro-
duced. In diploids grown in Uganda, pollen germination was 84% (Nyine and Pillay, 2007).
The method of in vitro germination uses various combinations of sucrose and micronutri-
ents; however, Nyine and Pillay (2007) suggested that better results could be obtained using
diluted banana nectar harvested from male flowers from newly opened bracts as the main nutri-
tive source. Pollen germination increased from 47% (range 17–50%) with sucrose to 84% (range
of 60–97%) with nectar. Pollen germination was better for M. acuminata (83%) than M. balbi-
siana (44%). Soares et al. (2008) achieved germination rates of 16–20% with a range of 2–90%
on regular pollen media. Banana pollen germinates readily, many within 1 hour from the start
of incubation. The main factors that affect germination are the genotype, culture medium and
incubation regime, sampling time, physiological development, and flower age. To a lesser extent,
the nutritional state of the parent, temperature, humidity, and photoperiod may be contributing
factors (Soares et al., 2008).
8.3 Seed Production
8.3.1 Seed Morphology and Anatomy
The family Musaceae contains three genera—Musa, Ensete, and Musella—although the generic
status of Musella is not universally accepted (De Langhe et al., 2009). All seeds of the Musaceae
contain two chambers, similar to the spiral gingers—Costus sp. (Zingiberaceae; Humphrey,
1896). The larger, proximal chamber contains the embryo and endosperm and the smaller, a
chalazal mass. The outer integument is thick, has a silicified exotesta (Graven et al., 1996) and
internally, the middle layers are sclerotic cells. The innermost layer of the outer integument is an
endotesta of thickened cells with a U shape. The tegmen or inner integument has two layers and a
thick cuticle on its inner surface. The cuticle originates partly from the inner layer of the tegument
and partly from the nucellus (Graven et al., 1996). The outer integument encloses the whole seed
while the inner integument encloses the large chamber (McGahan, 1961). Within the large cham-
ber, the embryo is wedged into the micropylar collar and its cotyledonary haustorium extends
towards the center of the seed and is surrounded by endosperm. The nucellar pad lies between
the embryo and the micropylar plug. Within the micropylar plug, which forms an operculum, the
micropyle can be detected. The external surface of the micropylar plug becomes the hilum. The
micropylar plug, which fills the only opening in the seed, is easily removed, at least in the seed
of Musa balbisiana studied by McGahan (1961). The chalazal mass protrudes towards the center
of the seed, creating the doughnut shape of the endosperm. The aril is trichomatous and in the
ovular stage surrounds the seed coat. Later it is reduced and may not always be detected on the
mature seed (Humphrey, 1896).
Broadly, Ensete has large smooth seeds with a rimmed hylar depression. Musella and Musa have
small seeds but the coats have a different texture. Seeds of Musella are smooth compared with the
rough coat of Musa (Graven et al., 1996). De Langhe (2009) presents a key to genera, species, sec-
tions and some subspecies based on seed shape, size, and the nature of the surface of the seed coat.
The seeds of the Musaceae are either cylindrical (for example, Musa violascens, section Callimusa)
or subglobular, angular, and flattened (section Eumusa) (Chin, 1996). Seed weight, adjusted to 10%
water content, varies from 27 mg for Musa ornata (section Rhodochlamys) to 71 mg for Musa vio-
lascens. Seeds of Musa acuminata, as recorded by Chin (1996), weigh about 45 mg. The seeds of
Ensete glauca are about 10 to 22 times heavier than the seeds of Musa spp. Wattanachaiyingcharoen
(1990) measured seed weight within a population of more than 2,000 seeds of an undescribed Musa
acuminata ssp. (similar to microcarpa; Turner and Hunt, 1984). Mean seed weight was 55 to 56 mg
over two seasons but the range was from 10 to 72 mg. Seed weight was not normally distributed,
with both skewness (mode > mean) and kurtosis (peaked > normal curve) being significant (p =
0.05). Seed size can vary significantly within a population of seeds of Musa sp.
The main features of mature dried seed of Musa balbisiana (McGahan, 1961) are similar to those
described by Humphrey (1896) for M. ornata. In a number of studies, summarized by McGahan
(1961), although different species of Musaceae were examined, the characteristics of the seed of
the family are clear. Graven et al. (1996) examined seeds of several Musa species (M. acumi-
nata, M. balbisiana, M. mannii, and M. paradisiaca [possibly French Plantain, AAB triploid clone,
Simmonds 1966], M. textilis, M. velutina), Ensete (E. ventricosum, E. glaucum), and Musella lasio-
carpa. They concluded that the seed coats were very similar in structure across the three genera and
so they presented only their data for Musa.
8.3.2 The Seed Coat

Musaceae grow in a range of environments in the tropics and subtropics that experience extremes
of water supply and a range of temperatures, although few below 0°C. In the wild species, the seed
carries the plant propagule, the embryo, through time and space. The complexity of the seed coat
of Musa was identified more than a century ago (Humphrey, 1896) and has been studied by numer-
ous people since, including a detailed study of M. balbisiana by McGahan (1961). More recently,
Graven et al. (1996) examined the structure and macromolecular properties of the testa of Musa,
Ensete, and Musella with the objective of determining the features that contributed to its ability to
protect the embryo as well as allow germination.
Under selection pressure, the seed coat has characteristics that increase the probability of sur-
vival of the new propagule and that allow germination. Graven et al. (1996) consider that survival
and germination place competing demands on the seed coat. In Musa, the fruit of wild species
are usually eaten by animals, mainly monkeys, birds, and bats (Simmonds, 1959, 1962). The seed
itself may not have appeal for animals, but the sweet flesh of the surrounding pulp tissue would
be attractive, thus consumption of seeds may be accidental. Graven et al. (1996) thought that the
presence of silica on the outer seed coat may assist the seed to pass through the alimentary tract of
animals. Simmonds (1959) fed ripe, seeded fruit of M. acuminata and M. balbisiana to adult pigs
and domestic fowl. The gastric acid of a pig has a pH of 1–2 (Manners, 1976). Passage through the
gut destroyed 90–96% of seeds fed to pigs and 99% of seed fed to fowl. Simmonds (1962) concluded
that survival of seed in the alimentary tract was possible, but not good. Passage of seed through the
gut of birds has different effects, depending on the species of bird and the species of seed (Traveset
et al., 2001), and so the results of Simmonds (1959) are indicative only.
Acid is used experimentally to scarify seed. Scarification with acid destroyed most seeds of M.
acuminata and M. balbisiana (Simmonds, 1952), more than halved the germination of M. balbisi-
ana (Stotzky et al., 1962), and halved the already low (18%) germination of Ensete seed (Tesfaye,
1992). A pH of 3.7 in the medium reduced germination of seed of M. balbisiana by 88%, compared
with germination at pH 5.2 to 6.3 (Perry and Boodley, 1980). Seeds of Musaceae are not particularly
resistant to acids, despite the presence of silica in the exotesta that might assist them to survive such
conditions (Graven et al., 1996). The animal gut applies high selection pressure to seed and the fact
that many don’t survive the experience suggests that over time, a significant proportion of seed are
dispersed by other means.
For germination, water needs to enter the seed and gas exchange needs to occur. Graven et
al. (1996) suggested that the silica in the exotesta could restrict water loss if the silica gel was
hydrated. They also suggest that the thick cuticle on the innermost side of the seed coat may resist
the diffusion of water, in addition to the outer layers. For interpreting experiments on scarifica-
tion, for example those of Stotzky et al. (1962), it is worthwhile distinguishing those techniques
that affect mainly the exotesta (such as acids and alkali; Stotzky et al., 1962) and those that pen-
etrate the inner cuticle (chipping; Stotzky et al., 1962), allowing more rapid access of water to
the endosperm and embryo. Chipping increased germination of Musa balbisiana from 0 to 81%,
whereas the other scarification techniques increased germination from 0 to 34% at most (Stotzky
et al., 1962). These data imply that the inner cuticle is a significant barrier for the flow of water
into the seed.
Removing the micropylar cap did not increase germination, and so it is unlikely that water penetrates
at this part of the seed, at least in M. balbisiana. Removal of the chalazal mass, which lies between the
inner and outer integuments, increased germination from 0 to 16%. Wattanachaiyingcharoen (1990)
measured imbibition of seeds of M. acuminata ssp. by weighing seed sequentially over several days. He
compared intact seed with that which had either been scarified using sandpaper or pierced. The scari-
fication ruptured some of the exotesta and piercing created a hole in the outer and inner integuments,
allowing water to enter the endosperm. Scarifying the seed coat did not significantly (p = 0.05) increase
the rate of water absorption or the final amount absorbed (23.4 mg/seed; Figure 8.9), but piercing the
45
Increase in seed weight as % of initial weight

40
35
30
25
Control
20
Scarified
15
Pierced
10
0
0 20 40 60 80 100 120
Time of soaking, h
Figure 8.9 Imbibition of seed of Musa acuminata ssp. that is either intact (control) or has been scarified or
pierced. Scarification ruptured the exotesta, piercing penetrated the inner cuticle allowing access of water to
the endosperm. The rate of increase in water content for the pierced seeds and the maximum weight reached
are significantly greater than the values for the intact or scarified seed, which do not differ (p = 0.05). (Data
from Wattanachaiyingcharoen, D., 1990, Viability, germination and dormancy of banana seed (Musa acumi-
nata ssp.), master’s thesis, The University of Western Australia.)
seed did increase the rate and the amount of water absorbed (29.6 mg/seed; Wattanachaiyingcharoen,
1990). Over the time span of this experiment (8 days) germination had not commenced.
8.3.3 Pollinators
Pollinators place selection pressure on the pollen source. Therefore, if there is variation between
pollinators within Musa, there will be differences in plant form and function (Inito et al., 1991).
Apart from birds and bats, pollinators include tree shrews (Tupaia sp.) and bees (Trigona sp.), but
ants, butterflies, flies, and spider-hunters (Arachnothera sp.) were unimportant in the experiments
conducted by Nur (1976). Musella is pollinated by bumblebees (Bombus sp.), honeybees (Apis sp.),
and wasps (Vespa sp.). Indeed, the insect pollination of Musella has contributed to its reproductive
isolation (Liu, Kress, et al., 2002). Features of Musa that favor pollination are: female flowers have
a longer flowering time than male flowers; the banana produces flowers all year, meaning that ver-
tebrate pollinators will find it attractive; and there are more male flowers than female flowers in a
population of Musa plants.
Itino et al. (1991) studied the bat and bird pollinators of Musa acuminata ssp. halabanensis (chi-
ropterophily) and Musa salaccensis (ornithophily) in the field. The timing of anthesis and the peaks
in nectar production by flowers are consistent with pollinators being present during the day (birds)
or at night (bats). Itino et al. (1991) concluded that seed set in these two species was pollinator lim-
ited and that this indicated competition among nectar-producing plants for the available vertebrate
pollinators. Musa itinerans in southern China is pollinated equally by birds and bats (Liu, Li, et al.,
2002). Musa itinerans has a peak of nectar production near midday and midnight but most flower
opening occurs near dawn and sunset. The total resource available to pollinators will be a function
of the rate of nectar production and the number of flowers open. Assuming that flowers stay open
for 1 day, the availability of the resource throughout the day coincides with the number of visits
from birds (day) or bats (night). The data of Liu, Li, et al. (2002) show that the greatest amount of
resource is available from 8 a.m. to 12 noon and from 10 p.m. to 2 a.m. This matches the timing of
most visits by birds or bats. Male flowers show a similar pattern of resource availability throughout
the day as do female flowers, but they provide less resource at the peak times. This is reflected in a
lower frequency of visits by pollinators, compared with female flowers.
Stephens and Tyson (1975) recorded the visits of nectar bats to bunches of Musa spp. (AAA,
Cavendish subgroup) cv ‘Valery’ in Panama. Almost all bunches within the large commercial plan-
tation were located by bats, as indicated by scratches on the young fruit. Bats did not visit bunches
in the first few nights of their opening, and so most of the scratches appear on hands towards the
bunch apex. The cv ‘Valery’ is sterile but if bats visited the bunches of fertile seeded Musa species
in the same way, then this may disadvantage the first few hands on a bunch in terms of pollination
and seed production.
Despite structural self-incompatibility, Nur (1976) observed that fruit of Musa velutina (section
Rhodochlamys) set seed even though all pollinators were excluded from the inflorescence. He con-
cluded that M. velutina was self-pollinating as it had hermaphrodite basal flowers, or some other
mechanism was responsible for seed set.
8.3.4 Factors Affecting Seed Set

For breeding, seed production is important because it provides the population from which desirable
progeny can be selected. On the other hand, the advantage of banana fruit for human consumption
is that they are sterile and parthenocarpic. An ongoing difficulty in breeding of Musa has been the
low seed set, and several studies have had the objective of identifying the factors contributing to
seed production. Many of these studies have been empirical (Table 8.3). Looking deeper into the
problem, Shepherd (1954) investigated the significance of pollen tube growth in contributing to the
variation in seed fertility. More recently Ortiz and Vuylsteke (1995b) gathered indirect evidence
that seasonal conditions affect the ploidy of the gametes of the female parents in French Plantains.
Ortiz (1997) obtained direct evidence that seasonal conditions affected the ploidy of pollen derived
from ‘Pisang Lilin.’ The value of these studies for breeding is that since seed set is very low, even
a small increase in seed production can significantly increase the population of progeny available
for selection.
8.3.4.1 Maximum Seed Set

In Jamaica, the ‘Gros Michel’ (AAA) × ‘Pisang Lilin’ (AA) crosses generally produced only 1 to 3
seeds per bunch but the maximum recorded was 60 seeds/bunch (Shepherd, 1954). In Nigeria, the
‘Bobby Tannap’ AAB × ‘Calcutta 4’ (AA derived from M. acuminata ssp. burmannicoides) cross
produces about 22 seeds per bunch, but has produced 219 seeds/bunch (Swennen et al., 1991). The
maximum values can be taken as indicating what is possible, and lower numbers of seeds encourage
the search for reasons for reduced seed fertility.
8.3.4.2 Bunch and Fruit Factors

Most cultivars of edible triploid bananas are largely male-sterile, but almost all will set seed if
pollinated with suitable pollen (Shepherd, 1954). If ovaries of cv ‘Gros Michel’ (AAA) are hand-
pollinated with material from the edible diploid ‘Pisang Lilin’ (AA), then bunches containing thin
fruit at maturity are more likely to have more seed than bunches containing well-developed fruits.
Bunches with mature fruit of 10 cm circumference had 1.7 seeds per bunch and those with a cir-
cumference of 15 cm or more had only 0.6 seeds per bunch. Shepherd (1954) thought this difference
was associated with the impact of growth regulators that suppressed seed fertility but promoted
growth in the larger fruit. In these experiments, differences in fruit diameter were associated with
differences in seasonal conditions and so the impact of environmental factors on seed fertility may
be confounded with the effects of fruit size.
In hand-pollinated bunches of cv ‘Gros Michel’ (AAA) pollinated with ‘Pisang Lilin’ (AA),
the proximal hands of female flowers are more fertile than the distal hands. In bunches that had
Table 8.3
Plant and Environmental Factors That Affect Seed Set in Musa spp.
Factor Magnitude of Effect Ref.
Bunch size Small bunches, 6–8 hands, produce fewer seeds (1.3 seeds/100 fruit) than 2
large bunches, 12–13 hands (5.2 seeds/100 fruit), cv ‘Gros Michel’
Position of hands within a 66–79% of seed within a bunch are located in the three proximal hands in 1
bunch bunches with 7–8 hands, cv ‘Gros Michel’
42–45% of seed in a bunch located in the three proximal hands, cvv ‘Bobby 8
Tannap,’ ‘Obino l’Ewai’
Fruit size Thin fruit at maturity produce more seeds (1.7 seeds/bunch) than full fruit 1
(0.6 seeds/bunch), cv ‘Gros Michel’
Position within the fruit In fruit 150 mm long, seeds tend to be located 30 mm from flower end. In 1
fruit 230 mm long, seeds tend to be 50 mm from flower end, cv ‘Gros
Michel.’
Style length Increasing length of style negatively correlated with number of seed set/fruit, 4
78 genotypes of East African Highland bananas
Style receptivity Pollination at stage 3 of stigma development was correlated with the greatest 5
number of seeds per hand, five genotypes of East African Highland bananas
Season of pollination across Consistent seasonal variation in seed fertility at two locations in Jamaica 1
locations (range 0–3 seeds/bunch), one location having high seed fertility from
November to February (4–6 seeds/bunch) compared with 1 seed/bunch at
other locations, cv ‘Gros Michel’
Season of pollination at one July better than September/October or January/February in Jamaica, cv ‘Gros 3
location Michel’
Bunches pollinated in February contain 65–70 seeds but at other times of the 6
year the number falls to 2–30 seeds, cv ‘Bobby Tannap’ (French Plantain)
with pollen from ‘Calcutta 4’
Time of day of pollination Morning pollination (6–10 a.m.) increased seed set by 12–100% compared 3
with afternoon (1 p.m.), cv ‘Gros Michel’
Pollination at 7 a.m. better (1.95 seeds/bunch) than later in the day (0.6–0.8 1
seeds/bunch), cv ‘Gros Michel’
Altitude Seeds occur in fruit of M. fehi grown at 900 to 1100 m in Tahiti, but not in the 7
lowlands
Fields at one location 1.83 seeds/bunch in one field compared with 6.82 seeds/bunch in another 3
field at the one location, cv ‘Gros Michel’
Soil fertility More seeds/bunch in section of field with higher P and K than other sections, 1
cv ‘Gros Michel’
Pruning of suckers No pruning produced 73% more seed/bunch than pruning to one sucker per 1
mat, cv ‘Gros Michel’
Sources: Data from (1) Shepherd, K., 1954, Seed fertility of the Gros Michel banana in Jamaica, J. Hort. Sci., 29, 1–11. (2)
Shepherd, K. 1960a. Seed fertility of edible bananas. J. Hort. Sci. 35:6−20. (3) Shepherd, K. 1960b. Seed fertility
of ‘Gros Michel’ bananas. Trop. Agric., Trinidad 37:211−221. (4) Ssebuliba, R.N., P. Rubaihayo, A. Tenkouano, D.
Makumbi, D. Talengera, and M. Magambo, 2005, Genetic diversity among East African Highland bananas for
female fertility, Afr. Crop Sci. J., 13, 13–26. (5) Ssebuliba, R.N., M. Magambo, D. Talengera, D. Makumbi, A.
Tenkouano, P. Rubaihayo, et al., 2006, Biological factors affecting seed production in East African Highland
bananas, J. Crop Improvement, 16:67–79. (6) Swennen, R., D. Vuylsteke, and K. De Smet, 1991, Season dependent
seed set in plantain, Banana Newslett., 14, 35–36. (7) Baker, J.G., 1893, A synopsis of the genera and species of
Museae, Ann. Bot., 7, 189–222. (8) Swennen, R. and D. Vuylsteke, 1990, Aspects of plantain breeding at IITA, in:
Sigatoka leaf spot diseases of bananas, R.A. Fullerton and R.H. Stover, eds., 252–266, Montpellier, INIBAP.
eight hands and were from several locations, 67–79% of the seed was in the proximal three hands
(Shepherd, 1954). This pattern of seed distribution within the bunch differs from that in the French
Plantain cvv ‘Bobby Tannap’ (AAB) and ‘Obino l’Ewai’ (AAB), pollinated with Calcutta 4 (AA),
where only 42 to 45% of the seeds in a bunch occur in the three proximal hands (Swennen and
Vuylsteke, 1990). Within fruit of cv ‘Gros Michel’ (AAA) that are pollinated by hand, seeds may
set as close to the stylar end of the fruit as 13 mm or as distant as 250 mm. However, the mean
distance is within 30 to 58 mm from the stylar end and the distance tends to increase with fruit size
(1.8 mm deeper into the fruit for each 10 mm increase in fruit length). These observations caused
Shepherd (1954) to examine pollen tube growth. Penetration of the style was assumed to take 1
hour and subsequent growth 3 mm h–1, based on observations on M. acuminata. The tubes of pol-
len from cv ‘Pisang Lilin’ (AA edible diploid allied to M. acuminata ssp. malaccensis; Simmonds,
1966) applied by hand to ovaries of cv ‘Gros Michel’ generally grew at a slower rate than might be
expected from the rate observed in Musa acuminata. No growth occurred in 26% of ovaries. Pollen
tube growth varied greatly between hands and between fruit (ovaries) within hands. In some hands
pollen grew in all fruit, while in others pollen tubes grew in only one or two fruit. The data avail-
able on pollen tube growth seem consistent with the observed distribution of seeds within fruit and
the low seed fertility of cv ‘Gros Michel’ (AAA) (Shepherd, 1954). There is a need to discover why
so many pollen tubes fail to grow, especially in the light of recent knowledge about this process
(Higashiyama and Hamamura, 2008).
8.3.4.3 Environmental Factors
Environmental conditions influence seed fertility in Musa. Baker (1893) observed that M. fehi
(Australimusa) in Tahiti is seedless when grown at low altitudes but contains seeds when grown at
900–1100 m. A number of environmental components change with altitude, such as temperature,
humidity, and cloudiness but this observation suggests a strong environmental component in the
fertility system of M. fehi. Alternatively, pollinators may be absent at low altitudes. Firmer evidence
supporting the influence of environment on the success of pollination comes from hand crosses
between ‘Gros Michel’ (AAA) with pollen from ‘Pisang Lilin’ (AA, parthenocarpic) from different
locations in Jamaica (Shepherd, 1954). At an exposed coastal site, 80% of pollinated bunches con-
tained fruit with seeds, whereas at a sheltered site in a nearby valley, only 20% of pollinated bunches
contained seeds. Maintaining humidity around bunches immediately after pollination increased the
proportion of fertile bunches from 30 to 48%, but had no significant effect on the number of seeds
per bunch (Shepherd, 1954). Despite large variation in seediness of bunches between localities
and seasons, Shepherd was unable to detect significant correlations with temperature, rainfall, or
humidity. Therefore, standard meteorological data could not be used to predict bunch fertility, under
these conditions.
A stronger link between environment and seed fertility has been reported by Swennen and
Vuylsteke (1990) and Swennen et al. (1991, 1992). Swennen et al. (1991) found a very distinct
effect, repeated over 2 years, of month of pollination on the seed fertility of French Plantain cv
‘Bobby Tannap’ (AAB) pollinated with ‘Calcutta 4’ (AA, M. acuminata ssp. burmannicoides).
Bunches pollinated in February contained 65–70 seeds per bunch. Bunches pollinated from
March to June produced fewer than 10 seeds/bunch and from July to January seed number var-
ied from 15 to 35 seeds/bunch. These data were further collected for 5 years (Figure 8.10) and
the data analyzed to determine any correlations between meteorological conditions and seed set.
For the AAB plantains, variations in humidity accounted for 32–51% of the variation in seed
set. Over the same time period, variations in rainfall, solar radiation, temperature, and humid-
ity accounted for 63% of the variation in seed set in the AAA and ABB bananas (Ortiz and
Vuylsteke, 1995b). Similarly, Ssebuliba et al. (2009) found that there were two major seed-set
periods in East African Highland banana within a year with a major peak occurring in March–
April and a minor peak occurring in September. Optimum seed set appeared to be related to
climatic variables.
60.0
50.0
Seed set per bunch
40.0
30.0
Bobby Tannap × Calcutta 4
20.0
Obino l’Ewai × Calcutta 4
10.0
0.0
0 1 2 3 4 5 6 7 8 9 10 11 12
Month of pollination
Figure 8.10 Seed set per bunch in two French Plantain (AAB) cultivars grown at Onne, Nigeria, and polli-
nated every day, but mean shown for each month, with pollen from ‘Calcutta 4’ (derived from Musa acuminata
ssp. burmannicoides), a wild-seeded diploid. The rainy season is from March to December. Vertical bars are
standard errors across years, n = 5. (Data from Ortiz, R. and D. Vuylsteke, 1995, Factors influencing seed set
in triploid Musa spp. L. and production of euploid hybrids, Ann. Bot., 75, 151–155.)
While these correlations are useful, they are specific for location and cultivar, or group of culti-
vars. The process of pollination and seed set is complex and season may influence any one or more
components. Deeper studies are required to identify which components are sensitive to environ-
mental perturbations. An example of this is the study of 2n pollen production by Ortiz (1997) who
found that the proportion of 2n pollen produced by ‘Pisang Lilin’ throughout the year in Nigeria
was positively correlated with the amount of solar radiation (r 2 = 0.72). Nonetheless, the empirical
relationships tell us that planting dates of female parents can be managed so that seed set can be
maximized, thus increasing the efficiency of breeding Musa spp. (Swennen and Vuylsteke, 1990).
Such information, if available, could also be used to select sites for breeding programs in bananas.
Time of day when pollination occurs strongly influenced bunch fertility in ‘Gros Michel’ in
Jamaica (Shepherd, 1954). When bunches were pollinated at 0700 h (compared with 1000, 1300, or
1600 h), 60% produced seed, compared with 30–40% at other times. As well as increasing bunch
fertility, pollinating at 0700 h increased the fertility per bunch by two- to threefold. Whether these
effects are caused by the environmental conditions at that time of day or the receptivity of the flow-
ers remains to be determined.
8.3.5 Seed Growth

Seeds stimulate the growth of the pulp in wild Musa species, partially parthenocarpic and, if
seeds are present, in parthenocarpic genotypes, according to Simmonds (1960). He investigated
this by applying plant growth regulators, or their antagonists, to growing fruit. In the wild-
seeded species, M. acuminata and M. balbisiana, each seed produced 0.23 cm 3 of pulp, about
four times the size of the seed itself. In parthenocarpic fruit, Simmonds (1960) estimated the
stimulatory effect of the seed on fruit volume to be 23 times the size of the seed. He concluded
that the stimulatory effect of the seed increased with increasing parthenocarpy. However, these
calculations ignore the two autonomous synthetic systems (Simmonds, 1960) that contribute to
growth of the pulp in partially or fully parthenocarpic fruit. A more likely scenario is that seeds
across the genetic spectrum in Musa have a more limited stimulatory effect on growth of the
pulp but in parthenocarpic fruit this is complemented by the independent growth of pulp linked
to the genes in the maternal tissues that contribute to parthenocarpy (Simmonds, 1953b). In
seedless mandarin (Citrus reticulata), endogenous gibberellins in the ovaries are linked to the
parthenocarpic development of fruit (Talon et al., 1992). Parthenocarpy in tomato (Lycopersicon
esculentum) is controlled by the deregulated production of gibberellins in the seedless fruit
(Olimpieri et al., 2007). These two examples indicate that the seed is not the only source of
growth regulator for fruit development, and it is likely that similar systems operate in Musa
fruits.
The size of mature fruit of wild bananas (M. acuminata and M. balbisiana) is linearly propor-
tional to the number of seeds in the fruit (Simmonds, 1953a, 1960). The regression coefficients are
0.23–0.24 cm3 per seed. A similar, but more limited, data set is available in the studies of Inito et
al. (1991) on pollination of M. salaccensis. Their data show a nonlinear relationship between seed
number per fruit and fruit size such that the contribution of each seed to fruit growth decreases with
increasing seed number. When a fruit contains fewer than 15 seeds, the contribution of the seed to
fruit weight is about 0.37 g/seed; but when there are between 15 and 62 fruit per seed, the contribu-
tion falls to 0.10 g/seed. Inito et al. (1991) measured whole-fruit weight, not the mass of the pulp, and
so the differences between the two coefficients may be linked to the high proportion of peel weight
likely to be present in small fruit where the pulp has not developed.
If insufficient ovules contain embryo sacs, then seed fertility will be affected, especially in
triploid cultivars. Shepherd (1954) measured the number of embryo sacs in ovaries obtained from
plantings of ‘Gros Michel’ (AAA) in Holland and Potosi, two locations in east Jamaica. At Holland,
18% of ovules contained embryo sacs but only 10% contained embryo sacs at Potosi. While this
is consistent with the observations on seed fertility observed at these locations 3 years earlier, at
Holland there were many embryo sacs with abnormal constitution. Shepherd (1954) concluded that
it was impossible to estimate the importance of embryo sac formation in controlling seed fertility,
based on the evidence available.
8.3.6 Seed Storage

In the forest the ripe fruit of seeded wild bananas are usually eaten by animals or birds and so to
recover seed from the field, mature green fruit need to be harvested as long as the seeds are black
and hard. Seed can be extracted from ripe fruit, washed, and cleaned. Initially the seed water con-
tent may vary from 30 to 45%. For storage, the seed need to be dried to 8–10% water content, placed
in a sealed container, and then they can be stored for 1 to 2 years at 5°C or at –18°C (Chin, 1996).
Banana seeds are thought to be intermediate in their storage behavior, although seeds of Ensete may
be orthodox (Seed Information Database, 2010). Seed water content strongly influences the viability
of banana seed in storage; seed with high water content loses its viability much more rapidly than
dried seed (Figure 8.11).
8.4 Seed Germination
8.4.1 Dormancy
The issues about dormancy in seeds of the Musaceae center on seed structure, imbibition, viability,
proportion of germinable seeds, experimental evidence for dormancy, changes in the definition of
dormancy, and differences between and within Musa, Ensete, and Musella.
Opinions about the nature of dormancy in the seed of Musaceae vary. Simmonds (1962) states
that banana seed are not inherently dormant as they will germinate as soon as they are extracted
from ripe fruit (Figure 8.12), and this is supported by the application of embryo rescue technology
(Cox et al., 1960; Vuylsteke et al., 1990). However, in the field they may lie viable for many years
and often germinate at the same time and in large numbers when the soil is disturbed or when veg-
etation is removed (Simmonds, 1962). Simmonds (1959) interpreted the delay in germination, under
100
90
80
70
Seed viability, %
60
50 10%
40
16%
30
29%
20
10
0
0 2 4 6 8 10 12 14
Time in storage, months
Figure 8.11 Effect of seed water content (10, 16, or 29%) on the viability of seed of Musa violascens
during 12 months’ storage at 22°C. (Data from Chin, H.F., 1996, Germination and storage of banana seed, in
New frontiers in resistance breeding for nematode, Fusarium and Sigatoka, E.A. Frison, J. Horry, and D. De
Waele, eds., 218–227, Montpellier, France, INIBAP.)
apparently favorable conditions, as a storage phenomenon, rather than dormancy. He thought that
the carbon dioxide concentration in the soil was sufficient to prevent germination and retain seed
viability, and he provided some experimental evidence to support this contention. Simmonds (1959)
also tested storage of seeds of M. balbisiana at different depths in the soil, compared with storage
of seed on the soil surface. Seed stored at depth maintained viability longer than seed stored on the
soil surface. Chin (1996) linked dormancy with seed water content, especially the desiccation of
the chalazal mass. If the chalazal mass was large, then seed usually germinated, but if it was desic-
cated, then the seeds became dormant. For example, freshly harvested seeds of M. gracilis (46%
water content) germinated (70%) after exposure to moist heat for 7 days. When dried to 12% water
content, these seed did not germinate after heat treatment, even after 3 months. Chin (1996) linked
germination of large numbers of banana seeds in the field after soil disturbance with the exposure
of these seed to diurnal changes in soil temperature. This is the interpretation reached by Stotzky
and Cox (1962) in their studies on the effect of alternating temperature on the germination of seed
100
90
80
Seed germination, %
70
60
50 Musa balbisiana
40
Musa acuminata
30
Musa balbisiana
20
y = –2.1414x2 – 3.0627x + 96.8
10
R2 = 0.7627
0
–8 –6 –4 –2 0 2 4 6
Time before or after full ripe (0), weeks
Figure 8.12 Relationship between the maturity of the bunch, expressed as weeks before or after the fruit
are fully ripe (week 0) and the germination of the seeds of Musa balbisiana and Musa acuminata. Seeds
were extracted and sown fresh and undried. The fitted equation is only for the M. balbisiana data. (Data from
Simmonds, N.W., 1952, The germination of banana seeds, Trop. Agric., Trinidad, 29, 35–49.)
of M. balbisiana. Removal of vegetation will also expose the soil to large variations in temperature,
without necessarily disturbing the soil.
Stotzky et al. (1962) state that difficulties in germination of seed of M. balbisiana lie in the seed
coat and that the embryo exhibits no dormancy because it is easy to culture aseptically (Cox et al.,
1960). On the other hand, Graven et al. (1996) believe that seeds of Musaceae show embryo dor-
mancy. De Langhe (2009) stated that seeds possess dormancy but its nature is unknown. In Uganda,
seeds of M. balbisiana were observed to germinate around the parental plants from which they fell,
while this was not observed in M. acuminata ‘Calcutta 4,’ suggesting that there was some inher-
ent dormancy required for ‘Calcutta 4’ (M. Pillay, personal communication). Whether seeds of the
Musaceae possess dormancy or not, they are often notoriously difficult to germinate. In the work on
banana breeding in Africa, usually only 1% of the hybrid seeds germinated (Swennen et al., 1992).
This can be increased to 12% if the embryos are extracted and cultured aseptically (Vuylsteke et
al., 1990).
There are a number of reasons why authors may have different opinions about dormancy in seeds
of the Musaceae. The concept of dormancy in relation to seeds has undergone revision during the
years that people have been working on banana seeds. Vleeshouwers et al. (1995) suggest that dor-
mancy should not be viewed simply as the absence of germination. Dormancy reflects a changing
internal status of the seed and it can vary on a continuous scale. The level of dormancy determines
the conditions needed for germination. If the environment external to the seed meets those require-
ments, then the seed germinates. They conclude that dormancy is a dynamic state, influenced by the
response of the seed to changes in the environment.
Baskin and Baskin (2004b) proposed a classification system for dormancy. Seeds may possess
dormancy that is physical, physiological, morphological, or combinations of these. In addition,
physiological dormancy and some of the combinations can have different levels and different types
of dormancy. Baskin and Baskin (2004a) present a dichotomous key, allowing the type of dormancy
to be identified providing the following are known: seed coat permeability, response to scarification,
embryo differentiation, and response to temperatures that simulate the seasonal variation in the
habitat. Using the classification of Baskin and Baskin (2004b) and bearing in mind the concepts of
dormancy proposed by Vleeshouwers et al. (1995), we can examine the experimental data available
for seeds of the Musaceae.
Baskin and Baskin (1998) included Musaceae in a list of families that contained at least some
species that had physical dormancy. This was based on the observation of Stotzky et al. (1962)
that scarification of seeds of M. balbisiana promoted germination and that the seed coat was
impermeable to water (Bhat et al., 1994). However, Baskin et al. (2000) removed Musaceae from
the group of families in which physical dormancy occurred because the seed coat was perme-
able, according to their interpretation of the comments of Stotzky and Cox (1962), and because a
water-impermeable layer of palisade cells could not be detected in the seed coat (Humphrey, 1896;
Graven et al., 1996).
Physical dormancy is characterized by an impermeable seed coat (Baskin et al., 2000). When the
seed coat becomes permeable to water through the action of factors such as high or fluctuating tem-
perature, freezing/thawing, drying, fire, or passage through the alimentary tract of animals, then the
embryo germinates under a wide range of temperatures and in the light as well as the dark (Baskin et
al., 2000). The feature that creates the high resistance to water flow across the seed coat is a layer(s)
of palisade cells. In addition, the comments of Stotzky and Cox (1962) on imbibition, which in intact
seed of M. balbisiana took 3 days but in chipped seed took 2 days, and both intact and chipped seed
absorbed the same amount of water, appear not to support physical dormancy in Musa.
A seed may functionally express physical dormancy, especially if a layer other than the palisade layer
prevented water entering the endosperm and embryo. Graven et al. (1996) suggest such a role for the
thick cuticle on the inner side of the tegument in seed of Musaceae. The data of Wattanachaiyingcharoen
(1990) (Figure 8.9) suggest that the inner cuticle prevents water movement into the chamber contain-
ing the endosperm and embryo in M. acuminata ssp. Piercing the cuticle allows an extra 7 mg of
water to flow into the seed, and this difference was maintained over the 8 days of the experiment.
Thus the thick inner cuticle provides the barrier to water flow to the embryo, providing a functional
expression of physical dormancy. However, Stotzky and Cox (1962) state that chipped seed absorbed
as much water as the intact seed. Chipping was done with a scalpel and differs from the technique
of piercing with a needle used by Wattanachaiyingcharoen (1990). Chipping cut through to the inner
cuticle and so would have removed a section of the seed coat. This loss of part of the seed coat would
reduce the amount of water that the seed could imbibe into the seed coat, but in chipped seed it could
be compensated with the water entering the chamber containing the endosperm and embryo. Thus the
comment by Stotzky and Cox (1962) that chipped and intact seeds absorbed the same amount of water
is not necessarily inconsistent with the measurements of Wattanachaiyingcharoen (1990) showing that
piercing the cuticle increases the amount of water absorbed.
The seed of Musa may well have a functional physical dormancy, where the barrier to water flow
is located at the inner surface of the seed coat, rather than the exterior as in Canna sp. (Graven et
al., 1997). Once the seed coat is imbibed, alternating temperatures increase the permeability of the
inner cuticle, allowing germination to proceed in M. balbisiana (Stotzky and Cox, 1962) and M.
acuminata ssp. (Wattanachaiyingcharoen and Turner, 1989a). Stotzky and Cox (1962) established
that alternating temperatures promote the germination of seed of M. balbisiana (Figure 8.13). In
their experiments, no germination occurred at constant temperature. Germination of M. balbisiana
increased as the difference between the maximum and minimum temperatures increased, until the
difference reached 15°C and beyond that germination was unaffected (Figure 8.13). Stotzky and Cox
(1962) excised the embryos of seeds of M. balbisiana and exposed them to constant and alternating
temperatures. In contrast to intact seeds, the excised embryos germinated at constant temperature
and did not require alternating temperature for germination or growth. Stotzky and Cox (1962) con-
cluded that the factor(s) that promoted germination that were affected by alternating temperatures
did not lie in the embryo. Thus the embryo of M. balbisiana meets another requirement of Baskin
90
y = –0.1909x2 + 7.5982x
80
R2 = 0.9869
70
60
Germination, %
50 Combined data
40 Outlier, 27/12
30 Combined data
20
10
0
0 5 10 15 20 25
Temperature diﬀerence, C°¯
Figure 8.13 Effect of the difference between maximum and minimum temperature on the germination of
seeds of M. balbisiana. Maximum temperatures were 27ºC, 32ºC, or 35ºC, minimum temperatures ranged
from 12ºC to 35ºC. The response of the seeds to the 27/12ºC treatment is about half that expected and is
presented as an outlier. (Data from Stotzky, G. and E.A. Cox, 1962, Seed germination studies in Musa, Pt. 2,
Alternating temperature requirement for the germination of Musa balbisiana. Am. J. Bot. 49:763–770.) The
fitted equation was forced through zero because no germination occurred at constant temperature.
et al. (2000) for physical dormancy: that the embryo can grow over a range of temperatures once
physical dormancy is broken.
Baskin et al. (2000) point out that for species with physical dormancy, the mechanism for break-
ing dormancy needs to be fine-tuned to the environment so that individuals may germinate, estab-
lish, and eventually reproduce. For M. acuminata ssp., the seed requires at least 4 days to reach
maximum water absorption (Figure 8.9) and for M. balbisiana, more seed germinates as the number
of alternating temperature cycles increases and while the maximum temperatures remain at about
32°C (Stotzky and Cox, 1962). In addition, Stotzky and Cox (1962) compared alternating tempera-
tures where the high temperature was maintained for 5 or 19 h and the minimum was 19 or 5 h,
respectively. The longer time at the maximum temperature reduced germination to less than 10%
and the shorter time at maximum temperature promoted seed germination (30–60%). In tropi-
cal conditions, it is more important for a seed to detect the wet or dry season, since temperatures
are reasonably moderate; nonetheless, the response of banana seed to temperature is quite precise
(Stotzky and Cox, 1962). Simmonds (1962) described the wild bananas as seral plants functioning
as “jungle weeds” that spring up in response to disturbance in vegetation or soil. Disturbance may
be clearing of forest by humans, land slips, or sections of forests damaged by storms. Exposure of
soil to sunlight and high temperature for a short time would more likely be a feature of the edge of
disturbed vegetation, rather than its center. Being able to detect this difference would allow banana
seeds to germinate adjacent to the forest but in a disturbed area.
8.4.2 Viability
Fresh wild banana seed can germinate at a high level (Figure 8.11) but as they dry seeds become
dormant (Chin, 1996). In studies on germination, knowledge of the viability of seed is important for
interpreting the response of seeds to environmental cues. If seeds do not germinate, are they viable,
or is there some other reason for the inability to germinate? Earlier studies on banana seed usually
separated them into “good” or “bad” seed depending on their appearance, hardness, and endosperm
content (Shepherd, 1954, 1960a, 1960b). However, in the analysis of data “good” and “bad” seeds
were combined because it was difficult to predict the behavior of the seed based on this classifica-
tion. Triphenyl tetrazolium salts have been widely used to determine viability of embryos in seed
and they are straightforward to use on seed of Musa spp. (Wattanachaiyingcharoen and Turner,
1989b). The tetrazolium salts form a red color when they react with dehydrogenases in living tissue.
If seed is killed by autoclaving, no red color appears. Embryos of Musa sp. can be taken as viable if
the red color exists on 90% of the embryo. The tetrazolium test for viability is destructive and other
ways of determining seed viability are worth exploring. In addition to problems of viability, a seed
may not germinate because it lacks an embryo or endosperm, or there is no functional connection
between the embryo and the endosperm.
Wattanachaiyingcharoen (1990) found that seed weight was a good predictor of viability, as
determined by the tetrazolium test. In M. acuminata ssp., seed below 20 mg fresh weight contained
no viable embryos and the increase in the proportion of seed in the population with viable embryos
(P%) increased linearly with seed weight (W) so that the largest seeds of 75 mg had 100% viable
embryos. The relationship was P% = 1.56W – 27.21, r 2 = 0.92. Floating was unable to separate viable
from nonviable seed. Most seed of less than 30 mg weight floated. Among the seeds that sank,
viability varied from 25 to 100% (Wattanachaiyingcharoen 1990). Tesfaye (1992) used floating to
separate seeds of Ensete sp.
8.4.3 Germination
The sequence of germination in Musa sp., as recorded by Simmonds (1959), is: exudation of a
brownish fluid from the micropyle, the micropylar lid or operculum is extruded, and the primary
root emerges from the micropylar canal. One week after the primary root emerges, lateral roots
dominate; after 2 weeks the endosperm has disappeared, after 4 weeks seedlings show nutrient
deficiency symptoms if germinated in sand. If the seed coat is ruptured, then the expanding embryo
will emerge from the seed through the injury, whether it is at the chalazal end of the seed (Stotzky
and Cox, 1962) or through a hole pierced in the coat (Wattanachaiyingcharoen, 1990). This implies
that in intact seed, the expanding embryo needs to push the micropylar cap from the seed in order
to emerge and will use an alternative route if it is available. This would provide a point for selection
pressure because in the wild, embryos that were not able to dislodge the micropylar plug would not
germinate. Selection based on in vitro culture of excised embryos, such as is used in banana breed-
ing technology, would avoid this selection and may therefore include embryos that are less fit than
those germinated from seed.
Fresh seed germinates (Simmonds, 1952; Figure 8.12). On drying, the seed enters functional
physical dormancy and this can be broken in M. balbisiana by alternating temperature, after the
seed coat has imbibed (Stotzky and Cox, 1962). The embryos can readily grow after excision from
the seed using embryo rescue technology (see, for example, Cox et al., 1960; Pancholi et al., 1995)
and are not dormant, although Graven et al. (1996) thought that they were. In the study of Pancholi
et al. (1995) on M. velutina seeds, sowing the seed in a propagating medium allowed 82% to ger-
minate, but the germination was spread over 10 months. Excising the embryos allowed the seed to
germinate (74%) in 14 days. Ellis et al. (1985) provide a list of the various treatments that either do
or do not promote germination in Musaceae. Alternating temperatures promoted germination in
intact seed of M. balbisiana (Stotzky and Cox, 1962) but did not promote germination in Ensete
sp. (Tesfaye, 1992). However, soaking in water at 40°C for 24–48 h did promote germination in
Ensete (Tesfaye, 1992). On the other hand, soaking seeds of M. acuminata or M. balbisiana in
water usually had deleterious effects (Simmonds, 1952). Seeds from different species within the
Musaceae do not appear to have the same requirements for germination and may not have the same
mechanism of dormancy, despite the similarities in the structure of the seed and seed coat (Graven
et al., 1996). In Ensete, the embryo is small (8 mm) in relation to the size of the seed (20–30 mm)
and so it needs to grow considerably within the seed before it can emerge (Tesfaye, 1992).
8.5 Conclusion
The inflorescence of most Musa species and cultivars is monoecious. The female flowers are first
formed and precede the male flowers in anthesis. Ovules are formed and are similar in the wild-
seeded species and the edible, sterile, and parthenocarpic landraces, at least up until anthesis.
Beyond this, numerous events combine to minimize seed development in cultivars. While many
of these are known, the importance of each in any situation is uncertain. There is a need to know
more about why so few seeds are produced, when there are records of high seed production, even in
cultivated genotypes. Several factors, such as receptivity of the female organs, pollen tube growth,
ovary position within the bunch, season of pollination, time of day, and location contribute to seed
production in banana. There is also an interaction between environment and fertility of female and
male components. These factors can be managed to manipulate seed production so that the immedi-
ate objective of crosses (such as production of triploids or tetraploids) can be met.
The clumped nature of the plant means that crossing in the wild-seeded species can be achieved
between inflorescences within the one clump, and outbreeding can occur if other clumps of bananas
are nearby. Heterozygosity is high among the edible diploids, which have been dispersed by people,
but somewhat less among the seeded wild species that tend to be geographically isolated, one spe-
cies from another.
Pollen production varies considerably between genotypes and locations, as does its viability.
There is not necessarily a correlation between the viability of pollen and the number of seeds
produced by receptive flowers. The production of 2n pollen is used to increase the production of
tetraploids in breeding schemes and it is believed that this was the process that produced the triploid
landraces used widely today.
Hybridization lies at the heart of breeding. There has been considerable effort to discover the
likely route by which the diploid and triploid landraces were produced. This opens the possibility
of determining which crosses might best be used to not only incorporate disease or pest resistance
in progeny but to also provide new cultivars that retain the desirable qualities that people selected
in the first place.
Selection of bananas for edibility has been a high priority for people for thousands of years and
the primary aim was to select plants with fruit that were seedless. Breeding bananas for resistance
to disease, tolerance of edaphic and environmental constraints, and desirable postharvest qualities
is a more recent activity spread over several decades. Breeding requires seed. Once seed are pro-
duced, few of them germinate and so seed production and germination are bottlenecks in banana
breeding. Seeds of the wild bananas germinate readily when they are fresh and as they dry they
enter a functional physical dormancy that can be broken by alternating temperatures when the seed
is hydrated. Many banana seeds do not germinate because they do not contain embryos. Many
embryos are not viable, further reducing germination. Viable embryos can be excised and cultured
aseptically to produce plants for selection.
In recent decades, knowledge of the reproductive biology of plants has progressed, but work on
banana has been slow. There is a need to reengage research on reproductive biology in Musaceae,
bringing to bear the new knowledge gained on “model plants” so that new, disease-resistant culti-
vars can contribute to sustaining the world’s population.
Acknowledgments
We are grateful to Dr. David Merritt, Professor Rony Swennen, and Jeff Daniells who made con-
structive comments on the text.
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9 Breeding Techniques
Abdou Tenkouano, Michael Pillay, and Rodomiro Ortiz
Contents
9.1 Introduction: Short History of Breeding Programs............................................................... 181
9.2 Breeding Objectives in Africa............................................................................................... 182
9.3 Breeding Schemes................................................................................................................. 183
9.3.1 Pollination and Seed Production............................................................................... 184
9.3.2 Seed Germination...................................................................................................... 185
9.3.3 Somaclonal Variation................................................................................................. 185
9.4 Breeding Philosophies........................................................................................................... 186
9.4.1 Evolutionary Breeding............................................................................................... 186
9.4.2 Reconstructive Breeding........................................................................................... 188
9.5 Occurrence and Potential of 2n Gametes in Banana Breeding............................................. 189
9.6 Selection................................................................................................................................. 189
9.6.1 Ploidy and Genome Analysis..................................................................................... 190
9.6.2 Phenotypic Evaluation............................................................................................... 190
9.6.3 Improving Selection Efficiency................................................................................. 192
9.7 Breeding Achievements......................................................................................................... 193
9.7.1 Substitutes for Commercial or Subsistence Production............................................. 193
9.7.2 Genetic Stocks for Breeding...................................................................................... 194
9.8 Breeding Constraints............................................................................................................. 195
9.9 Future Breeding Goals.......................................................................................................... 197
References....................................................................................................................................... 198
9.1 Introduction: Short History of Breeding Programs

Banana improvement formally started in response to the decimation of large commercial planta-
tions of dessert bananas by Panama disease caused by Fusarium oxysporum f. sp. cubense (Ploetz,
1992). No chemical or cultural method was available to control this fungal disease, which rapidly
spread from its area of first occurrence in Surinam to Honduras, the largest commercial banana
producer, in the early 1920s. By the 1950s, the disease had virtually wiped out the prevailing ‘Gros
Michel’ cultivar and threatened the mere existence of commercial dessert banana production. This
prompted the search for new cultivars, initially through exploration of existing accessions, to replace
the ‘Gros Michel’ cultivar. These were found in the Cavendish subgroup of AAA bananas, which
became and remain to date the predominant type of commercial dessert banana. The outbreak of
fungal Sigatoka diseases in the early 1970s and the financial and environmental cost of chemical
treatment gave additional impetus for genetic improvement because no existing cultivar had natural
resistance to the new diseases.
The earliest attempts to breed bananas were at the Imperial College of Tropical Agriculture
(Trinidad), but this program has virtually ceased to operate. The oldest breeding program in opera-
tion is that of the Fundación Hondureña de Investigación Agrícola (FHIA, http://honduras.com/
fhia/banana.htm, accessed December 27, 2009), which was established in 1958 by the United Fruit
181
Company in La Lima, Honduras. FHIA’s program initially focused on dessert bananas, but it gradu-
ally expanded to plantain and cooking banana for which it is most known. Other major programs in
existence were established from the mid 1970s to the mid 1990s. This includes the programs of the
Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD,
based in Guadeloupe for dessert bananas), the Centre Africain de Recherches sur les Bananiers et
Plantains (CARBAP, based in Cameroon for plantains), and the International Institute of Tropical
Agriculture (IITA, with two main programs established in Nigeria for plantains and in Uganda for
the East African Highland bananas).
Relatively smaller or domestically focused programs have been established in Austria
(International Atomic Energy Agency [IAEA]), Brazil (Empresa Brasileira de Pesquisa Agropecuaria
[EMBRAPA]), India (National Center for Research on Banana [NCRB], Tamil Nadu Agricultural
University [TNAU], and Kerala Agricultural University [KAU]), Taiwan (Taiwan Banana Research
Institute [TBRI]), Uganda (National Research Organization [NARO]), and Côte d’Ivoire (Centre
National de Recherches Agronomiques [CNRA]). The program based in Taiwan is focused on the
search for somaclonal variants using induced mutagenesis as the main avenue for creating genetic
diversity. All other programs, except that of CNRA, use the array of tools available for creating
diversity, with a preponderance of cross-pollination.
In 1985, the International Network for Improvement of Banana and Plantains (INIBAP, France)
was established to coordinate the efforts of the various institutions involved in the development and
dissemination of new cultivars and to facilitate access to genetic stocks and new cultivars through
the International Musa Testing Program (IMTP). INIBAP established offices in Africa, Asia, and
Latin America and coordinates several networks of banana researchers involved in various disci-
plines contributing to genetic improvement. The INIBAP International Transit Center operating
at the Katholieke Universitat Leuven (KULeuven, Belgium) remains as the main ex situ, in vitro
gene bank of bananas in the world. To date, INIBAP, nowadays included in one of the programs
of Bioversity International (Italy), constitutes the largest source of information on banana research.
The number of breeding programs remains small, despite the place of banana in world trade and as
a global staple crop (Escalant and Panis, 2002).
9.2 Breeding Objectives in Africa

Banana and plantain (Musa spp. L.) constitute major export crops in some countries, predominantly
in Latin America and the Caribbean and a few countries in Western and Central Africa. However,
only about 10% of world production enters international trade. Banana and plantain are major staple
foods for rural and urban consumers and an important source of income for rural populations in the
production areas, particularly in the equatorial belt of Africa, where more than 70 million people
derive in excess of 25% of their daily calorie intake from plantains in West and Central Africa
(Robinson, 1996). Seventy-three percent of the world crop of plantain is grown and consumed in
Western and Central Africa (WCA), although increasing quantities of plantain are being exported
to the United States and Europe (Marin et al., 2003).
Despite their introduction to Africa only about 3,000 years ago (De Langhe, 1995), a remarkable
morphological diversity now exists for both banana and plantain in this region, with a multitude of
vernacular names for the prevailing cultivars, superimposing linguistic diversity to genetic and eco-
logical diversity of the cultivars in any given area (Rossel, 1998). Similar interactions between lin-
guistics and genecology occur throughout Asia, the geographical origin and primary diversification
center of the Musaceae. Many indigenous cultivars are susceptible to a range of pests and diseases.
The most serious threat to the production of plantains is caused by the Sigatoka leaf diseases: yellow
Sigatoka (Mycosphaerella musicola) in the highlands and black Sigatoka (M. fijiensis) in the low-
lands. Black Sigatoka is the most damaging and costly disease of bananas and plantains because its
control accounts for 27% of total production costs in commercial plantations (Stover, 1980). Other
long-standing threats to banana and plantain production are caused by Fusarium wilt (Fusarium
Breeding Techniques 183
oxysporum f. sp. cubense [E.F. Smith] Snyder and Hansen), banana weevil (Cosmopolites sordi-
dus Germar), and a complex of plant parasitic nematodes (Pratylenchus goodeyi Sher and Allen,
Helicotylenchus multicinctus [Cobb] Golden, and Radopholus similis [Cobb] Thorne), but bacterial
wilt (Xanthomonas campestris pv. musacearum) has recently emerged as the most damaging dis-
ease of bananas in the highlands and Great Lakes region of East Africa while banana bract mosaic
virus has become a cause of concern in Central Africa and to a lesser extent in West Africa.
Black Sigatoka was accidentally imported into Africa in the late 1970s (Wilson and Buddenhagen,
1986) and quickly reached epidemic proportions. When unimpeded, the disease induces leaf decay,
thereby reducing the photosynthetic area, and considerable reduction in yield or complete crop
failure may ensue (Mobambo et al., 1993). Because none of the indigenous cultivars had natural
resistance, black Sigatoka has since reduced the yields of these cultivars to less than half what they
were before the arrival and spread of this disease.
Black Sigatoka has been especially devastating to the food security for the people in the
affected regions, essentially for two reasons: (1) there were inadequate supplies of food even
before this new disease further reduced production, and (2) prior to the arrival and spread of this
disease, plantains and bananas were one of the most dependable foods since these crops were not
as vulnerable to production uncertainties (droughts, floods, diseases, insects, and storage losses)
as the basic grains.
Black Sigatoka turned plantain into a delicacy in urban Nigeria and other countries in Africa,
due to the high cost of the fruits subsequent to the reduced production caused essentially by the
disease. Chemical protection is relatively effective but expensive and detrimental to the environ-
ment, leaving host-plant resistance as the most practical option for sustainable control of the
disease for resource-poor farmers of the developing world where the bulk of the production takes
place. In particular, the thousands of small-scale plantain farmers in Africa do not have access to,
and cannot afford, the fungicides used by banana exporters and commercial producers for control
of black Sigatoka; the only sustainable solution is to plant hybrids with genetic resistance to this
disease.
It is against this background that Musa breeders aim to develop new disease- and pest-resis-
tant cultivars that also retain the organoleptic properties of the traditional cultivars. Besides
resistance to biotic threats, genetic improvement of Musa also aims at developing hybrids
that are high yielding per unit area and time (Buddenhagen, 1996). Hence, improved hybrids
should be photosynthetically efficient, early to mature in the first production cycle, and dis-
play minimum delay between consecutive harvests (Eckstein et al., 1995; Ortiz and Vuylsteke,
1994a). Other desirable characteristics include short stature and strong roots for optimal nutri-
ent uptake and greater resistance to wind damage. These breeding objectives are summarized
in Figure 9.1.
9.3 Breeding Schemes

There are two basic steps in plant breeding: (1) access natural variation or artificially create genetic
diversity, and (2) select individuals with the desirable gene combinations from existing or artificially
created populations. These principles have been successfully applied to the improvement of sexual,
seed-propagated crop species using conventional breeding techniques, which are commonly viewed
as inappropriate for banana and plantain improvement. It seems that the distinction between “con-
ventional” or “classical” and “nonconventional” breeding lies in differences in the methods of gene
shuffling and in the origin of the genes being manipulated.
Nevertheless, the methodological principles applied for sexually propagated crops can be, and
have been, applied to banana and plantain breeding, largely with the aid of nonfield methods of
recovering viable progeny and identifying those progenies with putatively desirable gene combina-
tions. Thus, banana and plantain breeding is neither strictly conventional nor strictly non-conven-
tional, which will be illustrated in the subsequent sections of this chapter.
P F H
Breeding Objectives
High and stable yield
Fruit quality (taste, shelf life, processing)
R-Black Sigatoka
R-Nematodes
R-Wilts (Fusarium, Bacterial)
R-Weevil
R-Viruses
Earliness
Short H-H intervals
Short stature
Good roots
Figure 9.1 Illustration of breeding objectives for banana and plantain: increased frequency of harvests of
high and stable yield with good fruit quality through resistance to an array of pests and diseases and greater
biological efficiency. The letters P, F, and H indicate planting, flowering, and harvesting events, respectively.
The letter R indicates resistance.
9.3.1 Pollination and Seed Production

Breeding starts with effective pollination to produce seeds. Pollination is the transfer of pollen from
the anther to stigma either within a flower or between flowers of the same genotypes (autogamy) or
between flowers of different genotypes (allogamy). This mechanism guarantees genetic recombina-
tion and preserves variability within populations. An efficient pollination scheme can be measured
by the proportion of seed set (Cruden, 1977) and the diversity of genes that can be incorporated into
individual plants within limited time and resources.
In banana and plantain, the male gametophyte is easily affected by the environment because the
pollen grains must be transferred from one plant to another by pollinating agents. This is compen-
sated for by the production of a large number of pollen grains. In contrast, the female gametophyte
develops within the ovary and is less affected by the environment. Hence, smaller numbers of
female gametophytes are observed across species. Pollination success also depends on the ability
of the male gamete to access the ovule and the ability of the female to provide adequate resources
for seed and fruit development. The duration of flowering, flower arrangement, and the pattern of
anther dehiscence are also important aspects that determine pollination success.
A common practice in hand pollination in most crops is to start in the morning as soon as pollen
is shed, because pollen viability declines with time. Thus, the male and female flowers of bananas
and plantains are covered shortly before anthesis to prevent contamination by alien pollen. At
anthesis, pollen grains from male flowers are collected and brushed against female flowers, usually
between 7 a.m. and 10:30 a.m. (Swennen and Vuylsteke, 1993). Pollination can be carried out over
a period of 7 to 15 days depending on the number of hands present in the bunch under pollination.
It usually takes from 60 to 90 days for the pollinated bunch to reach maturity, following which the
fruits are subjected to rapid ripening before seeds are mechanically extracted.
Pollination can be carried out year round, but Ortiz and Vuylsteke (1995) found that periods of
high temperature, high solar radiation, and low relative humidity were most favorable for seed pro-
duction in banana and plantain. Hand pollination is a tedious and relatively inefficient process, con-
sidering the time required, the poor seed set, and the quality of the seed produced. The potential of
open pollination for improvement in Musa has been suggested (Ortiz et al., 1995). Among the open
pollination methods, the polycross approach has been widely used in forage and maize to develop
superior genetic recombinants (Allard, 1960). Although attractive, this scheme requires effective
control over prospective parents, notably by choosing prospective parents on the basis of their com-
bining ability and floral synchrony prior to growing them in isolated crossing blocks where natural
agents would effect pollination.
At maturity, the seeds are black or dark brown stony bodies of variable size and shape with a
rough seed coat and a complex structure that makes germination very difficult to achieve (Kiew,
1987; Chin, 1995; Graven et al., 1996).
9.3.2 Seed Germination

Regardless of the approach, most cultivated cultivars display relatively high infertility levels, which
is the most important obstacle to overcome in order to create genetic variability. Cross-pollination
usually produces sexual embryos that can develop into an array of genetically varied offspring,
but the embryos often abort or develop into seeds that lack enough autonomy to develop into seed-
lings. The embryos must be rescued using tissue culture, which has become an integral component
of banana and plantain breeding (Vuylsteke, 1989). With the small number of seeds recovered
from crosses (typically a few per pollinated bunch), Musa breeders disproportionately allocate their
resources for producing, rather than evaluating, progenies.
9.3.3 Somaclonal Variation

Tissue culture not only serves as a tool for producing progenies from crosses but also stands as a
diversity-generating option on its own. Tissue culture has the potential of generating somaclonal
variation, which is genetic in nature and can be stably passed onto progeny (Larkin and Scowcroft,
1981). Vuylsteke et al. (1988) have reported that variations in inflorescence morphology occur in
populations of ‘False Horn’ plantains derived from micropropagation, with a reversion to a typical
French plantain bunch type accounting for 40–100% of the total variability. Somaclonal varia-
tion prevents clonal uniformity and can be a serious nuisance, particularly for populations used
to establish commercial banana plantations (Damasco et al., 1996; Grillo et al., 1998). However,
this phenomenon may also provide a useful source of genetic variability to the plant breeder as
was first demonstrated in sugarcane with the in vitro selection of a commercial cultivar resistant
to Fiji disease (Heinz and Mee, 1969). Likewise, Hwang and Ko (1987) obtained somaclonal vari-
ants of bananas with putative field resistance to Fusarium wilt (Fusarium oxysporum Schlect. f. sp.
cubense [E.F. Smith] Snyder and Hansen) while Trujillo and Garcia (1996) selected a variant of the
Cavendish subgroup resistant to yellow Sigatoka (M. musicola Leach).
The resistant somaclones often had inferior horticultural characteristics, but these examples
suggest that somaclonal variation arising in culture may produce useful variants for the genetic
improvement of Musa spp. However, to date all studies concerning somaclonal variation of plan-
tains suggested that this source of variation may not readily provide useful agronomic improve-
ments in this crop (Vuylsteke et al., 1991; Vuylsteke, 1998; Nwauzoma et al., 2002). Understanding
the molecular basis of such variations could provide a more deterministic approach for using
somaclonal variation as a breeding tool. A recent study at IITA assessed differences in genomic
regions varying within clones grown naturally or in vitro, using 52 simple sequence repeats (SSR)
markers and 6 EcoRI/MspI-AFLP primer combinations (Vroh et al., 2010). Sequencing the poly-
morphic regions at one SSR locus with significant similarity to an arcelin gene revealed a deletion
in a subculture regenerant. Out of 390 amplified fragment length polymorphism (AFLP) bands, 24
(6.15%) accounted for within-clone variations, among which 0.5% and 5.65% occurred in conven-
tionally and micro-propagated plants, respectively. Homology searches revealed that most poly-
morphic AFLP sequences were related to cytochrome P450, cell-wall biosynthesis, and senescence
genes. Thus, somaclonal variation may result from mutagenic events occurring in hot spots that
might not be associated with the expression of traits of horticultural value.
Tissue culture also offers opportunities for using physical or chemical mutagenesis as well as
genetic transformation based on site-directed or unspecific gene insertion, disruption, or substitu-
tion to create genetic variability, with varying degrees of success (Mohan Jain and Swennen, 2004;
Tripathi et al., 2008). There has been no assessment of the comparative efficiency of alternative
schemes of generating, managing, and exploiting genetic diversity (induced mutation breeding, for-
tuitous somaclonal variation, genetic transformation, and biotechnology-assisted crossbreeding).
However, each approach has produced lines that are now reaching farmers, either as experimental
materials in the case of genetic transformation or as confirmed cultivars for the other approaches.
The rest of this chapter will focus on crossbreeding.
9.4 Breeding Philosophies

Breeding for resistance to M. fijiensis is a fairly simple process in theory, but there are two compet-
ing philosophies that are not mutually exclusive. Both are based on a good understanding of the
evolution of banana and plantain from interspecific hybridization and polyploidization involving
derivatives of M. acuminata Colla and M. balbisiana Colla that are the diploid (2n = 2x = 22)
ancestors of present-day cultivars to which they contributed the A and B genomes, respectively
(Simmonds and Shepherd, 1955).
Two critical events subtend the evolutionary pathway leading to the emergence of cultivated
cultivars from the seminiferous ancestors. The first event is the appearance of vegetative parthe-
nocarpy and female sterility in M. acuminata, allowing for production of pulp without seeds as
evidenced by the occurrence of parthenocarpic and seedless diploid M. acuminata. The second
event includes crosses within M. acuminata or between M. acuminata and M. balbisiana coupled
with female restitution and haploid fertilization, which produced two groups of natural hybrids. One
group comprises autoploid and homogenomic hybrids that are essentially AAA dessert and East
African Highland bananas. The other group contains the alloploid and heterogenomic AAB plan-
tains and the ABB cooking bananas. Since these cultivars produce no seeds and cannot reproduce
sexually, only vegetative propagation is possible, implying that survival in nature and geographical
dispersal do not occur without human intervention. Consequently, secondary diversification in areas
devoid of wild Musa plants must be due to somatic mutations of the introduced materials. It is gener-
ally accepted that edibility of mature fruits in interspecific hybrids comes from the A genome while
starchiness and acid taste come from the B genome, causing AAB plantain to be starchier but less
sweet and less palatable when raw than the AAA dessert bananas (Simmonds, 1962).
9.4.1 Evolutionary Breeding
One school of thought, which we may call evolutionary breeding, attempts to mimic the evolutionary
development of the Musa species complex (Rowe and Rosales, 1996; Ortiz, 1997a). In this process,
female fertile triploid (2n = 3x) landraces of banana (AAA genomes) or plantain (AAB genomes)
are crossed to diploid (2n = 2x) accessions of M. acuminata (AA genomes) or M. balbisiana (BB
genomes) that are resistant to black Sigatoka, having coevolved with M. fijiensis in their area of
origin in Southeast Asia. The 3x × 2x crosses produce primary hybrids with considerable pheno-
typic variation subtended by ploidy and genome polymorphisms, among which 2x and tetraploid (2n
= 4x) progenies that are disease resistant and agronomically desirable are selected and intermated
via 4x × 2x crosses to produce high-yielding secondary 3x hybrids (Vuylsteke et al., 1993a). When
4x and 2x offspring take into consideration the genetic diversity of their 3x progenitors, this process
allows for the establishment of genetic bridges between banana or plantain landraces that cannot be
crossed directly due to reproductive barriers (Tenkouano, 2001). Ideally, therefore, plantain genes
from different sources may be brought together in a 3x background where the resistance genes
lacking in the traditional cultivars will have been restored, resulting in an improved plantain hybrid
that retains the preferred processing properties of the landraces (Tenkouano, 2001). Completing
this scheme is recurrent 2x breeding to produce 2x stocks that retain the disease resistances of
their 2x progenitors with improved agronomic characteristics. It is widely accepted that improved
2x would be better parents for the improvement of 3x landraces (Tezenas du Montcel et al., 1996).
It has been reported that traits of economic importance in banana and plantain—for example, pest
resistance, increased bunch weight, and reduced time interval between flowering and harvest—are
more predictably inherited from a 2x male background than from parents with a higher ploidy sta-
tus (Tenkouano et al., 1998a, 1998b). Furthermore, fewer chromosomal imbalances occur during
meiosis in 2x compared to polyploids due to the disomic nature of inheritance in 2x, thereby mak-
ing genetic analysis more attractive in 2x than in polyploids. This fact gives in retrospect a justi-
fication for past and current investment in the development of 2x breeding stocks with a balanced
combination of resistance and good agronomic features, as pursued by major programs worldwide
(Ortiz and Vuylsteke, 1996). Breeders following this school of thought have used a limited pool of
accessions in their work, due to reproductive barriers caused by the low fertility of existing triploid
cultivars used as candidate parents (base generation) for improvement. At IITA, for example, the
base generation consisted mainly of a few triploid landraces (‘Bobby Tannap,’ ‘Mbi Egome,’ and
‘Obino l’Ewai’) of the Medium French subgroup of AAB plantains (Swennen, 1990; Swennen et al.,
1995). The sources of resistance were the diploid AA accessions M. acuminata ssp. burmannicoides
‘Calcutta 4,’ M. a. ssp. malaccensis ‘Pisang Lilin,’ and M. a. ssp. microcarpa ‘Tjau Lagada.’
The accession ‘Calcutta 4’ is a nonedible wild accession from Myanmar (De Langhe and Devreux,
1960) that produces many true-seeded fleshless fruits because it lacks one of the three dominant
complementary genes for parthenocarpy (Simmonds, 1953). ‘Calcutta 4’ is abundantly pollenifer-
ous and resistant to black Sigatoka and several nematode species, including R. similis and P. cof-
feae (Viaene et al., 2000). ‘Calcutta 4’ is morpho-taxonomically related to plantain (De Langhe,
1969), justifying its extensive use in crosses to improve plantain landraces (Swennen and Vuylsteke,
1993; Vuylsteke et al., 1993b; Vuylsteke and Ortiz, 1995). The accession ‘Pisang Lilin’ originated
in Malaysia (Stover and Simmonds, 1987) and is described as a translocation heterozygote with
edible parthenocarpic fruits and high resistance to black Sigatoka. It is moderately female fertile
but highly male fertile and known to frequently produce 2n or 4n pollen, depending on the season
(Ortiz, 1997b). Both ‘Calcutta 4’ and ‘Pisang Lilin’ are reference accessions of the International
Musa Testing Program (Orjeda, 2000). ‘Tjau Lagada’ was originally collected in the Java island of
Indonesia (Stover and Simmonds, 1987) with an incomplete resistance to black Sigatoka that may
best be described as a slow-lesion development type. It is moderately female and male fertile, with
a characteristic long bunch with many hands, and moderately parthenocarpic fruits. We denote
the triploid cultivars as 3x0 and the diploid accessions as 2x0. The primary diploid descendants
derived from crossing these accessions to triploid cultivated landraces or to other 2x0 diploid acces-
sions constitute the first generation, which we refer to as 2x I hybrids. Likewise, primary 3x or 4x
descendants from the 3x0 × 2x0 crosses are denoted as 3x I and 4x I, respectively. However, the most
interesting descendants in the first generation were 2x I and 4x I hybrids. The second generation is
obtained by crossing the primary generation hybrids in various combinations (2x I × 2x I, 4x I × 2x I, or
2x I × 4x I). The most frequent combinations have been the 2x I × 2x I and 4x I × 2x I crosses, producing
Generation 0: 3x0 X 2x0
[OL, BT, ME] [C4, PL, TL]
Generation 1: (2xI, 3xI, 4xI) X (2xI, 3xI, 4xI)
[4xI X 2xI] or [2xI X 2xI] or [2xI X 4xI]
Generation 2: (2xII, 3xII)
Figure 9.2 Schematic representation of the plantain breeding process whereby initial crosses are carried
out between triploid (2n = 3x) landraces (predominantly ‘Obino L’Ewai’ [OL], ‘Bobby Tannap’ [BT], and ‘Mbi
Egome’ [ME]) and diploid (2n = 2x) sources of resistance (predominantly ‘Calcutta 4’ [C4], ‘Pisang Lilin’
[PL], and ‘Tjau Lagada’ [TL]), followed by intermating best primary diploid, triploid, and tetraploid (2n =
4x) progenies using 2x × 2x, 4x × 2x, and 2x × 4x crosses to produce secondary diploid (2x II) or triploid (3x II)
descendants. Diploid improvement can also be concomitantly carried out as a complementary scheme.
predominantly 2x II and 3x II descendants, respectively. The 2x I × 4x I crosses have been less frequent,
but they produce predominantly 2x II descendants (Oselebe et al., 2006).
9.4.2 Reconstructive Breeding
The second school of thought may be described as reconstructive breeding, whereby breeders
attempt to reenact the landraces by first determining their most likely ancestors from the pool of
2x species and subsequently using only such putative ancestors or closely related derivatives in
crosses aiming at synthesizing improved variants of the susceptible landraces (Carreel et al., 2002).
Proponents of this school of thought advocate the use of colchicine to induce tetraploidization of 2x
accessions prior to crossing with another 2x (Tezenas du Montcel et al., 1996). Colchicine prevents
the formation of mitotic spindles, causing mitotic restitution in treated cells. However, colchicine
treatment may not affect uniformly all cells in multicellular meristems, causing cytochimeras that
may not be easy to dissociate (Roux et al., 2001). Thus, efficient methods for in vitro dissociation of
chimeras and selection of the desired cells are required before such cells can be cultured to regener-
ate a plant that will now be used for crossbreeding. Furthermore, the use of colchicine could result
in increased inbreeding, reduced vigor, and reduced genetic variability (Ortiz et al., 1992).
Despite these limitations, Bakry et al. (2007) have successfully derived stable, nonchimerical,
colchicine-induced 4x variants from several diploid M. acuminata accessions. Crossing the induced
4x to 2x clones resulted in the production of 3x, as anticipated (Bakry et al., 2007). This approach
now forms one of the pathways used by the breeding program of the Centre de Coopération
Internationale en Recherche Agronomique pour le Développement (CIRAD). CIRAD’s program
aims at creating commercial cultivars of AAA dessert bananas for the banana industry operating
in the Caribbean islands of Guadeloupe and Martinique. Whether this approach could be used for
other Musa groups is an attractive idea that needs to be explored.
However, it is conceivable that the identification of putative 2x ascendants of the present-day
cultivars could also be followed by a breeding process that could be the same as for evolutionary
breeding, albeit with initial crosses of the landraces with their putative ascendants, as described
above. With the tremendous developments in the use of molecular or cytological tools, it is possible
to clarify the phylogenetic relationships in the Musaceae, allowing for inferences about gene pools
and the search for novel genes. Thus, AFLP analysis of total DNA revealed that the M. acuminata
complex of about 50 morphological subspecies can be ascribed to only three genetic subspecies
(burmannica, malaccensis, and microcarpa) while M. balbisiana was shown to contain two forms
(‘Singapuri,’ ‘I-63’), suggesting that there might be at least three A genomes and two B genomes
(Ude et al., 2002a, 2002b). Furthermore, it seems that the A genomes in the plantains came from
the subspecies microcarpa while the B genome was from the ‘I-63’ form of M. balbisiana. More
recently, Ge et al. (2005) also traced variation in M. balbisiana to two main clades, based on SSRLP
and cpDNA analysis.
A major retroactive justification for this school of thought was the discovery in the early 1990s
of banana streak badnavirus (BSV) and its association with genetically variable but otherwise qui-
escent integrated forms in the B genomes that are activated by factors located in the A genomes
(Lockhart and Olszewski, 1993; LaFleur et al., 1996; Dahal et al., 1998). Thus, interspecific hybrid-
ization may trigger genome interactions that are favorable to the transcriptional recombination of
dispersed BSV sequences into virulent episomes. Therefore, breeding emphasis would be on avoid-
ing bringing compatible genomes together in hybrid cultivars (Tenkouano et al., 2001).
9.5 Occurrence and Potential of 2n Gametes in Banana Breeding

Triploid breeding can take advantage of the occurrence of 2n gametes via chromosome doubling
during microsporogenesis, which has been observed in some diploid Musa accessions and is appar-
ently under genetic control (Ortiz, 1997b). Fertilization of a normal egg (n = x = 11) from a 2x
individual by a 2n pollen grain (n = 2x = 22) from another 2x plant produces a 3x progeny (2n = 3x
= 33), which is a process termed unilateral sexual polyploidization (USP). A major advantage of the
USP-based breeding is that genetic analyses will be essentially carried out at the 2x level, avoiding
complex inheritance patterns involving ploidy polymorphisms within and across generations.
The analysis of breeding populations at IITA revealed that nearly 21% of these population pro-
duced 2n pollen with highly significant (χ 2 = 597.8, P = 0.001) differences among genotypes.
Furthermore, 3x progenies were obtained from 2x × 2x crosses. Preliminary data suggests that such
3x hybrids may prove to be agronomically superior to their 2x counterparts derived from same or
similar crosses. For example, the cross ‘Wh-o-gu’ (2x) × ‘Calcutta 4’ (2x) has produced a 3x geno-
type ‘1968-2’ which is early maturing and has shown field resistance to black Sigatoka, weevils, and
nematodes. Another example is 25287-S28 (1448-1 × 1448-1) that displayed a bigger bunch associ-
ated with increased fruit length and circumference, compared to the diploid hybrids (Table 9.1).
This could have been a consequence of higher order interactions, leading to better exploitation of
recombinative heterosis.
In practice, breeding 3x hybrids via USP requires (1) population improvement at the 2x level to
accumulate favorable alleles in the 2x background, (2) screening of the 2x breeding populations for
2n pollen production, and (3) synchronizing pollination with periods that are favorable for 2n pollen
production. Hence, 4x × 2x crosses, coupled with recurrent 2x breeding to make better 2x sources of
resistance or other desirable traits, remain the predominant 3x breeding scheme.
9.6 Selection
Selection is the process whereby individuals carrying the genes of interest are identified on the basis
of the level of expression of the traits subtended by the genes. While this may seem straightforward
for many crop species, efficient selection in banana and plantain breeding requires sieving through
a diversity of ploidy and genome configurations.
Table 9.1
Performance of USP-Derived Triploid Hybrid (25287-S28) Compared to Diploid
Hybrids in a Preliminary Yield Trial at IITA High Rainfall Station, Onne,
Southeastern Nigeria
Bunch Weight Fruit Fruit
(kg) (no. of hands) (no. of fingers) Length (cm) Girth (cm)
Hybrid P R P R P R P R P R
Triploid
25287-S28 17.0 14.4 9 8 182 179 17.8 15.4 9.7 9.6
Diploids
25291–1A 6.5 15.8 8 11 167 291 10.7 14.4 7.5 9.4
25291-S26 10.2 – 8 – 177 – 16.8 – 9.1 –
25291-S32 15.1 14.5 13 14 262 287 16.8 14.0 8.3 9.0
25291-S41 5.6 10.3 10 12 151 226 12.2 14.2 8.1 8.0
25291-S62 9.1 14.0 10 12 168 246 18.6 17.2 9.2 8.6
25447-14 6.9 15.6 9 11 193 257 12.1 14.4 7.6 9.3
LSD (P = 0.05) 1.7 3.9 1 2 27 43 6.7 2.8 1.2 1.3
Note: P = plant crop; R = ratoon crop; LSD = least significant difference.
The multiploidy and heterogenomic structure of breeding populations results in unpredictable

variation in genome size and structure across and within generations (Tenkouano, 2001). This causes
complex inheritance patterns and complicates phenotypic selection for most yield and growth traits
(Ortiz and Vuylsteke, 1996). Breeders would gain in efficiency if they were able to assign segregat-
ing progenies to ploidy and genome classes putatively indicative of their potential use as plantain,
dessert, or cooking banana, prior to field evaluation.
9.6.1 Ploidy and Genome Analysis

Methods for unambiguous detection of the ploidy and genome status of Musa breeding popula-
tions have been extensively described. The most accurate and user friendly of these methods are
flow cytometry (Dolezel et al., 1994, 1997) and genome-specific DNA markers (Pillay et al., 2000,
2004; Nwakanma et al., 2003). The application of these methods is best carried out at the nursery
stage in juvenile plants, allowing for sorting the progenies into ploidy and genome classes, prior to
field establishment for performance evaluation, which typically takes place approximately 9 to 10
months following the date the cross was made.
9.6.2 Phenotypic Evaluation

Field evaluation follows a rather classical approach of phenotypic selection. Thus, seedlings from
crosses usually undergo a series of trials, starting with early evaluation trials (EET) that are mostly
nonreplicated trials in which different numbers of plants per genotype are evaluated during two
production cycles (1 cycle = year). This is followed by two cycles of clonal evaluation in preliminary
yield trials (PYT). At IITA, the PYT are replicated trials with each test genotype being represented
by four plants in each of three replications. Implication of farmers in field evaluation and selection at
this stage followed by on-farm validation of selected clones under farmers’ field conditions, together
with replicated multilocational reference evaluation trials (MET) at three to five locations, completes
the hybrid development process. The MET follows the same experimental details as the PYT.
Hybridization of newly introduced exotic material and

improved diploid, triploid, and tetraploid hybrids from
Musa breeding program in IITA and across the world
Black Sigatoka resistance,

Early Evaluation Trial bunch size, fruit parthenocarpy,
and dwarfness
As above plus earliness,

ratooning, pest and disease
Preliminary Yield Trial resistance, post harvest quality,
and durability
Multilocational As above plus stability of yield

Evaluation Trial and black Sigatoka resistance
International Musa
Regional Musa
Testing Program
Yield Trial
Coordinated by INIBAP
Nationality
Coordinated Trial
Cultivar Release
Figure 9.3 Typical flow diagram of the banana and plantain selection process: Each step may take approx-
imately 2 years, so that about 15 years may be required from crossing to release of an improved cultivar.
Furthermore, each plant occupies about 6 m2 of field space and less than 1% of the plants in the EET typically
make it to the next evaluation steps.
A variant of the MET is the evaluation of the best bet selections, usually through a farmer-
participatory approach outside research stations, to comply with requirements for official cultivar
release in nationally coordinated trials (NCT). The characteristics of the NCT are country specific.
Optional steps are regional or international yield trials that are carried out outside the countries
where the new cultivars have been bred. Figure 9.3 summarizes the salient features of the selection
process followed by the breeding programs operating in Africa, notably those of IITA.
The selection criteria are earliness, short plant stature, resistance to black Sigatoka, resistance to
nematodes, bunch weight significantly higher than the mean of the triploid breeding population, and
good plant vigor. Both ‘Calcutta 4’ and ‘Pisang Lilin’ are routinely included in the EET as resistant
controls. For 4x breeding, the EET and PYT also include the most popular landrace (for example,
‘Agbagba’ for plantain) as susceptible control and the best first-generation tetraploid hybrid (for
example, ‘PITA14’ for plantain) as the reference control. Specifically, breeders aim to maintain or
improve yield and resistance to black Sigatoka above the levels of the best tetraploid hybrids, reduce
plant stature to enhance the ability of the plant to withstand wind damage and reduce the need for
propping, and improve fruit quality (size, shape, and color) and the nutritional value of fresh and
processed fruits, using the most popular landrace as the target reference.
Genotype response to black Sigatoka infection is assessed under natural field conditions using
the youngest leaf spotted (YLS) method of Vakili (1968). Increasing YLS values indicate the pres-
ence of more healthy leaves on the plant and, hence, greater resistance to black Sigatoka. To adjust
for genetic differences in number of standing leaves (NSL), the index of nonspotted leaves (INSL)
is calculated as INSL = 100(YLS – 1)/NSL.
Nematode-resistance screening is carried out in parallel field trials using a method that is based
on the inoculation of individual roots (De Schutter et al., 2001). Screening trials usually include two
reference cultivars with known responses to R. similis, namely, ‘Valery’ (Musa AAA, Cavendish
subgroup), which is very susceptible to R. similis and ‘Yangambi Km 5’ (Musa AAA group), which
is resistant to R. similis (Sarah et al., 1992; Price, 1994; Fogain and Gowen, 1998). ‘Yangambi Km5’
is also used as a reference cultivar of the International Musa Testing Program (Orjeda, 2000). Based
on the assumption that susceptible plants allow a higher reproduction ratio of the nematodes than
resistant plants, test genotypes are classified as resistant (resp. susceptible) when the final nematode
population density is not significantly different from that of the resistant (resp. susceptible) check
but significantly different from that of the susceptible (resp. resistant) check. Otherwise, the test
genotype is considered partially resistant when its final nematode population density is significantly
different from those observed on both checks (Dochez et al., 2009).
Growth and yield variables are evaluated at flowering and at harvest, as described by Swennen
and De Langhe (1985). For each plant, data are recorded on bunch weight (BWT, kg) and its com-
ponents, days to flowering (DTF), days for fruit filling (that is, the number of days elapsed from
flowering to harvest [DFF]), plant height (PHT, cm), height of tallest sucker (HTS, cm), and total
number of leaves (TNL) at flowering. Three other variables are often calculated from collected data
in 6 m2 plots: yield (YLD = 1.667 × 365 × BWT/[DTF + DFF], t ha–1 yr–1), sucker growth index
(SGI = HTS/PHT), and leaf emission rate (LER = DTF/TNL, the number of days required for the
production of one leaf). The number of days to flowering is calculated as the time interval between
planting and flowering in the plant crop, and the interval between harvest of the previous crop and
flowering of the next in ratoon crops.
Postharvest characteristics are assessed using the methods of Dadzie and Orchard (1996). Of
recent, breeders have been interested in biofortification of banana and plantain by assessing micro-
nutrient content of fresh and processed fruits from field-grown plants, including total carotenoids,
iron, and zinc (Adeniji and Tenkouano, 2007; Honfo et al., 2007).
The completion of an evaluation cycle may take about 15 years from crossing to release of an
improved cultivar. Furthermore, each plant occupies about 6 m2 of field space and less than 1% of
the plants in the EET typically make it to the next evaluation steps. Thus, traditional banana and
plantain breeding has been quite inefficient in utilizing resources, and breeders have developed a
keen interest in improving the breeding process.
9.6.3 Improving Selection Efficiency

Essentially due to the lack of large segregating populations, early breeding efforts focused on
hybrid development rather than genetic analysis, except for a few qualitative traits. However,
knowledge of genetic parameters is essential in (1) designing crosses targeting the improvement of
specific traits, based on combining ability and heterosis, and (2) predicting the expected advance
from selection for these traits, based on heritability and genetic correlations among traits. However,
several advances have been made in understanding the inheritance of important traits in banana
and plantain and it is now possible to design crosses on the basis of combining ability (Ortiz,
1997a; Tenkouano et al., 1998a). Likewise, information on the meiotic behavior of prospective
parental stocks, their inbreeding status, and genetic relatedness have been estimated using a com-
bination of pedigree and molecular analyses, increasing the prospects for designing crosses with
maximum recombinative heterosis (Tenkouano et al., 1999; Tenkouano, 2005). For example, it
was observed that nongenetic effects accounted for a large proportion of the phenotypic variation
observed among hybrids from 4x × 2x crosses for most traits, except bunch weight (Tenkouano et
al., 1998b). General combining ability (GCA) effects were exclusively associated with the female
parents for plant height, number of leaves, suckering behavior, and fruit circumference, suggesting
that selection for these traits should be carried out in the prospective 4x female parents. Conversely,
GCA effects were primarily of paternal origin for fruit filling time, bunch weight, and fruit length.
Thus, breeding for these traits would be more efficient when carried out in prospective males at the
2x level. Specific combining ability (SCA) effects were observed for all traits, except fruit filling
time, suggesting that additional genetic gain could be achieved through recombinative heterosis for
these traits.
Most growth and yield characteristics of Musa display complex inheritance and genetic associa-
tion patterns and are subject to genotype-by-environment interactions. Powerful statistical genetics
tools have been used to further our knowledge of the phenotypic and genetic correlations between
indirectly and directly selected traits, in order to help determine whether an indirectly selected trait
will increase, decrease, or remain constant in advanced cycles of selection (Tenkouano et al., 2002).
It was suggested that selecting for specific adaptation based on site-specific selection indices con-
structed from site-specific genetic correlations may significantly enhance Musa breeding. A neu-
tral genotype-by-environment (GxE) interaction effect on the relationships between bunch weight
and yield components was reported, suggesting that indirect selection for bunch weight could be
achieved by selecting for yield components. Conversely, the predictive value of phenological traits
for yield was considered negligible, the only exception being the relatively high genetic correlations
between bunch weight and the number of leaves, which was not surprising given the role of leaves
in photosynthesis and in reaction to black Sigatoka.
Selection for specific rather than broad adaptation has been credited with the potential of achiev-
ing greater genetic gains (Simmonds, 1981). This is of particular relevance for farmers who often
are more interested in crop cultivars that ensure reliable performance from year to year in their local
environment and bother less about what happens at other locations.
Clearly, selection for most of these traits, particularly those that are expressed late in the plant
cycle or strongly influenced by the environment, would benefit from the use of molecular markers,
but progress in developing trait-specific molecular markers has been very limited. Nevertheless, a
considerable amount of genomic resources is becoming available (Vilarinhos et al., 2003; Kaemmer
et al., 2002; Safar et al., 2002).
9.7 Breeding Achievements

9.7.1 Substitutes for Commercial or Subsistence Production
The initial focus of Musa breeding was to develop improved dessert banana cultivars of commercial
value, but progress has been rather limited, partly due to genetic drag from the sources of resistance
to disease used in the breeding process. However, recent breakthroughs have been obtained by the
CIRAD program, based on their reconstructive breeding approach (Bakry, personal communica-
tion). Nonetheless, it is speculated that the banana industry would massively embrace the new cul-
tivars only if this does not impose drastic modification of the production to delivery logistics that
have been so well tuned to the existing Cavendish cultivars.
In contrast, past investments in breeding stand to benefit smallholder producers of banana and
plantain, with several new cultivars becoming available, particularly in the last decade (Tenkouano
and Swennen, 2004; Tenkouano et al., 2006). Undoubtedly, the most successful breeding program
has been that of the Fundación Hondureña de Investigación Agrícola (FHIA) based in Honduras.
The most publicized cultivars from this program are ‘FHIA-01,’ ‘FHIA-21,’ and ‘FHIA-25,’ which
18
Yield performance of 16
BLS-sensitive cultivars
and BLS-resistant 14
hybrids under natural 12
field conditions with
high disease pressure. 10
8
Even with fungicide
treatment, the farmer 6
cultivars produce less 4
than the hybrids that do
not require treatment. 2
0
Landrace Landrace Hybrid
not treated treated not treated
Figure 9.4 Illustration of progress in breeding for resistance to black Sigatoka.
have been tested worldwide and have been released for cultivation in several countries, notably in
Ghana and Uganda.
The oldest breeding programs in Africa are those of CARBAP and IITA, which both started
by assembling a considerable pool of local cultivars (more than 100 from various countries) and
sources of resistance to black Sigatoka (more than 200 from Southeast Asia where bananas and
the disease originated from) at their research stations in Cameroon and Nigeria, respectively. The
first IITA cross to transfer resistance to the local cultivars was made in 1987 (Swennen, personal
communication). A decade later, several improved plantain-derived hybrids with resistance to black
Sigatoka had been produced and tested in many countries, revealing the superior performance of the
hybrids under situations of high black Sigatoka (BLS) prevalence (Figure 9.4).
These efforts earned IITA the CGIAR’s 1994 King Baudouin Award. The most promising of
the first-generation plantain-derived hybrids has been ‘PITA14’ in Nigeria and neighboring coun-
tries. First-generation hybrids of the East African bananas did not perform better than the lan-
draces. Secondary 3x derived from the first-generation plantain or East African banana hybrids
were obtained, displaying lesser propensity to forming seeds and greater culinary resemblance to
the prevailing landraces (Tenkouano et al., 2006), but adoption prospects only increased with the
distribution of the new cultivars through a scheme that was nondisruptive to the farmers’ practice
of cultivar mixtures and through the promotion of novel postharvest processing options. Apart from
the hybrids’ high yields and disease resistance, farmers appreciate their capacity for rapid multipli-
cation (a result of excellent proliferation of the rhizomes, or “suckers,” used for propagation), their
good taste and cooking qualities, and their resistance to other diseases and pests. Some farmers who
have adopted the hybrids are generating significant income from the sale of suckers, in addition to
boosting returns from the sale of fruit (Tenkouano et al., 2006). This is discussed in more detail
in Chapter 16 of this book. The CARBAP program has also developed a series of first-generation
hybrids, among which the most promising has been ‘CRBP39,’ but these 4x hybrids presented sev-
eral defects that prevented their diffusion, and some of them are being used as intermediate prod-
ucts for further improvement (Noupadja et al., 2007).
9.7.2 Genetic Stocks for Breeding

The limited success of the first-generation hybrids was largely attributed to defects they inherited
from their wild 2x parents. Thus, major efforts were devoted to developing improved 2x by stacking
desirable alleles while decreasing the extent of wildness. Such improved 2x could then be used for
transfer of resistance genes to the landraces without the genetic drag of undesirable traits.
Notorious progress in this area has been achieved (Ortiz and Vuylsteke, 1995; Krishnamoorthy
and Kumar, 2005), but the products from these efforts have not been widely distributed outside the
program that developed them. To date, only the improved 2x stocks from the IITA program have
been placed in the public domain and have been accessible worldwide.
Genetically, many of the 2x stocks of the first generation displayed excellent levels of resistance
to black Sigatoka but had unappealing agronomic or fruit characteristics (Figure 9.5 top). However,
it is possible to correct this situation and bring together productivity and resistance traits (Figure 9.5
bottom) to produce 2x stocks with greater potential for use in the improvement of 3x cultivars.
9.8 Breeding Constraints

Despite these advances, many problems specific to the biology of plantains and bananas impede
rapid breeding progress, including low reproductive fertility, triploidy, and slow propagation
(Vuylsteke et al., 1997). Thus, breeders may not be able to access all available variation, largely due
to the high sterility of many cultivated cultivars. The conundrum of banana breeding is that seed
set is required to produce seedless cultivars. Improving female or male sterile accessions can only
be achieved by circumventing reproductive barriers, for example, by deliberate mutagenesis or by
direct gene transfer through genetic engineering. Ideally, genetic engineering would provide a more
deterministic approach for substituting undesirable genetic materials with those that would enhance
the performance of the recipient line or cultivar with respect to a particular constraint.
Additionally, the available germplasm may not harbor the desired genes with respect to an emerg-
ing threat, which is clearly the case for the bacterial wilt caused by Xanthomonas campestris pv.
musacearum that is threatening livelihoods in the Great Lakes region of Central and Eastern Africa.
There is little that crossbreeding can do to address such a threat in the near future, leaving genetic
engineering as the best alternative to control the bacterial wilt epidemics through host resistance.
The crossbreeding approach is time consuming, given the long generation time of the crop; it
is also technically complex, since it requires both ploidy and genome selection. With the advent of
flow cytometric analysis of nuclear DNA content (Dolezel, 1997) and molecular markers for the A
and B genomes (Pillay et al., 2000), ploidy and genome selection have become easier. However, pre-
dicting progeny performance remains a major challenge when ploidy and genome variation occurs
within and across generations (Tenkouano et al., 1999a, 1999b). Until very recently, there were few
assessments on parent–offspring relationships so that parental contribution to offspring and ensuing
patterns of ploidy segregation were not well understood.
Microsporogenis studies (Oselebe et al., 2001) revealed that 2x individuals produced essentially
haploid gametes (n = 1x =1C), whereas 4x individuals produced mostly hypoploid gametes with half
the chromosome content of the haploid class that would have been expected with normal meiosis,
which would only result from double reduction (2n = 4x = 4C � n = 2x = 2C � 0.5n = 1x = 1C).
Other findings by the IITA research team were that euploid gametes with two or four times the basic
chromosome set were also produced by the 2x individuals, presumably through first divisional res-
titution (FDR) or second divisional restitution (SDR) or both (double restitution), the first two events
producing 2n gametes and the last event 4n gametes. Progeny analysis showed that 4x × 2x crosses
mostly produced 3x progenies, against mostly 2x progenies for 2x × 4x and 2x × 2x crosses, with very
low frequencies of 4x and 5x progenies. It was concluded that parental contribution to progenies was
unequal with respect to 3x progeny (n = 2x = 2C by the 4x parent plus n = 1x = 1C by the 2x parent),
but equal with respect to the parents, both contributing half of their endowment in 4x × 2x crosses.
Conversely, parental contribution was equal with respect to the progeny (n = 1x = 1C plus n = 1x =
1C) but unequal with respect to the parents in 2x × 4x crosses, with the 2x parent donating half of
its endowment and the 4x parent a quarter of its endowment. In 2x × 2x, 2x progeny would result
from equal parental contribution with respect to both parental and progeny endowment, whereas, 3x
progeny obtained by unilateral sexual polyploidization (2n pollen) would be attributed to unequal
parental contributions with respect to both parental and progeny endowments. These studies further
PC1 = 53%, PC2 = 20.5%, Sum = 73.5%

1.6 Transform = 0, Scaling = 1, Centering = 2, SVP = 2
Bc4
1.2
HI
0.8
FLT
0.4 Bc9 Bc2
CI Bc8 FCR
PC2
Bc3
0.0 INSL Bc6 BWT
Bc5 Bc7
DFF
–0.4
Bc10 Bc1
–0.8 LRI
Bc11
PHT
–1.2
PGT
–1.6 –1.2 –0.8 –0.4 0.0 0.4 0.8 1.2 1.6 2.0 2.4
PC1
PC1 = 40.6%, PC2 = 22%, Sum = 62.6%

Transform = 0, Scaling = 1, Centering = 2, SVP = 2
1.6
PGT
LRI
1.2
S9
PHT
BWT
0.8 S7
S4
PC2
INSL
S2 S3 FLT
0.4
DFF
FCR
0.0
S13
S10
S12 HI
–0.4 S6
S1
S11
S8 S3 CI
–0.8
–1.6 –1.2 –0.8 –0.4 0.0 0.4 0.8 1.2 1.6 2.0 2.4
PC1
Figure 9.5 GGE biplot analysis of trait association in diploid banana and plantain hybrids. Primary hybrids
derived from the cross ‘Bobby Tannap’ × ‘Calcutta 4’ often display resistance traits in opposition relation to
productivity traits (top). Crossing selected primary hybrids with unrelated stocks from the same generation
(e.g., ‘Tjau Lagada’ × ‘Calcutta 4’) produces secondary hybrids that combine resilience and productivity traits
(bottom). (From Daniel N. Igili. Progress in Breeding Diploid Genetic Stocks of Banana with Resistance to
Black Sigatoka, master’s thesis, University of Nigeria, Nsukka.)
suggested that 3x progeny could also be derived from 4x × 4x crosses, a scheme that has not been
frequently used but is now being considered for the improvement of banana and plantains (Pillay,
unpublished). Macrosporogenesis was not directly examined but has been assumed to be essentially
normal in both 2x and 4x individuals, attested by the predominantly 2x nature of progenies from
2x × 2x crosses and the reduced frequency of 2x progenies from 4x × 2x crosses. Knowledge of the
meiotic behavior of breeding stocks could thus allow for designing informed crosses with highest
probability of generating 3x lines with putative plantain, dessert, or cooking banana labels.
9.9 Future Breeding Goals

Despite the evolving nature of biotic constraints, black Sigatoka remains at the forefront of the con-
straints to commercial and subsistence production of banana and plantain. However, overcoming
this problem through genetics is no longer a challenge. Breeders have been looking at refined ways
of utilizing the genes embedded in the available improved stocks. Likewise, the focus of breeding
is gradually shifting from addressing existing constraints to assessing the risk potential of emerging
threats and preparing to respond to these.
For decades, banana and plantain research has centered mainly on the development of tech-
nologies that can help boost and stabilize production. As farmers gain better access to new germ
plasm and knowledge generated by that research, they will be better able not only to enhance
crop productivity but also to strengthen their ties with markets for surplus production. New
opportunities to accomplish both ends are rapidly emerging. A major challenge is to find ways of
ensuring that small farmers are able to capture benefits from such developments through stronger
market links.
Thus, breeding is now conceived as a component of a larger, integrated effort aimed at devel-
oping and promoting market-preferred cultivars. This genetic-improvement undertaking is done
through the following options, which constitute emerging orientations of the existing programs:
• Preemptive breeding (germplasm diversification) to counteract emerging and shifting bio-

logical and environmental constraints, in close association with:
• Development of upstream prebreeding methods (e.g., anther or ovule culture for
production of haploid lines, haplo-diploidization with support from cytogenetics) to
recover useful progenies while minimizing wildness
• Search for novel genes (drought, bacterial, and viral diseases) through judicious assem-
bly and mining of agro-biodiversity of crop species and biotic threats
• Deployment of pest and disease management and monitoring schemes (increased alertness
to threats), coupled with:
• Development of cost-effective alternatives that combine environmental friendliness
with human health safety for prevalent production systems
• Design of early alert systems based on predictive analysis of biological shifts in pest
and pathogen populations
• Seed systems, including technical and policy-related issues, combined with germplasm
(source populations and genetically stabilized cultivars) testing through networks
• Development of standards and protocols for cost-effective dissemination of improved
germplasm, including efficient cross-border exchange of plant propagules (seeds, veg-
etative materials)
• Promotion of traceability mechanisms that protect intellectual property and ensure
visibility, e.g., facilitating cultivar release processes that are more agile to ensure effec-
tive delivery, facilitating the emergence of a commercial seed system
• Crop utilization and product development, including health-related issues (mycotoxins,

nutrition) and trade-related issues (commodity policy) with emphasis on:
• Development of sustainable systems for intensification of production as more effort is
directed to industrial use
• Assessment of the distribution of the toxic organisms and promotion of remedial tech-
nologies and standards
The purpose of Musa breeding has been to develop new cultivars resistant to diseases. However, Musa
breeders very well know that, even with widespread adoption of new cultivars by farmers, decreases in
soil fertility would still remain an obstacle to the profitable cultivation of banana and plantains.
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Research Highlights 7:3.
10 Mutations and Cultivar
Development of Banana
Shri Mohan Jain, Bradley Till,
Prasnna Suprasanna, and Nicolas Roux
Contents
10.1 Introduction...........................................................................................................................203
10.2 Mutation Induction................................................................................................................204
10.2.1 Methods on Mutation Induction................................................................................204
10.2.2 Choice of Plant Material............................................................................................206
10.2.2.1 Shoot Tips...................................................................................................207
10.2.2.2 Embryogenic Cell Suspensions...................................................................207
10.2.3 Establishment of a Radiosensitive Curve..................................................................208
10.3 Postmutagenesis.....................................................................................................................209
10.3.1 Dissociation of Chimera............................................................................................209
10.3.2 In Vitro Selection....................................................................................................... 210
10.4 Reverse-Genetic Strategies for Banana Using Induced Mutations........................................ 211
10.5 Conclusions and Prospects.................................................................................................... 214
References....................................................................................................................................... 214
10.1 Introduction
Spontaneous genetic variation has contributed to the origin of present-day Musa. Spontaneous
mutations are considered to have played a role in the origin of almost all of the edible banana and
plantain varieties (Buddenhagen, 1987). A spontaneous Cavendish mutant, resistant to Fusarium
wilt (race 1), that originated in Vietnam replaced ‘Gros Michel’ in 1950s–60s (Ploetz, 1994) as the
major export banana and saved the commercial banana industry from collapse. Development of new
banana clones with improved agronomic features has been slow due to the complex and polyploid
nature of the Musa genome as well as sterility barriers and other obstacles to conventional breeding
approaches (Pillay and Tripathi, 2006, 2007). Breeding efforts have produced only a few cultivars
through selection of improved dessert and cooking bananas and plantains (Rowe, 1984; Vuylsteke
et al., 1995). Concerted efforts are being made to develop new banana cultivars by a number of
breeding programs.
In vegetatively propagated crops like banana that have a narrow genetic base (Pillay et al.,
2001), it is essential to generate additional genetic variability to facilitate selection of desirable
traits. Many techniques involving cellular and molecular biological tools such as in vitro plant
regeneration, mutagenesis, transgenics, and molecular markers have been deployed for crop
improvement (Jain and Swennen, 2004). Induced mutagenesis and in vitro selection for desired
traits offer several advantages such as mutagenizing the plant parts, uniform mutagen treat-
ment, handling large number of samples in a short time span, rapid production of large popula-
tions to separate chimeras, and facilitating in vitro selection (Van Harten, 1998). Currently, it
203
is believed that in vitro mutation breeding programs can deliver more promising results (Smith
et al., 2005, 2006).
There is rapid accumulation of genome sequence information for many crop species, including
banana. This has allowed the development of hypotheses for gene function based on homologies
between different organisms. Reverse-genetic techniques target disruptions in specific loci, provid-
ing in vivo testing of gene function and the development of crops with novel traits (for example,
Enns et al., 2005; Slade et al., 2005). Targeting induced local lesions in genomes (TILLING) is a
general reverse-genetics strategy utilizing traditional mutagenesis and high-throughput mutation
discovery that has been applied to many species (McCallum et al., 2000; Colbert et al., 2001).
TILLING provides a method to combine the power of induced mutations with expanding sequence
information for functional genomics and crop improvement projects. Challenges exist for the effi-
cient adaptation of TILLING for banana, and several approaches are being evaluated. While the
development of large mutant populations in banana can be considered a bottleneck, reverse-genetics
may provide a practical means for genetic studies and plant improvement.
10.2 Mutation Induction
The combination of mutation breeding and in vitro culture (also called in vitro mutagenesis) makes
the induction and selection of induced somatic mutations more effective. This method can also
aid in further propagation, which will ensure the formation of periclinal chimeras or homohistont
individuals. Furthermore, increased recovery of plantlets through decreased somatic competition
can be obtained by modifying culture conditions. Plant growth regulators, and in particular a cyto-
kinin-enriched medium, can increase the recovery rate of mutated cells. Hence, the combined use
of mutation induction and in vitro technology is more efficient because it speeds up the production
of mutants as a result of an increased propagation rate and a greater number of generations per unit
time and space (Morpurgo et al., 1997).
10.2.1 Methods on Mutation Induction

Mutagens cause random changes in the nuclear or cytoplasmic DNA, resulting in gene, chromo-
somal, or genomic mutations (Jain and Maluszynski, 2004). Both physical and chemical mutagens
have been employed for mutagenesis in banana for generating useful mutations (see Table 10.1 and
Table 10.2). Physical mutagens such as gamma- and x-rays have been useful for mutation induction
(Kulkarni et al., 1997; Roux et al., 2004). Physical mutagens, especially gamma rays, show reason-
able reproducibility and have a high and uniform penetration of multicellular systems. However,
selection of a mutagen is dependent on the type of plant material, such as organs (shoot tips, mer-
istems, axillary buds), cell suspension cultures, or protoplasts. Although gamma rays have been
commonly used for physical mutagenesis, ion-beam techniques have recently been described. Ion
beams integrate the factors of mass, energy, and charge, inducing damage to the biological materi-
als, thereby displacing, recombining, and compounding the biological molecules and atoms. Ion
beams can frequently produce large DNA alterations such as inversions, translocations, and large
deletions rather than point mutations. Reyes-Borja et al. (2007) were the first to report the use of
ion-beam irradiation for mutation breeding in banana by selecting lines tolerant to black Sigatoka.
The effect of irradiation doses on the regeneration of plantlets was investigated in Japan, and the
variation of the black Sigatoka response under field conditions was evaluated in Ecuador (Reyes-
Borja et al., 2007). Chemical mutagens can also be used, producing a high density of predominantly
point mutations. The problems involved in using physical mutagens are the high cost of radiation
source requirement and the simultaneous induction of chromosomal and gene mutations. Chemical
mutagens are carcinogenic in nature and penetrate multicellular tissues nonuniformly.
Mutations and Cultivar Development of Banana 205
TABLE 10.1
Studies on Mutation Induction Using Chemical and Physical Mutagens in Banana
Cultivar/
Genomic Observed Responses/ Mutant/Clones
Target Material Group Mutagen Dose Mutation(s) Developed Reference
Excised shoot tips SH-3362 (AA); EMS 24.69 mM Number of newly — 1
GN-60A initiated adventitious
(mutant of buds decreased with
Grande Naine, increased EMS
AAA) concentrations
Shoot tips of in Highgate, AAA -Sodium azide Tolerance to Fusarium Tolerant clones for 2
vitro grown Group (NaN3) 2.3 oxysporum f. sp. field screening
cultures mM cubense
-diethyl sulphate
(DES)-20 mM
-EMS 200 mM.
Shoot tips Grande Naine, Gamma rays Earliness Early flowering 3
AAA putative mutant
designated
GN-60A
Shoot tips Gamma rays 25 Bunch size and Klue Hom Thong 4
Gy cylindrical shape KU1
Shoot tips Dwarf Parfitt, an Gamma rays 20 Improved agronomic Improved lines with 5
extra-dwarf Gy characteristics (taller productivity and
Cavendish plant size, increased resistance
banana yield and no choking)
Shoot tips Highgate, AAA Gamma rays Tolerance to Fusarium Tolerant clones for 6
8–20 Gy oxysporum f. sp. field screening
cubense
Protocorm like Nanicao, AAA Gamma rays Aluminum tolerance Tolerant selections 7
bodies 2kR
In vitro cultured Diploid and Gamma rays 25 Diploid clones were Plants with 8
shoot tips tetraploid Gy more sensitive than morphological and
clones tetraploids physiological traits
Tissue cultured Dwarf Gamma rays Morphological traits 22 clones for 9
shoot tips Cavendish, 8–20 Gy different
AAA morphological
traits
In vitro cultured Basrai, AAA Gamma rays Morphological traits Clones for different 10
shoot tips morphological
traits
Excised shoot tips Basrai, AAA Gamma rays Differential response of — 11
in vivo and in in vivo and in vitro
vitro plant materials
In vitro Williams and Carbon ion Fungal disease Resistant plants to 12
propagated shoot Cavendish beam 0, 0.5, 1, resistance black Sigatoka
tips Enano 2, 4, 8, 16, 32,
64 and 128 Gy
(continued)
TABLE 10.1 (Continued)
Studies on Mutation Induction Using Chemical and Physical Mutagens in Banana
Cultivar/
Genomic Observed Responses/ Mutant/Clones
Target Material Group Mutagen Dose Mutation(s) Developed Reference
In vitro shoot Basari, AAA, Gamma rays Morphological traits Morphological 13
cultures Chakkarakela, 30Gy variations: thick
AAB, and (recurrent shiny dark green
Rasthali, AAB dose) leaves, ovate leaves
and a dwarf with a
rosette of leaves
Sources: Data from (1) Omar, M.S., F.J. Novak, and H. Brunner, 1989, In vitro action of ethyl-methanesulphonate on banana
shoot tips, Sci. Hort., 40, 283–295. (2) Bhagwat, B. and E.J. Duncan, 1998, Mutation breeding of banana cv.
Highgate (Musa acuminata, AAA group) for tolerance to Fusarium oxysporum f. sp. cubense using chemical muta-
gens, Sci. Hort., 73, 11–22. (3) Roux, N., R. Afza, H. Brunner, R. Morpurgo, and M. van Duren, 1994, Complementary
approaches to cross-breeding and mutation breeding for Musa improvement, 213–218, Proceedings of the first
global conference on International Musa Testing Program, FHIA, Honduras. (4) List of new mutant cultivars; Musa
sp. (banana), 1990, Mutation Breed. Newslett., 35, 32–41. (5) Smith, M.K., S.D. Hamill, P.W. Langdon, J.E. Giles,
V.J. Doogan, and K.G. Pegg, 2006, Towards the development of a Cavendish banana resistant to race 4 of Fusarium
wilt: Gamma irradiation of micropropagated Dwarf Parfitt (Musa spp., AAA group, Cavendish subgroup), Aus. J.
Exp. Agri., 46, 107–113. (6) Bhagwat, B. and E.J. Duncan, 1998, Mutation breeding of Highgate (Musa acuminata,
AAA) for tolerance to Fusarium oxysporum f. sp. cubense using gamma irradiation, Euphytica, 101, 143–150. (7)
Matsumoto, K. and H. Yamaguchi, 1990, Selection of aluminium-tolerant variants from irradiated protocorm-like
bodies in banana, Trop. Agric. (Trinidad), 67, 229–232. (8) Novak, F.J., R. Afza, M. Van Duren, and M.S. Omar,
1990, Mutation induction by gamma irradiation of in vitro cultured shoot-tips of banana and plantain (Musa cvs),
Trop. Agric. (Trinidad), 67, 21–28. (9) Miri, S.M., A. Mousavi, R. Mohammad, A. Naghavi, M. Mirzaii, A.R.
Talaei, et al., 2009, Analysis of induced mutants of salinity resistant banana (Musa acuminata cv. Dwarf Cavendish)
using morphological and molecular markers, Iranian J. Biotech., 7, 86–92. (10) Kulkarni, V.M., T.R. Ganapathi, P.
Suprasanna, V.A. Bapat, and P.S. Rao, 1997, Effect of gamma irradiation on in vitro multiple shoot cultures of
banana (Musa species), J. Nucl. Agric. Biol., 26, 232–240. (11) Karmarkar, V.M., V.M. Kulkarni, P. Suprasanna,
V.A. Bapat, and P.S. Rao, 2001, Radio-sensitivity of in vivo and in vitro cultures of banana cv. Basrai (AAA),
Fruits, 56, 67–74. (12) Reyes-Borja, W.O., I. Sotomayor, D. Garzón, M. Vera, B. Cedeño, A. Castillo, et al., 2007,
Alteration of resistance to black Sigatoka (Mycosphaerella fijiensis Morelet) in banana by in vitro irradiation using
carbon ion-beam, Plant Biotech., 24, 349–353. (13) Mishra, P.J., T.R. Ganapathi, P. Suprasanna, and V.A. Bapat,
2007, Effect of single and recurrent gamma irradiation on in vitro shoot cultures of banana, Int. J. Fruit Sci., 7,
47–57.
10.2.2 Choice of Plant Material

Both in vivo and in vitro cultures have been used to induce mutations in banana. Earlier studies
used seeds and suckers of plantains and bananas, including the diploid Musa balbisiana, and dem-
onstrated that gamma rays affected the rates of seed germination and seedling survival (Stotzky
et al., 1964). The chemical mutagen ethyl methanesulphonate (EMS) was used in seeds of diploid
M. acuminata (Menendez, 1973). Irradiation of suckers, prior to excision of shoot tips for in vitro
culture, generally gives a low yield of mutagenized material for further screening. De Guzman et al.
(1976) reported that viable shoots could be obtained from suckers irradiated with a dose of up to 100
Gy. In situ sucker irradiation before initiation of tissue culture was not effective and yielded only a
low number of regenerants in the M1V1 generation. In a comparative study on the radiosensitivity of
different in vivo and in vitro planting materials, Karmarkar et al. (2001) observed a decrease in per-
cent survival of irradiated material with increasing irradiation dose. The hardened plants were more
sensitive than suckers, while individual in vitro shoots were least sensitive to increases in doses of
Table 10.2
Examples of Desirable Variants/Putative Mutants Identified for Release or Further
Confirmation Trials
Country Parent/Selection Traits Technique Place of Induction
Cuba SH3436 (AAAB)/‘SH3436 Reduced height Gamma rays Cuba
Parecido al Rey (AAA)/ Reduced height Gamma rays IAEA
Parecido al Rey 6.44
Malaysia Pisang Rastali (AAB)/Mutiara Tolerance to Foc race 4 Somaclones United Plantation
Bhd., Malaysia
Grande Naine GN-GoA Tolerance to Foc race 4 Somaclones
(AAA)/Novaria
Pisang Berangan Early flowering and Somaclones
reduced height
Pisang Berangan Tolerance to Foc race 4 Gamma rays IAEA
Pisang Mas Tetraploid Colchicine
Philippines Lakatan (AAA) Reduced height and Gamma 40 Gy IAEA
earliness
Latundan (AAB) Large fruit size and 3 Gy fast
reduced height neutrons
Sri Lanka Embul (AAB)/Embul Earliness and reduced Gamma rays Sri Lanka
height
Source: Jain, S.M. and M. Maluszyuynski, 2004, Induced mutations and biotechnology in improving crops. In: In vitro
application in crop improvement, A. Mujib, M. Cho, S Predieri and S. Banerjee, Eds., 169–202. Science Publishers;
Plymouth, UK. With permission.
gamma irradiation. Irradiation of in vitro multiple shoots adversely affected multiplication, except
for doses of 10–20 Gy, which were observed to significantly enhance multiplication.
10.2.2.1 Shoot Tips
Shoot tips propagated in vitro have also been used for chemical mutagenesis. Omar et al. (1989)
studied the effects of EMS on the growth and development of excised shoot tips of two banana
clones: ‘SH-3362’ (AA) and ‘GN-60A,’ a mutant of ‘Grande Naine’ (AAA). The effects of EMS
on fresh weight and on the number of newly initiated adventitious buds of cultured shoot tips were
evident after 30 days of incubation. On the basis of different studies, a suitable level of EMS treat-
ment for banana clones appears to be 12.41–37.23 mM for 1 to 3 h. Bhagwat and Duncan (1998a)
compared the effect of three chemical mutagens—namely sodium azide (NaN3), diethyl sulphate
(DES), and EMS—at various concentrations on shoot tips of in vitro grown cultures of the cultivar
‘Highgate’ (AAA). On the basis of the number of apices that survived the treatment and the num-
ber of regenerated shoots, the authors found that the mutagens differed in their mutation induction
efficiency. The highest factor of effectiveness (7.8%) was obtained with NaN3 resulting in 63.3%
explant survival and 58.9% shoot regeneration, while DES gave 65.5% survival and 38.2% shoot
regeneration, and EMS gave 5.8%, 80% explant survival, and 31.6% shoot regeneration.
10.2.2.2 Embryogenic Cell Suspensions

Embryogenic cell suspension (ECS) cultures can also be used for the induction of mutations with
physical (Kulkarni et al., 2004, 2007) and chemical mutagens. Since ECS is of single-cell origin,
the number of regenerated chimeric plants is reduced, allowing rapid generation of homohistonts or
nonchimeric plants. Over a million embryogenic cells per ml can be treated with mutagens, directly
producing mutant somatic embryos that regenerate to mutant plantlets. In vitro direct selection
pressure can be applied on mutagen-treated cells for the selection of mutant somatic embryos. The
initiation of somatic embryogenic cultures can commence by culturing a wide variety of explants
including zygotic embryos, nucellus, parts of seeds and fruits, inflorescences, leaf pieces, stem seg-
ments, protoplasts, and microspores. Embryogenic callus is subcultured in a liquid medium on a
shaker to develop fine cell suspensions (Kulkarni et al., 2007). Superior quality embryogenic cell
suspensions are usually characterized by the presence of clusters of round and densely stained cells,
and absence or minimum number of enlarged, elongated, and vacuolated cells and debris (Roux
et al., 2004). The embryogenic cells are uniformly spread on agar-solidified culture medium for
physical mutagen treatment and subsequent somatic embryo formation. Well-developed somatic
embryos are germinated to obtain complete plants (somatic seedlings), which can be acclimatized
and transferred to the field. Embryo formation is not synchronous, and therefore large and small
embryoids coexist. The uniformity of populations can be improved by culturing on media contain-
ing high levels of sucrose and/or low levels of abscisic acid. High levels of sucrose and abscisic acid
induce reversible dormancy in somatic embryos and thus might be used to temporarily suspend the
growth for synchronous development.
10.2.3 Establishment of a Radiosensitive Curve

One of the first steps in mutagenesis experiments is to determine the appropriate mutagen dose.
For physical mutagens, this is generally based on understanding the radiosensitivity of the tissues/
cells. Theoretically, the highest frequency of mutations can be expected from a mutagen treatment,
killing about 50% of the treated materials (LD50) (Van Harten, 1998), and hence the LD50 dose
of the mutagen is obtained from a radiosensitive curve (Figure 10.1). The plant material (shoot
multiples, embryogenic calli, or cell suspensions) are treated over a wide range of doses, and data
are recorded on traits such as fresh and dry weight gain, survival of treated cultures, number of
120
GRANDE NAINE
(AAA)
100
80
Percentage of control
60
40
20
0
0 20 40 60 80 100 120
Radiation dose (Gy)
Figure 10.1 Radiosensitivity test curve illustrating the effect of increasing dose of gamma rays on survival
rate of shoot tips from the cultivar ‘Grande Naine’ (AAA). Data are expressed in percentage of control (nonir-
radiated shoot tips). (Reprinted from N. Roux, A. Toloza, J. Doležel, and B. Panis, Usefulness of embryogenic
cell suspension cultures for the induction and selection of mutations in Musa spp., Banana improvement:
Cellular molecular biology and induced mutations, Enfield, NH: Science Publishers, 2004, 33–34. With
permission.)
complete plantlets regenerated, and LD50 values estimated (Jain, 2009; Predieri, 2001). Different
genotypes respond differently to mutagenesis of shoot tips, and optimal dose (LD50) has been found
to vary considerably. Roux (1997) recommended the following doses for different ploidy levels and
banana genomic groups:
• 10–20 Gy of γ rays for diploid clones ‘Calcutta 4’ (AA) and ‘Tani’ (BB).
• 30–40 Gy of γ rays for the triploids ‘Three Hand Planty’ (AAB), ‘Grande Naine’ (AAA),
‘Williams’ (AAA).
• 40–50 Gy of γ rays for the triploid ‘Cachaco’ (ABB).
Less is known about optimal dosages for chemical mutagenesis, but TILLING provides a method to
directly assay the density and spectrum of induced nucleotide changes in direct response to mutagen
type and dose (see Section 10.4).
10.3 Postmutagenesis
Postmutagenesis handling of mutagenized populations is critical as in vitro mutagenesis of mul-
ticellular meristems of Musa spp. leads to a high degree of chimerism. In general, mutated cells
are difficult to monitor. However, mutations that result in a change in chromosome numbers are an
exception. The mutagen treatment of plant organs often leads to chimeras that require dissociation
by subsequent subcultures of shoot cultures up to the M1V4 generations (Kulkarni et al., 2007). The
number of subcultures depends primarily on the genotype, LD50 dose, and other factors such as
proliferation rate, number of plants to be field evaluated, and so forth. In vitro shoots can also be
directly rooted in the greenhouse under 70–90% humidity to avoid the additional step of in vitro
rooting. The number of plants developed is dependent on the greenhouse facilities.
10.3.1 Dissociation of Chimera
In an effort to dissociate chimeras, Roux et al. (2001) assessed three different propagation systems
(shoot-tip culture technique, multi-apexing culture technique, and corm-slice culture technique).
The average percentage of cytochimera was reduced from 100% to 36% after three subcultures
using shoot-tip culture, from 100% to 8% after the same number of subcultures using the multi-
apexing technique, and from 100% to 24% when propagating by the corm-slice culture technique.
Although none of the systems studied eliminated chimerism completely, the study showed that the
possibilities of reducing chimeras depend on the type of shoot produced (axillary or adventitious)
and the multiplication rate (number of new shoots produced per subculture). Nevertheless, in all
cases, after three subcultures the proportion of chimeras tends to stabilize. This is due to the forma-
tion of periclinal chimeras, which are difficult to eliminate using multicellular propagation systems.
In a multicellular propagation system, it is expected that more than three to four subcultures will
not significantly increase the dissociation of chimeras, and it will not be possible to reach 0% chi-
mera as long as mitotic divisions maintain cell layer identity. Although embryogenic cell suspen-
sions (ECS) can be a useful system for producing mutants in banana, the system has to be fully
optimized. Compared to shoot-meristem cultures, which often yield chimeras, ECS allow large cell
populations to be used for mutagenesis under controlled conditions, and chimerism can be avoided
as somatic embryos are mostly derived from single cells. Roux et al. (2007) demonstrated that
colchicine treatment of ECS followed by flow cytometry of regenerated plants produced no mixop-
loid plants (chimeric). ECS fresh weight gain and regeneration capacity indicated that the optimal
irradiation dose ranged from 50 to 75 Gy for ‘Williams’ (AAA, Cavendish subgroup) and ‘Three
Hand Planty’ (AAB, plantain subgroup). Xu et al. (2008) found that plant regeneration capacity also
decreased with an increase in radiation dosage in ECS of ‘Beida Aijiao’ (Musa AAA). At the dose
of 60 Gy, ECS did not survive on embryo regeneration medium. It was shown that plant regeneration
capacity of a growing cell mass was much higher than that of ECS directly radiated with gamma ray
at four radiation dosages, suggesting that cultures of ECS on embryo regeneration medium could
reduce the sensitivity of ECS to gamma radiation. Somaclonal variation, probably associated with
chromosome instability, can also occur among plantlets produced from ECS and could interfere
with this approach for producing mutants in banana. Variable DNA ploidy was detected in ECS and
in regenerated plants (Roux et al., 2007). The random nature of mutation warrants the screening of
thousands of individuals when taking a forward-genetic approach. The application of banana ECS
in mutagenesis research should be able to increase the efficacy of mutation induction and this could
revolutionize the isolation and screening of new and useful mutants.
10.3.2 In Vitro Selection

Mutagenesis combined with in vitro selection is generally regarded as an appropriate tool for
enhancing and accelerating induction, selection, and recovery of desirable mutants (Suprasanna
et al., 2008). Based upon the information generated on radio sensitivity of different banana cul-
tivars, researchers have attempted to employ irradiated in vitro cultures for in vitro selection, for
example, for aluminum stress (Matsumoto and Yamaguchi, 1990). In another study, Roux and
Toloza (2002) applied radiation-induced mutagenesis to select plants resistant to black Sigatoka.
Four batches (100 meristems/batch) of the variety ‘Grande Naine’ were irradiated at 35 Gy and
further propagated during four subcultures. The plants were then acclimatized in the greenhouse.
The early mass screening method was adopted by using infiltration of juglone, a toxic metabolite of
Mycosphaerella fijiensis. After screening approximately 4,000 plants, 15 putative mutants showed
tolerance (Figure 10.2). Table 10.2 shows various banana mutant varieties/lines have been released/
isolated in several countries. In Malaysia, ‘Mutiara’ (Figure 10.3) and ‘Novaria’ banana mutant
cultivars have been released commercially by United Plantation Bhd.
(a) (b)
Figure 10.2 Regenerated plants from irradiated shoot tips of the cultivar ‘Grande Naine’: (A) Susceptible
to 25 ppm juglone (5-hydroxy-1,4-naphthoquinone), a toxic metabolite of Mycosphaerella fijiensis, the fungus
responsible for the black Sigatoka disease. (B) Putative mutant, tolerant to 25 ppm juglone. Note the increased
content of anthocyanin. (Reprinted from N. Roux et al. (2003). Mutagenesis and somaclonal variation to
develop new resistance to Mycosphaerella leaf spot diseases on Musa. In: Proceedings of 2nd International
Workshop on Mycosphaerella Leaf Spot Diseases of Bananas, San Jose, Costa Rica, 20−23 May 2002:
239−250. With permission.)
Figure 10.3 Tolerant ‘Mutiara’ banana plants surviving in Fusarium wilt-infected hot spot, developed in
Malaysia.
10.4 Reverse-Genetic Strategies for Banana

Using Induced Mutations
A reverse-genetic strategy that combines traditional mutagenesis with high-throughput mutation
discovery can be considered for banana functional genomics and crop improvement. Reverse-
genetic approaches promise enhanced efficiencies over forward-genetic approaches described above.
Commonly referred to as TILLING (for targeting induced local lesions in genomes), the reverse-
genetic method first developed for Arabidopsis thaliana has since been applied to a large number
of seed-propagated crops, including maize, rice, soybean, and wheat (McCallum et al., 2000; Till et
al., 2003, 2007; Till, Reynolds, et al. 2004; Slade et al., 2005; Cooper et al., 2008b). For TILLING,
a mutant population is prepared in advance, typically comprised of several thousand individuals.
DNA is collected from each plant and the germplasm is stored. To identify mutations, PCR is per-
formed with gene-specific primers and the resulting amplicons evaluated for induced mutations via
enzymatic mismatch cleavage or alternative mutation discovery technologies (Till, Burtner, et al.,
2004). Mutant individuals are then recovered from the germplasm bank for further molecular and
phenotypic characterization. Since only a small number of plants are expected to harbor deleterious
mutations in a target gene, the method can greatly reduce phenotyping efforts compared to forward-
genetic screens. It is further advantageous because mutant alleles can be obtained from a population
that by themselves do not produce a phenotype but will when combined with others, as for example
with duplicated genes or homoeologues in polyploids. The strength of the approach will continually
improve as genomic sequences from more species are made available and hypotheses for plant gene
function become more refined.
The choice of mutagen, tissue, and optimal dose of treatment are important factors when devel-
oping a TILLING population. Many mutagenesis strategies produce chimeric tissues with different
cells having different genotypes in the first (M1) generation. As described above, chimeras need
to be dissociated before mutation discovery is performed so that inheritance of mutations can be
ensured and phenotypes accurately measured. In seed-propagated plants, chimeras are easily dis-
sociated through sexual crossing. A structured population is typically created whereby M1 plants
are self-fertilized and a single-seed descent strategy is followed with tissue collected for mutation
discovery in the M2 generation. M3 seed from a self-cross of the M2 is collected and stored for future
phenotypic analysis (see, for example, Till et al., 2003; Till et al., 2007; Caldwell et al., 2004). What
has emerged from TILLING studies is a large data set of mutations caused by chemical mutagenesis.
Chemicals such as EMS have been shown to cause single nucleotide changes randomly throughout
the genome (Greene et al., 2003). This means that with the right combination of mutation density
and population size, multiple mutations in any gene in the genome can be obtained. The resulting
point mutations include changes expected to knockout gene function (nonsense mutations and those
affecting RNA splicing) as well as missense changes that can have varying consequences on pro-
tein function. Thus TILLING can provide knockouts and less severe mutations for more nuanced
studies of gene function and for the study of essential genes where a knockout would be lethal.
Computational tools have been developed that make predictions on the effect of missense muta-
tions, and many steps of the TILLING procedure can be automated and converted to high-through-
put methods, allowing the development of large-scale community mutation screening services (Ng
and Henikoff, 2003; Taylor and Greene, 2003; Till et al., 2003; Cooper et al., 2008a). Less is known
about the spectrum and density of induced mutations from physical irradiation, such as treatment
with gamma rays, but a broader spectrum of changes has been reported, including single nucleotide
changes and small indels (Sato et al., 2006).
Many challenges arise when TILLING is considered for banana. Widely consumed triploid
varieties are largely sterile, infertile, and/or parthenocarpic, thus requiring vegetative propagation,
making seed mutagenesis impractical. Mutation induction using in vitro material can therefore be
considered. Embryogenic cell suspensions have been prepared in banana, and mutagenesis of cell
suspensions is potentially highly efficient because each cell in the suspension accumulates dif-
ferent mutations and each cell can produce a single plantlet, rapidly providing many nonchime-
ric plants immediately suitable for mutation screening (Strosse et al., 2003). Mutagenesis is also
performed using isolated shoot apical meristems (shoot tips) with the resulting adult plant (M1V1)
being chimeric. As described, chimerism can be reduced through successive rounds of meristem
isolation, followed by cutting meristems and allowing plantlets to regenerate (Roux, 2004). Through
this process the number of totipotent stem cells in the central zone of the meristem that produce
adult plant tissue is reduced, therefore reducing the genotypic complexity of the resulting plantlet.
A population of ~1500 EMS mutagenized ‘Grande Naine’ AAA plantlets subcultured six times
(M1V6) to remove chimeric sectors and obtain enough plant material was recently prepared. A pilot
TILLING screen with this population revealed a spectrum of mutations as expected for EMS and
a density of mutations expected for a triploid species (Joanna Jankowicz-Cielslak, Chikelu Mba,
and Bradley J. Till, unpublished). The identification of mutations stably inherited in M1V6 suggests
that induced mutations and reverse genetics can be considered as realistic options for vegetatively
propagated species. It also provides genotypic evidence for the dissolution of chimeras at the level of
single-nucleotide mutations. A major genetic bottleneck in this strategy, though, is the expectation
that most single-nucleotide changes will produce recessive, loss of function alleles, and mutations
must be homozygous before phenotypes can be observed. The ratio of recessive to dominant gain of
function phenotypes in the M1 generation, however, may be lower in vegetatively propagated banana
compared to other plants because of the lack of meiosis, recombination, and selective pressure to
remove deleterious alleles from the population. Thus a situation may exist where two of three alleles
have naturally accumulated deleterious mutations, and mutagenesis of the third allele would reveal
a phenotype. The frequency of such events is currently unknown. Another perhaps important con-
sideration is that mutagenized and vegetatively propagated plants are unique in that they represent
nonchimeric multicellular eukaryotes that have not undergone meiosis or fertilization. Therefore,
potentially dominant large structural chromosomal differences that are meiotically lethal would be
maintained in the population. Analysis of such changes would require different mutation discovery
assays such as array comparative genome hybridization (Bruce et al., 2009; Rios et al., 2008). Such
an analysis would produce knowledge of the full spectrum of mutations caused by a particular
mutagen and the types of changes are filtered out through meiosis, gametogenesis, and fertilization.
Careful controls, however, are needed to separate induced changes from large chromosomal aber-
rations that might occur due to propagation by tissue culture (Veilleux and Johnson, 1998). It is also
worth considering that a particular mutagen and dose may be optimal for producing phenotypes in
Mutagenesis Diploid banana (M1V6)

Heterozygous mutations
Meristem
DNA extraction/
Mutation discovery
M1V1
Candidate mutant
selection and clonal
amplification
Field propagation and

6x self-fertilization
Recovery and analysis of

M1V6
homozygous mutants
M2
DNA extraction, mutation discovery +/– +/+ –/– –/+
(a) (b)
Figure 10.4 Meristematic mutagenesis and TILLING strategies for banana. Meristematic tissue is isolated
and treated with mutagen (A). The resulting M1V1 generation plant is chimeric because different cells in the
meristem accumulated different mutations. Plants are made genotypically homogeneous through six rounds of
meristem isolation, cutting the meristem in two, and generating two new plants. Tissue is collected for DNA
extraction and mutation screening at the M1V6 generation. TILLING allows for preselection of candidate
mutants for self-fertilization in sexually propagating diploids (B). To overcome low fecundity, clonal ampli-
fication can be used prior to sexual crossing, allowing for sufficient seed production for segregation analysis
in the M2 generation.
the M1 generation, perhaps those conditions favoring large chromosomal aberrations. At some level,
however, too many genes are disrupted and reverse-genetic approaches become impractical.
More straightforward is the consideration of reverse genetics in sexually fertile diploid bananas.
Here, a strategy can be envisioned whereby vegetative propagation and TILLING are combined
to make an efficient system in varieties that are normally recalcitrant to genetic investigation and
improvement due to issues of low fecundity and a heavy demand on field resources (Figure 10.4).
In normal forward-genetic strategies including mutation breeding, thousands of M2 or higher lines
must be screened for a reasonable chance in finding a deleterious mutation in a gene capable of caus-
ing a desired phenotype. Also required is the recovery of sufficient seed from a self-cross to ensure
recovery of homozygous mutant alleles. Thus, forward genetics is labor intensive and inefficient for
banana. Reverse-genetic strategies provide a method for making genetic studies tractable. Using the
strategy described for triploid banana, thousands of nonchimeric diploid plants can be produced
and maintained in tissue culture. Each plant will harbor unique heterozygous mutations. TILLING
can then be performed to identify deleterious mutations in candidate genes. With knowledge of
mutation densities in many diploid populations, the expectation is that screening a population of
3,000 would result in the discovery of several, but fewer than 10, potentially deleterious alleles per
target screening region (Greene et al., 2003; Ng and Henikoff, 2003). Field labor could therefore be
reduced by as much as three orders of magnitude. Furthermore, preknowledge of the plant with a
candidate mutation allows clonal propagation of that plant prior to sexual fertilization. Intercrossing
between clonal siblings to produce a suitable amount of M3 seed to recover homozygous alleles then
becomes possible. Mutagenized populations of diploid ‘Calcutta 4’ AA for TILLING are currently
being developed at INIVIT in Cuba as part of an FAO/IAEA Coordinated Research Project, where
mutagenesis of cell cultures is also being investigated (J. López, personal communication). With
the complete banana genomic sequence expected in the near future, many candidate genes will be
available to exploit this approach. For example, ‘Calcutta 4’ is resistant to Mycosphaerella fijiensis,
the causative agent of black Sigatoka disease (Mobambo et al., 1997). Identification of knockouts
in candidates’ resistant genes could allow for the identification of the gene(s) conferring resistance.
The ‘Calcutta 4’ resistance gene(s) could then, for example, be used in cis- or transgenic approaches
to improve resistance in edible triploid varieties. Another interesting topic is the identification of
mutations in genes causing parthenocarpy. In both cases TILLING is advantageous because inter-
esting mutations can be propagated in vitro in a heterozygous form in near perpetuity. Finally, it
is also worth considering the use of reverse genetics for the improvement of parental material for
breeding programs.
10.5 Conclusions and Prospects

Considerable research efforts have been made to speed up in vitro mutagenesis for banana improve-
ment. Although shoot apical meristems have been used, somatic embryogenic cell suspension cul-
tures are highly suitable for mutation induction in banana genetic improvement strategies. In vitro
selection systems are at the forefront for the isolation of disease resistant mutants, such as toler-
ance to black Sigatoka, caused by M. fijiensis, a devastating banana disease. So far, putative black
Sigatoka–tolerant banana mutants have been isolated (Figure 10.2), with confirmation of disease tol-
erance still in progress. Such mutants may or may not possess the required resistance, but the mutant
germplasm will be useful for genetic studies and in gene discovery. In vegetatively propagated crops
like banana, mutation induction should be adopted as a useful genetic improvement strategy that
can contribute to banana improvement programs.
The gains in our understanding of basic plant biology and in crop improvement using traditional
mutagenesis and forward genetics have been immense. Over 3,000 induced mutant varieties are
registered in the FAO/IAEA Mutant Variety and Genetic Stock Database (http://mvgs.iaea.org/).
A number of bottlenecks exist in banana, making progress slower than in sexually propagating
crops like cereals. Through studies described here, knowledge has been gained with regards to
overcoming bottlenecks such as the induction of chimeric plant sectors and limitations of vegetative
propagation. The development of new mutagenesis methods promise to increase allelic diversity.
Furthermore, projected acquisition of whole genome sequence for banana is expected to provide
gene sequences hypothesized to be important for key agronomic traits. By applying this knowledge
along with new strategies such as reverse genetics, gains in the efficiency of using induced mutations
for functional genomics and breeding can be envisioned. This is timely as changes in climate and an
increasing world population are expected to put new pressures on global agricultural production.
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11 Biotechnology
Improvement
in Musa
Leena Tripathi
Contents
11.1 Introduction........................................................................................................................... 219
11.2 Recent Advances in Biotechnology....................................................................................... 220
11.3 Application of Tissue Culture for Banana Improvement....................................................... 220
11.3.1 Micropropagation...................................................................................................... 220
11.3.2 Embryogenic Cell Culture......................................................................................... 221
11.3.3 Embryo Culture......................................................................................................... 221
11.3.4 Anther Culture........................................................................................................... 221
11.3.5 Germplasm Conservation.......................................................................................... 221
11.4 Genomics for Banana Improvement...................................................................................... 222
11.4.1 Genomics for Banana Improvement.......................................................................... 222
11.4.2 Molecular Markers in Musa...................................................................................... 223
11.5 Transgenic Technology for Banana Improvement.................................................................224
11.5.1 Transformation Procedures.......................................................................................224
11.5.2 Disease Resistance..................................................................................................... 225
11.5.3 Pest Resistance........................................................................................................... 227
11.5.4 Enhanced Micronutrients.......................................................................................... 228
11.6 Challenges for Development of Transgenic Bananas............................................................ 228
11.7 Conclusions............................................................................................................................ 230
References....................................................................................................................................... 231
11.1 Introduction
Biotechnology can greatly impact the genetic improvement of vegetatively propagated crops.
Bananas and plantains (Musa spp.) are staple foods for millions of people in the tropical and sub-
tropical regions of the world. The annual banana production in the world is estimated at 1.3 × 1011
kg, of which less than 15% enters the international commercial market, indicating that the crop is
far more important for local or domestic consumption than for export (FAO, 2008). Almost 87% of
the bananas grown worldwide are produced by small-scale farmers for consumption and for local
markets, leaving only 13% for international trade. Nonetheless, banana is an extremely important
export commodity, especially in Latin America and the Caribbean. Musa cultivation is affected
by many pests and diseases, including black Sigatoka (Mycosphaerella fijiensis), Fusarium wilt
(Fusarium oxysporum f. sp. cubense), bacterial wilt (Xanthomonas campestris pv musacearum),
viruses (banana bunchy-top virus, banana streak virus), nematodes, and weevils. Biotechnology
could provide solutions to some of the limitations of conventional banana breeding, such as sterility,
long generation times, and limited genetic variability. This chapter reviews the progress and chal-
lenges in tapping the potential of biotechnology for the genetic improvement of banana.
219
11.2 Recent Advances in Biotechnology

Plant biotechnology, like all aspects of the life sciences, has changed very rapidly over the past
20 years (Flavell, 2003). The new ways of analyzing genes, molecules, and processes constitute,
together with established plant breeding systems, new platforms for discovery, design, and synthe-
sis of novel products (Flavell, 2003). Such products include improved sources of food, feed, fiber,
energy, chemicals, and drugs. Knowledge of the ways plants develop their architecture; survive
abiotic and biotic stresses; utilize fertilizer, water, and CO2; and make complex secondary metabo-
lites and macromolecules will increase more rapidly in the next few decades (Flavell, 2003). More
importantly the genes and alleles that program these attributes will become known rapidly from the
combination of genomics, genetics, and plant breeding, particularly for intensively studied species
(Flavell, 2003). Due to the sterility of most triploid banana varieties, plant propagation has been
achieved traditionally by vegetative multiplication using naturally occurring plant offshoots (Haicur
et al., 1998). In vitro multiplication of banana is mostly performed through proliferation of vegetative
meristems, which has the advantage of producing healthy homogeneous plants in large numbers.
Micropropagation of banana is now an industrial process and up to 50 million tissue-cultured plants
are produced annually (Teisson and Cote, 1997). The recent development of embryogenic suspen-
sion cultures has paved the way for future mass production of banana plants at low cost (Haicur et
al., 1998). Suspension cultures can also be used for cryopreservation and for genetic transformation
(Haicur et al., 1998). Plant regeneration from protoplasts has been achieved (Ducreux et al., 2001;
Xiao et al., 2008). Protoplast fusion is particularly promising for banana improvement, as most
cultivars are related, triploid, and sterile. Fusion between haploid plants and selected diploid geno-
types could facilitate the production of new triploid bananas (Xiao et al., 2009). The first transgenic
banana plants were produced in 1995 through particle bombardment of embryogenic suspension
cultures (Sagi et al., 1995).
Agrobacterium-mediated transformation has also been achieved (Hernandez, 1999; Ganapathi
et al., 2001; Khanna et al., 2004). Transformation has made it possible to introduce valuable agro-
nomic traits for pest and disease resistance, fruit maturation, and storage into banana (Haicur et al.,
1998). Thus, at the onset of the 21st century, improvement of banana and plantain through biotech-
nology should help ensure food security by stabilizing production levels in sustainable cropping
systems geared towards meeting domestic and export market demand (Haicur et al., 1998; Rout et
al., 2000).
11.3 Application of Tissue Culture for Banana Improvement

Besides the production of healthy planting material, tissue culture has been used in crop improve-
ment since the 1940s to create genetic variability and to increase the number of desirable germ-
plasm (http://anrcatalog.ucdavis.edu.). Specifically, in vitro techniques for the culture of protoplasts,
anthers, microspores, ovules, and embryos have been used to create new genetic variation in the
breeding lines, often via haploid production with crop improvement potential (Brown and Thorpe,
1995). The culture of single cells and meristems can be effectively used to eradicate pathogens from
planting material and thereby dramatically improve the yield of established cultivars (Badoni and
Chauhan, 2010). Tissue-culture techniques, in combination with molecular techniques, have been
successfully used to incorporate specific traits through gene transfer.
11.3.1 Micropropagation
Traditionally, bananas and plantains are propagated vegetatively from suckers or corms. Unlike
seeded commercial cultivars of other crop species, production of planting materials through suckers
takes a long time and very few suckers are produced per mother plant (Pillay and Tripathi, 2007). In
Biotechnology in Musa Improvement 221
vitro propagation has many advantages, such as higher rates of multiplying, pest- and disease-free
planting material, and less space required to multiply large numbers of plants (refer to Chapter 15).
11.3.2 Embryogenic Cell Culture

Recently, much progress has been made in the establishment of embryogenic cell culture from
banana explants from a variety of cultivars. Novak et al. (1989) established embryogenic cell sus-
pensions (ECS) from somatic tissues such as leaf sheaths and corm sections of the cultivar ‘Grand
Nain’ while Dheda et al. (1991) cultured highly proliferated shoot-tip cultures of ‘Bluggoe.’ Young
male flowers are the most responsive explants for initiating embryogenic cultures of ‘Grand Nain’
(Escalant et al., 1994), ‘Rasthali’ (Ganapathi et al., 2001), and the hybrid cultivar ‘FHIA-21’ (Daniels
et al., 2002). Suspension cultures can be regenerated into plantlets through somatic embryogenesis
at high frequencies and grown in the field (Dheda et al., 1991; Novak et al., 1989). The plant recov-
ery frequencies were as high as 81.5% (Daniels et al., 2002). Plant regeneration from cell suspension
cultures was investigated for its potential in mass propagation and as a tool in genetic transforma-
tion using recombinant DNA technology. However, most of the procedures are still laborious and
genotype dependent.
11.3.3 Embryo Culture
Embryo culture plays an important role in Musa breeding. Embryo culture has been used to rescue
hybrid plants from wide crosses, which often fail to produce mature viable seeds (refer to Chapter 15).
11.3.4 Anther Culture

Anther culture is generally used in the production of F1 hybrid varieties (Drew, 1997). The first step
is to develop inbred parental lines by repeated self-pollination. This can be a very slow process in
banana, which normally requires more than a year to flower. However, by culturing pollen grains
from diploids, haploid plants that contain only one copy of each chromosome can be produced.
These plants can be induced to double their chromosome number by a chemical means, resulting
in plants that have two identical sets of chromosomes, or are completely inbred (homozygous).
This process facilitates the selection of recessive traits. Assani et al. (2003) reported the produc-
tion of haploid plants of Musa balbisiana (BB). Callus was induced from anthers in which the
majority of the microspores were at the uninucleate stage. The frequency of callus induction was
77%. About 8% of the anthers developed androgenic embryos and of the 147 plantlets obtained,
41 were haploids (n = x = 11), but the frequency of regeneration was low (1.1%). The technique is
genotype specific.
11.3.5 Germplasm Conservation
Tissue culture is useful in germplasm conservation of bananas since most cultivars are seedless.
The main disadvantage of this method is the propensity for somaclonal variation (Oh et al., 2007).
Germplasm collections in fields require extensive use of labor, large space requirements, and are
costly. Field-grown plants are exposed to pests and diseases and can be lost. The propagation of
various cultivars of banana and plantain by conventional methods is laborious and time consum-
ing. In vitro collections offer a safer and cheaper alternative. In addition, in vitro plantlets are the
materials of choice for the international exchange of germplasm, simplifying quarantine procedures
as they are pest- and disease-free, safer and easier to handle than bulky suckers. Shoot-tip culture
and disease-indexed cultures for germplasm exchange have been widely adopted for conserving and
distributing important banana and plantain collections.
The international Musa germplasm collection of INIBAP (International Network for the
Improvement of Banana and Plantain) Transit Centre at K.U. Leuven, Belgium, uses in vitro culture
for germplasm storage. The collection also contains improved materials from breeding programs.
The in vitro maintenance of Musa germplasm is constrained by the occurrence of somaclonal
variation (Vuylsteke et al., 1991). As the frequency of somaclonal variation could be linked to
multiplication and growth rates, among other factors, Musa germplasm is now stored under slow
growth conditions as well as at ultralow temperatures. Shoot-tip cultures maintained at low tem-
peratures (15–18°C) have been used for the slow-growth storage of Musa germplasm (Banerjee and
De Langhe, 1985).
Cryopreservation is considered to be the only valid alternative for long-term preservation of
Musa germplasm because ultralow temperatures arrest physical and chemical reactions and elimi-
nate time-related changes. Cryopreservation techniques have been developed for more than 80 dif-
ferent plant species cultivated under various forms including cell suspensions, calluses, apices, and
somatic and zygotic embryos (Engelmann, 1997; Panis et al., 1990). Cryopreservation is not feasible
in all cultivars since the production of somatic embryos and cell suspensions is cultivar depen-
dent. In the case of banana, slow freezing and vitrification were both totally ineffective, while the
encapsulation-dehydration method resulted in a survival rate of 8.1% (Panis et al., 1990). A simple
technique was recently developed for cryopreservation of meristem cultures, which involves precul-
ture on high-sucrose medium followed by rapid freezing (Panis et al., 1996). This method was tested
on seven banana cultivars of different genomic groups and resulted in viability rates of 12% to 72%
depending on the cultivar. This method with improvements can be used as a routine cryopreserva-
tion method for banana gene banks.
11.4 Genomics for Banana Improvement

11.4.1 Genomics for Banana Improvement
Recent breakthroughs in genomics in plants include the whole genome sequencing of many crop
plants. The application of genomics in banana and plantain improvement is still in its infancy. The
genome size of Musa is 550–600 Mbp (Dolezel et al., 1994). This is larger than the genomes found
in species such as rice or Arabidopsis thaliana (between 150 and 500 Mbp), but much smaller than
the Triticeae cereals (5,500 Mbp in barley, 9,000 Mbp in rye, and 17,000 Mbp in wheat) (Heslop-
Harrison and Schwarzacher, 2007).
The Musa genome display several characteristics that could be exploited for gaining fundamen-
tal insights into the genomes of other species (Vilarinhos et al., 2003).
The small size of the haploid genome, the different ploidy levels, and the coexistence of various
genome combinations, as well as the combination of parthenocarpy with sterility and vegetative
propagation, place the banana as a good model to study gene expression in different chromosomal
environments (Musagenomics, 2007). To reinforce banana genomic research, The Global Genomics
Consortium has been established (http://www.musagenomics.org).
One of the first tools necessary to develop banana genomics research is a banana bacterial arti-
ficial chromosome (BAC) library (Shizuya et al., 1992). Several BAC clone libraries were devel-
oped from both A and B genome diploid Musa species (Vilarinhos et al., 2003; Safár et al., 2004;
Musagenomics, 2007) with a total genomic coverage of more than 30 times. The Musa Genomics
Resource Centre was established in the Czech Republic to distribute these resources, and by 2007,
had 42 BACs totaling 3·3 Mbp (Aert et al., 2004) and 4·5 Mbp of BAC end sequences (Cheung
and Town, 2007). These allow an overview of the Musa genome showing that it has 47% guanine-
cytosine (GC) content and, together with the development of large numbers of polymerase chain
reaction (PCR) based markers, a comparison of genes and genomic structures could be made with
other species (Heslop-Harrison and Schwarzacher, 2007).
Several thousand expressed sequence tags (ESTs), important for examination of gene expres-
sion, responses and differentiation of the plants, and examination of diversity, have been published
(Santos et al., 2005) and many more are becoming available.
Comparisons of EST libraries are very valuable for identification of genes that are differentially
expressed under stress conditions. Santos et al. (2005) made libraries from plants grown in cold
(5°C) and hot (45°C) conditions, and found that about 30% of the genes in their library had been
identified in other species as being involved in responses to environmental stress and that there were
substantial differences in the expression between the two libraries. Coemans et al. (2005) reported
on alternative method for gene-expression profiling: SuperSAGE (super-serial analysis of gene
expression), which they suggest will be very useful for transcript profiling and gene discovery.
Like all plant genomes, the Musa genome consists of repetitive DNA and single-copy sequences,
and understanding the composition and organization of the genome at the large-scale level will be
helpful to allow gene isolation and to understand long-term and short-term evolutionary processes
(Schmidt and Heslop-Harrison, 1998). Valarik et al. (2002) cloned and characterized many repeti-
tive DNA sequences and located those that were not related to rDNA or retroelements in the centro-
meric region of the chromosomes.
Retroelements, class I transposable elements or transposons, are abundant in the Musa genome
(Balint-Kurti et al., 2000), as in other species (Heslop-Harrison et al., 1997). Automated annotation
of the BAC libraries shows that more than a third of the open reading frames are related to retro-
elements (Musagenomics, 2007). The analysis of two BACs by Aert et al. (2004) revealed that one
BAC consisted of 45 kb of gene-rich sequence without retroelements, followed by 28 kb containing
mostly transposon-like sequences and repetitive DNA. BAC-end sequencing, allowing a survey of
the whole genome, showed that 36% of the BESs contained sequences homologous to transposable
elements (Cheung and Town, 2007). The evidence suggests that Musa has repeat-rich regions in the
centromeres and perhaps elsewhere, and there may be gene-rich regions, as suggested in other spe-
cies (Heslop-Harrison, 1991). Cheung and Town (2007) compared pairs of BAC end sequences from
single BACs and found that a small number also mapped to adjacent regions of the rice genome,
indicating conserved microsynteny over a larger taxonomic range and showing how part of the
Musa genome can be anchored to rice. This provides a cost-effective and efficient way to understand
Musa genes and the genome by informatics and conserved synteny with the model reference species
rice and Arabidopsis thaliana (Heslop-Harrison and Schwarzacher, 2007).
11.4.2 Molecular Markers in Musa

There is rapid progress in the development of molecular markers and gene mapping in crop plants.
A number of molecular markers such as restriction fragment length polymorphism (RFLP), simple
sequence repeat (SSR), random amplification of polymorphic DNA (RAPD), and amplified fragment
length polymorphism (AFLP) were applied in banana and plantain mostly for diversity analysis and
genetic mapping (Faure et al., 1993; Ude et al., 2002a, 2002b; Creste et al., 2004; Oreiro et al., 2006).
Ferreira et al. (2004) employed RAPD markers to characterize banana diploids (AA) with contrasting
levels of black Sigatoka (Mycosphaerella fijiensis) and yellow Sigatoka (M. musicola) resistance.
A putative RAPD marker for Sigatoka resistance has been identified at the National Research
Center for Banana (NRCB), India. The marker has been cloned, sequenced, and converted into a
sequence characterized amplified region (SCAR) marker and is being validated using contrasting
parents for expression of Sigatoka (Mycosphaerella musicola) disease resistance and their prog-
enies. Parallel studies have led to the identification of a putative RAPD marker for nematode resis-
tance (Uma, personal communication). Another RAPD marker has been identified for salt-tolerance
clones of cv ‘Dwarf Cavendish’ obtained through induced mutagenesis (Miri et al., 2009). Attempts
are under way at NRCB towards the development of resistant gene analogue-cleaved amplified
polymorphic (RGA-CAP) markers, which would facilitate the genetic mapping of candidate resis-
tant gene(s) in Musa (Backiyarani et al., 2010).
New markers such as Diversity Arrays Technology or DArT markers (Jaccoud et al., 2001), a
microarray technology that can detect and type DNA variation at several hundred of genomic loci
in parallel without prior knowledge of sequence information has been applied in banana (Amorim
et al., 2009).
11.5 Transgenic Technology for Banana Improvement

Transgenic technology has become an important tool for crop improvement. Genetic engineering—
that is, the introduction and stable integration of genes into the nuclear genome and their expression
in a transgenic plant—offers a better alternative for the genetic improvement of cultivars not ame-
nable to conventional crossbreeding, such as Cavendish bananas and False Horn plantains. Due to
lack of cross-fertile wild relatives in many banana-producing areas, as well as the male and female
sterility of most edible cultivars and clonal mode of propagation, gene flow is not an issue for this
crop, making a transgenic approach even more attractive.
The successful genetic transformation in plants requires the production of normal, fertile plants
expressing the newly inserted gene(s). The process of genetic transformation involves several dis-
tinct steps: identification of useful gene, the cloning of the gene into a suitable plasmid vector, and
delivery of the vector into plant cell, followed by expression and inheritance of the foreign DNA
encoding a polypeptide. With the advent of plant biotechnology and the rapid development of gene
transfer techniques, the potential to introduce desirable character traits is no longer restricted to
those occurring in close relatives. Despite technical difficulties of transforming a monocot spe-
cies, transformation protocols are available for many banana cultivars (Table 11.1). Development
of stable and reproducible transformation and regeneration technologies opened new horizons in
banana breeding (Table 11.2).
11.5.1 Transformation Procedures

Transformation protocols are available for most banana cultivars. Genetic transformation using
microprojectile bombardment of embryogenic cell suspension is now routine and has been used for
direct gene transfer in cooking banana cultivar ‘Bluggoe,’ plantain ‘Three Hand Planty’ (Sagi et al.,
1995), and Cavendish banana ‘Grand Nain’ (Becker et al., 2000).
Agrobacterium-mediated transformation offers several advantages over direct gene transfer
methodologies, such as the possibility to transfer only one or few copies of DNA fragments carrying
Table 11.1
Summary of Transformation of Various Cultivars of Banana and Plantain
Cultivar of Banana/Plantain Method of Transformation Reference
Bluggoe Microprojectile Bombardment Sagi et al., 1995
Three Hand Planty Microprojectile Bombardment Sagi et al., 1995
Cavendish banana cv Grand Nain Microprojectile Bombardment; Becker et al., 2000; Khanna et al., 2004;
Agrobacterium May et al., 1995
Rasthali Agrobacterium Ganapathi et al., 2001
Lady Finger Agrobacterium Khanna et al., 2004
Agbagba Agrobacterium Tripathi et al., 2005
EAHBa cv Mpologoma Agrobacterium Tripathi et al., 2008
EAHB cv Nakitembe Agrobacterium Tripathi et al., 2008
Gonja Manjaya Agrobacterium Author’s lab
a EAHB: East African Highland banana.

Table 11.2
Summary of Genetic Modification of Banana for Important Traits
Method Transgene Trait Reference
Microprojectile bombardment BBTV Resistance to BBTV Becker et al. 2000
Agrobacterium MSI-99 Resistance to fungal diseases Chakrabarti et al., 2003
Agrobacterium hrap Resistance to Xanthomonas wilt Tripathi et al., 2009, 2010
Agrobacterium pflp Resistance to Xanthomonas wilt Tripathi et al., 2009
Agrobacterium Chitinase Resistance to black Sigatoka disease Kiggundu, 2007
Agrobacterium Anti-apotosis Resistance to Fusarium wilt Paul, 2009
Agrobacterium Cystatin Resistance to nematode Atkinson et al., 2004
the genes of interest at higher efficiencies with lower cost and the transfer of very large DNA frag-
ments with minimal rearrangement (Shibata and Liu, 2000). Banana was generally regarded as
recalcitrant for Agrobacterium-mediated transformation, until Hernandez et al. (1999) reported that
A. tumefaciens was compatible with banana. Agrobacterium-mediated transformation of embryo-
genic cell suspensions of the banana cultivars ‘Rasthali,’ ‘Cavendish,’ and ‘Ladyfinger’ has since
been achieved (Ganapathi et al., 2001; Khanna et al., 2004). Banana functional genomics and plant
improvement initiatives demand higher transformation frequencies and a standard protocol that can
be used to transform all banana genomic groups. Khanna et al. (2004) described centrifugation-
assisted Agrobacterium-mediated transformation protocol developed using banana cultivars from
two economically important genomic groups (AAA and AAB) of cultivated banana.
Although most transformation protocols use cell suspensions, establishing cell suspension is a
lengthy process and is cultivar dependent. Protocols have also been established using meristematic
tissues from various cultivars of banana (May et al., 1995; Tripathi et al., 2005, 2008). This tech-
nique is applicable to a wide range of banana cultivars irrespective of ploidy or genotype (Tripathi
et al., 2003, 2005, 2008). This process does not incorporate steps using disorganized cell cultures
but uses micropropagation, which has the important advantage that it allows regeneration of homo-
geneous populations of plants in a short period of time. This procedure offers several potential
advantages over the use of embryogenic cell suspensions, as it allows for rapid transformation of
banana species, and meristematic tissues have the potential to regenerate plants from many different
cultivars, unlike somatic embryogenesis, which is restricted to only a few cultivars. The transforma-
tion of meristematic cells may result in chimeric plants when only one or a few cells receive T-DNA.
To obtain uniformly transformed plants, two steps of selection and regeneration are performed to
avoid regeneration of any nontransformed cells.
11.5.2 Disease Resistance
Recent advances in genetic engineering offer ways to transfer a resistance gene found in any plant
into crop varieties without changing other favorable traits. Plant defense genes from other plants and
antimicrobial proteins are now a potential source of plant resistance. One of the strategies to control
highly destructive fungal diseases like black Sigatoka in banana is the production of transgenic
disease-resistant plants based on expression of genes encoding antimicrobial peptides (AMPs). The
AMPs usually have a broad-spectrum activity against fungi and bacteria, and most are nontoxic to
plant and mammalian cells. Examples of AMPs are cecropins (Boman and Hultmark, 1987), magai-
nin (Bevins and Zasloff, 1990), and plant defensins (Broekaert et al., 1997). The cecropin (Alan
and Earle, 2002) and its derivatives (D4E1; Rajasekaran et al., 2001) have been found to inhibit
the in vitro growth of several important bacterial and fungal pathogens. Transgenic tobacco plants
expressing cecropins have increased resistance to Pseudomonas syringae pv tabaci, the cause of
tobacco wildfire (Huang et al., 1997). Similarly, magainin is effective against plant pathogenic fungi
(Kristyanne et al., 1997). Chakrabarti et al. (2003) reported successful expression of this synthetic
peptide and enhanced disease resistance in transgenic tobacco and banana.
The AMPs of plant origin may be potential candidates for fungal resistance in banana as they
have high in vitro activity to Mycosphaerella fijiensis and Fusarium oxysporum f. sp. cubense, two
major fungal pathogens of banana, and are nontoxic to human and banana cells. Several AMPs
isolated from radish and dahlias are toxic to both fungal pathogens. A large number of transgenic
lines of plantain expressing defensin-type AMPs has been developed at the Catholic University of
Leuven (Remy, 2000). Many transformed lines have been generated and screened under screen-
house conditions in Belgium for disease resistance, and the most promising lines of transgenic
bananas and plantains were evaluated in the greenhouse and under field conditions in Cuba and
Costa Rica (Agnet, 2004). The transgenic banana containing antifungal chitinase genes is also
being tested for resistance against black Sigatoka in a confined field trial in Uganda.
Plants have their own mechanisms of defense against plant pathogens and include a vast array
of proteins and other organic molecules produced prior to infection or during pathogen attack.
Pathosystem-specific plant resistance (R) genes have been cloned from several plant species. R
genes cloned from resistant varieties can be transferred to susceptible cultivars of the same plant
species, making them resistant to pathogens. It is also possible to transfer R genes from one plant
species to another.
A series of resistance gene analogues have been isolated from banana, using degenerate PCR
primers targeting highly conserved regions in proven plant resistance genes. The disease-resistant
genes were isolated from the somaclonal mutant ‘CIEN-BTA-03’ (resistant to both M. fijiensis and
M. musicola) and the parent ‘Williams’ (Kahl, 2004). All the resistance genes were fully sequenced,
and eight of them were also transcribed in the mutant, its parental genotype, ‘Pisang Mas’ and a
diploid M. acuminata. The R gene candidate (RGC-2) from Musa acuminata ssp. malaccensis, a
wild diploid banana segregating for resistance to Fusarium oxysporum f. sp. cubense (Foc) race 4,
has been isolated and completely sequenced.
Recently, scientists at the Queensland University of Technology (QUT) inserted this R gene for
resistance to Fusarium wilt, or Panama disease, into the banana genome (Dale et al., 2004). The
scientists at QUT also introduced the anti-apoptosis gene into the banana genome of two com-
mercially important banana cultivars ‘Grand Naine’ and ‘Lady Finger’ for developing resistance
to fusarium wilt, or Panama disease (Paul, 2009). The gene stops cells dying when attacked by the
disease. These transgenic plants are under evaluation in a confined field in Queensland, Australia
(Science Alert, 2008).
Hypersensitive response-assisting protein (HRAP) is a novel plant protein that can intensify
the harpinPss-mediated hypersensitive response in plants (Chen et al., 2000). The pflp has been
shown to delay the hypersensitive response (HR) induced by Pseudomonas syringae pv syringae
in nonhost plants through the release of harpinPss. Transgenic rice carrying the pflp gene showed
enhanced resistance to Xanthomonas oryzae pv oryzae (Tang et al., 2001). The pflp has also been
shown to enhance resistance in transgenic orchids against E. carotovora (Liau et al., 2003). The
elicitor-induced resistance is not specific against particular pathogens, so it could be very useful
strategy for developing broad-spectrum resistance. This is the strategy pursued by IITA, in col-
laboration with the National Agriculture Research Organization (NARO, Uganda) and the African
Agricultural Technology Foundation (AATF). This research aims at “designing” a genetically
modified banana that is resistant to the most devastating bacterial disease, banana Xanthomonas
wilt (BXW) (Biruma et al., 2007; Tripathi, 2008). Hundreds of transgenic lines with pflp or hrap
genes have been developed using a protocol based on the Agrobacterium tumefaciens technology
(Tripathi et al., 2009, 2010). These transformed lines of various cultivars have been validated via
PCR assay and Southern blot analysis. They have been tested for disease resistance under labora-
tory conditions. Most promising lines will be evaluated for efficacy against BXW in confined fields.
The transgenic lines will also be tested for environmental and food safety in compliance with target
country regulations.
The most promising transgenic strategies to control ssDNA viruses like BBTV is to express a
defective gene that encodes an essential virus life-cycle activity. For instance, the replication of
the virus can be encoded in the replication gene or genes (Rep) whereby the resultant Rep protein
may retain the ability to bind to its target viral DNA without the functions of the Rep (Brunetti et
al., 2001). The defective Rep protein binds to the invading viral DNA and is thought to outcom-
pete the native viral Rep protein, thus reducing or eliminating virus DNA replication. Lucioli et
al. (2003) expressed the first 630 nucleotides of the Rep gene of tomato yellow leaf curl Sardinia
virus to generate resistance. The duration of the resistance was related to the ability of the invading
virus to switch off transgene expression through post-transcriptional gene silencing (PTGS). Many
researchers are trying to develop transgenic plants of Musa resistant to BBTV, targeting the PTGS
mechanism using mutated or antisense Rep genes.
11.5.3 Pest Resistance

There are several possible approaches for developing transgenic plants with improved weevil and
nematode resistance. A variety of genes are available for genetic engineering for pest resistance
(Sharma et al., 2000). Among these are proteinase inhibitors, Bacillus thuringiensis (Bt) toxins,
plant lectins, vegetative insecticidal proteins (VIPs), and alpha-amylase inhibitors (AI). Proteinase
inhibitors contribute to host-plant resistance against pests and pathogens (Green and Ryan, 1972).
They operate by disrupting protein digestion in the insect mid-gut via inhibition of proteinases.
The two major proteinase classes in the digestive systems of phytophagus insects are the serine
and cysteine proteinases. Coleopteran insects, including the banana weevil, mainly use cysteine
proteinases (Murdock et al., 1987) and studies indicate a combination of both serine and cysteine
proteinases is useful for insect control (Gerald et al., 1997). These inhibitors have already been used
for insect control in transgenic plants (Leple et al., 1995). Presently, cysteine proteinase activity has
been identified in the mid-gut of the banana weevil and in vitro studies have shown that cysteine
proteinases are strongly inhibited by both a purified recombinant rice (oryzacystatin-I [OC-I]) and
papaya cystatin (Abe et al., 1987; Kiggundu et al., 2003).
The use of proteinase inhibitors (PIs) as nematode antifeedants is an important element of natu-
ral plant defense strategies (Ryan, 1990). This approach offers prospects for novel plant resistance
against nematodes and reduces use of nematicides. The potential of PIs for transgenic crop protec-
tion is enhanced by a lack of harmful effects when humans consume them in seeds such as rice and
cowpea. Cysteine PIs (cystatins) are inhibitors of cysteine proteinases and have been isolated from
seeds of a wide range of crop plants, including those of sunflower, cowpea, soybean, maize, and
rice (Atkinson et al., 1995). Transgenic expression of PIs provides effective control of both cyst and
root-knot nematodes. The cystatins were shown to mediate nematode resistance when expressed in
tomato hairy root (Urwin et al., 1995), rice (Vain et al., 1998), pineapple (Urwin et al., 2000), and
potato (Urwin et al., 2001). The partial resistance was conferred in a small-scale potato field trial
on a susceptible cultivar by expressing cystatins under control of the CaMV35S promoter (Urwin et
al., 2001). The enhanced transgenic plant resistance to nematodes has been demonstrated by using
dual proteinase inhibitor transgenes (Urwin et al., 1998). The resistance using cystatins has also
shown to be effective in banana (Atkinson et al., 2004). In partnership with the University of Leeds,
transgenic plants with proteinase inhibitors and repellent genes are being developed by IITA for
plantain (Tripathi, 2009).
The expression and biological activity of the Bt toxins has been investigated in genetically modi-
fied (GM) plants for insect control. Bt gene technology is currently the most widely used system for
lepidopteran control in commercial GM crops (Sharma et al., 2000). The expression of a selected Bt
gene for weevil resistance may be a rather long-term strategy since no potential Bt gene with high
toxic effects against the banana weevil has been identified as yet (Kiggundu et al., 2003). Some Bt
proteins are also effective against saprophagous nematodes (Borgonie et al., 1996). The Cry5B pro-
tein is toxic to wild type Caenorhabditis elegans; other C. elegans mutants are resistant to Cry5B
but susceptible to the Cry6A toxin (Marroquin et al., 2000). The approach using Cry genes has
potential for plant nematode control (Wei et al., 2003).
Alpha-amylase inhibitors (AI) and chitinase enzymes might also have a future potential for wee-
vil control. Alpha-amylase inhibitors operate by inhibiting the enzyme alpha-amylase, which breaks
down starch to glucose in the insect gut (Morton et al., 2000). Transgenic adzuki beans are produced
with enhanced resistance to bean bruchids, which are Coleopteran insects like weevils (Ishimoto
et al., 1996). Chitinase enzymes are produced as a result of invasion either by fungal pathogens or
insects. Transgenic expression of chitinase has shown improved resistance to Lepidopteran insect
pests in tobacco (Ding et al., 1998).
11.5.4 Enhanced Micronutrients
Nutritionally enhanced crops could make a significant contribution to the reduction of micro-
nutrient malnutrition in developing countries (Bouis et al., 2002). Biofortification (the devel-
opment of nutritionally enhanced crops) can be advanced through the application of several
biotechnologies in combination. Genomic analysis and genetic linkage mapping are needed
to identify the genes responsible for natural variation in nutrient levels of common foods.
These genes can then be transferred into familiar cultivars through conventional breeding and
marker-assisted selection or, if sufficient natural variation does not occur within a single spe-
cies, through genetic engineering.
Vitamin and mineral deficiencies are a major cause of child mortality and morbidity every year
in the developing world, but this can be easily prevented by adding just a few key nutrients to staple
foods. Genetic modification of banana has also been considered as a path towards increasing the
value of this crop to health and nutrition in developing countries. As a crop that is widely consumed
as a weaning food by children and as a starchy staple by all sectors of the community in some coun-
tries, banana has been advocated as a source of carotenoids that can counteract debilitating vitamin
A deficiency. Although much of the necessary technology is now available, these applications have
yet to advance to the stage of practical evaluation. However, recent works in engineering rice with
genes for β-carotene biosynthesis and the development of golden rice (Ye et al., 2000) have shown
the feasibility of enriching foods with vitamin A through biotechnology. Recently iron has been
enhanced in rice by the introduction of the ferritin gene driven by endosperm-specific promoter
(Vasconcelos et al., 2003). Researchers at QUT and NARO are developing biofortified bananas
using a number of genes for synthesis of provitamin A or iron, under the control of constitutive or
fruit-specific promoters (Dale and Tushemeirewe, 2008). These transgenic bananas are currently
being regenerated for field trials in Australia and Uganda.
11.6 Challenges for Development of Transgenic Bananas

The production of genetically modified banana plants is routine in many laboratories. The initial
technical hurdles have been overcome but new challenges that must be addressed if the technology
is to move forward are becoming apparent, from proof of concept to product development, deregu-
lation, and commercial release. The new challenges include intellectual property rights (IPR), an
underdeveloped regulatory environment, and a lack of biosafety infrastructures within target coun-
tries, besides some technical and sustained funding issues.
Most innovations in biotechnology are developed using the knowledge or technologies gener-
ated from previous innovations. Many plant biotechnology products or techniques are “modu-
lar” in that they are assembled from a number of previously developed technologies/transgenes,
each of which may be subject to a separate patent. The commercialization of many proprietary
biotechnology products is typically contingent on other proprietary biotechnology products or
processes, and in particular on agreements between IPR holders regarding the relative contri-
butions of different proprietary technologies to the product in question. Many biotechnology
products (for example, transgenic seeds or transgene constructs) now have a complex IPR pedi-
gree because a large number of proprietary products or processes are involved in developing
the product.
Some public sector agricultural research institutions and universities have become involved in the
“defensive” patenting of technologies they develop that may have commercial value or might have
some value in the future. Many biotechnology discoveries and enabling technologies (for example,
Agrobacterium and biolistic transformation methods) generated with public funding in research
institutions and agricultural universities are also protected and no longer being treated as “public
goods.” Such discoveries now primarily flow from the public sector to the private sector and, for
use in public research institutes, usually such technologies come under material-transfer agreements
(MTAs) that significantly restrict their use, usually for research purposes only, and often include
reach-through provisions to capture results of future research.
The IP issues are becoming a major factor, limiting the deployment of transgenic technologies
in developing countries, but this problem can be overcome if the correct procedures are followed
early in the product delivery program. Institutions with large portfolios of relevant IP are in a better
negotiating position to access the IP of others. As IPRs are in greater use by the private sector than
the public sector, it is likely that public sector research institutions such as the Consultative Group
on International Agricultural Research (CGIAR), the National Agricultural Research Stations, and
individual university researchers will not be in a strong negotiating position regarding access to
useful proprietary plant biotechnologies. The CGIAR is establishing a biotechnology transfer unit
with expertise in IPR law in an effort to strengthen its negotiating position with other IPR holders
of useful biotechnologies.
While there are still many nonproprietary biotechnologies available, the cost of access to patented
technologies is likely to be a growing issue for many public sector research institutions. It is illustra-
tive that commercially oriented research in many biotechnology companies has to now follow the
research route of least cost in terms of royalty payments to other companies for enabling technolo-
gies used to develop a commercial product (Mascarenhas, 1998). Unfortunately, no public sector
body has yet compiled a directory of which useful plant biotechnologies are freely accessible in the
public domain, especially for scientists in developing countries. Conversely, there is a corresponding
lack of publicly available studies on what the current patent situation is for key enabling biotech-
nologies. However, in 1998 the CGIAR Panel on Proprietary Science and Technology conducted a
study of proprietary science and technology within the CGIAR system (Cohen et al., 1998). This
CGIAR study included an initial review by International Service for National Agricultural Research
(ISNAR) of the extent of use of proprietary plant biotechnology tools in each of the International
Agricultural Research Centers (IARCs).
The current generation of transgenic bananas and their testing, however, highlights some prob-
lems that need to be avoided in future. Some genes of agronomic interest were owned by the indus-
try, and it took much effort by the Catholic University of Leuven before these genes could be used
freely for plantain and cooking bananas (Tollens et al., 2004). Therefore, it is urgent that a mecha-
nism be put in place whereby an authority at the global level interacts with industry to negotiate
access to protected technologies for developing countries. This is in contrast to the utility patent
system that extends protection to the seed and progeny of patented plants so breeders cannot legally
use protected varieties as breeding material.
The African Agricultural Technology Foundation (AATF) has been instrumental in facilitating
technology transfer negotiations whereby proprietary biotechnologies have been made available
to Africa. Recently, IITA has negotiated a royalty-free license from the patent holder Academia
Sinica, Taiwan, through the AATF for access to the pflp and hrap genes for production of com-
mercial banana varieties resistant to bacterial wilt in sub-Saharan Africa. The AATF has signed
the licensing agreement with Academia Sinica and granted a sublicense to IITA for developing the
improved varieties. Further, IITA has signed tripartite agreement with NARO and AATF for devel-
oping disease-resistant transgenic bananas and joint ownership of the developed material.
Given the novelty of commercial application of genetic engineering techniques, the regulatory
frameworks around the world are in the process of being formulated and reformulated in response
to, for example, consumer reactions to these new products. Existing regulations differ substantially
both in scope and stage of implementation, varying from very restrictive regulations in certain
industrial countries to nonexistent in certain developing countries.
Many developing countries are beginning to enact regulations related to genetically engineered
products. Furthermore, operational field-testing regulations have been implemented in, for example,
Argentina, Brazil, Mexico, Chile, Costa Rica, Cuba, India, the Philippines, and Thailand. But still
the majority of developing countries currently do not have a regulatory system for genetically modi-
fied organisms (GMOs) in place. Developing a regulatory framework may be a costly and time-con-
suming process involving extensive consultation and effort. Many countries in sub-Saharan Africa
are now establishing national biosafety committees and biosafety regulations regarding the use of
GMOs. There is also need to harmonize biosafety regulations at the regional level.
Although many developing countries have assembled the regulatory structures required to carry
out field trials, the existing legislation is often outdated and the process itself has either never been
put into practice or is too slow. This situation obviously presents significant obstacles for those wish-
ing to develop a product within such regions. The programs such as Plant Biosafety Systems (PBS)
and Agricultural Biotechnology Support Project II (ABSPII), both funded by the U.S. Agency of
International Development, are trying to modernize the regulatory infrastructure and build capacity
of regulatory bodies in developing countries. Thus, as a record of safe field trials is established, the
process will become progressively simplified and routine.
Desired traits within a transgenic plant must be expressed at the required level under natural cul-
tivation conditions. Presently, very little data is available regarding how transgene expression will
be affected when transgenic banana plants are cultivated in the field. Since the aim is to enhance
existing farmer-preferred germplasm without altering their desirable traits, it is essential that capac-
ity to produce genetically transformed plants is expanded into the agronomically most important
varieties within the major banana-growing regions.
Rather, studies in East Africa suggest that varieties modified for pest or disease resistance will
be incorporated into the range of varieties already grown as part of a strategy to reduce risk, pro-
vide multiple products, and satisfy varying tastes (http://archives.foodsafety.ksu.edu/agnet-archives.
htm). In the meantime, various biotechnologies are already contributing to conventional breeding
efforts and are expected to become even more effective in this area as genetic maps and markers are
refined. The use of tissue-culture plants is already contributing to the development of novel produc-
tion systems for smallholder farmers. Indeed, tissue culture is expected to be much more widely
used in increasing the productivity and sustainability of such systems as part of a balanced program
of deploying biotechnologies cost effectively in developing countries in the future.
11.7 Conclusions
Bananas are seriously threatened by several diseases and pests. Thus resistance to biotic stresses is
an important part of regional or national efforts. Since the major cultivated varieties of banana are
sterile and therefore do not set seed, traditional breeding is more difficult than genetic transformation
using molecular techniques. Attempts to produce transgenic bananas are proceeding slowly, but
public acceptance of these novel plants and their products will depend on sound information and
risk assessment. There is major public concern for the transfer of transgenes from transgenic field
material to wild species, but the chances for this happening in banana are expected to be negligible
in view of the sterility of many cultivars. The scope for further improvement of banana is large;
along with other methods of crop improvements, transgenic technology should provide fast and
effective methods.
Currently, no transgenic bananas and plantains are commercially available. However, many
research institutes, organizations, and universities are concentrating on the development of pest- or
disease-resistant varieties and improving the nutritional contents of banana and plantain. The num-
ber of experimental transgenic bananas is continuously increasing. Transformation protocols, includ-
ing tissue culture techniques, suitable transformation constructs with modified promoters driving
one or more transgenes, appropriate transformation techniques, the detection of the transgenes, and
characterization of their insertion sites are well developed.
Musa genomics can open up new avenues for more efficient breeding of the crop. It is impor-
tant to investigate the possibilities by which the primary production and other uses of banana
can be promoted for the benefit of the growing world’s population. Strategies for future genom-
ics research in Musa include the development of molecular markers, construction of genetic
and physical maps, identification of genes and gene expression, and whole genome sequencing.
Sequencing of other plant genomes such as A. thaliana and O. sativa has provided an enormous
amount of data that could reveal unknown features of their genomes. Such data could also be
generated for Musa. A Global Musa Genomics Consortium was established in 2001 with the
goal of ensuring the sustainability of banana as a staple food crop by developing an integrated
genetic and genomic understanding, allowing targeted breeding, and transforming and efficiently
using Musa biodiversity. Basically, the consortium aims to apply genomics to the sustainable
improvement of banana and plantain. The consortium believes that genomic technologies such as
analysis and sequencing of the banana genome, identification of its genes and their expression,
recombination, and diversity can be applied for the genetic improvement of the crop (Frison et
al., 2004).
There is enormous potential for genetic manipulation of bananas for disease and pest resistance
using the existing transformation systems using the genes isolated from the Musa genome. The use
of appropriate gene constructs may allow the production of nematode, fungus, bacterial, and virus-
resistant plants in a significantly shorter period of time than using conventional breeding, especially
if several traits can be introduced at the same time. It may also be possible to incorporate other
characteristics such as drought tolerance, thus extending the geographic spread of banana and plan-
tain production, and contributing significantly to food security and poverty alleviation in developing
countries. Long-term and multiple disease resistance can be achieved by integrating several genes
with different targets or modes of action into the plant genome.
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12 Genotype by Environment
Interaction and Musa
Improvement
Rodomiro Ortiz and Abdou Tenkouano
Contents
12.1 Introduction........................................................................................................................... 237
12.2 Phenotypic Stability in Banana and Plantain........................................................................ 238
12.2.1 Multienvironment Testing......................................................................................... 238
12.2.2 Stability Analysis....................................................................................................... 238
12.3 Effect of Genotype by Environment Interactions on Musa Breeding................................... 239
12.3.1 Genotype by Environment Interactions and Trait Heritability and Repeatability.... 239
12.3.2 Indirect and Multitrait Selection, Selection Index, and Ideotype Breeding.............. 239
12.3.3 Genotype by Crop Management Interactions............................................................240
12.3.4 Black Sigatoka Host-Plant Resistance Interactions with the Environment............... 241
12.3.5 Genotype by Environment Interactions on Root and Reproductive Traits................ 241
12.3.5.1 Roots........................................................................................................... 241
12.3.5.2 Reproductive Traits..................................................................................... 241
12.4 Genotype x Environment Interactions on Reaching the End User........................................ 242
12.4.1 Market Potential: Beyond the Farm Gate.................................................................. 242
12.4.2 Defining Best Bets for Dissemination....................................................................... 243
12.4.3 Nondisruptive Dissemination....................................................................................244
References....................................................................................................................................... 247
12.1 Introduction
This chapter provides the state of knowledge on genotype by environment interactions (or GxE
hereafter) in Musa and components of phenotypic stability for some traits (especially bunch
weight and host-plant resistance to black Sigatoka) in this crop. This chapter reviews how to
manage GxE to have efficient selection schemes and multilocation testing prior to release of
improved cultivars.
The clonal phenotype, which corresponds to a specific genotype, can vary from year to year in
the same location and or from location to location within an agro-ecozone in the same year (Ortiz
and Ekanayake, 2000). This phenomenon, which affects genotype ranking in different environ-
ments, is known as genotype by environment interaction.
Plantain and banana breeding programs aim to identify genotypes that have both a high and a
stable yield in a range of environments across a targeted a region (Vuylsteke et al., 1997). In the
presence of a significant GxE, both the stratification of environments according to their agro-cli-
matological similarities and the determination of stability parameters for genotypes across environ-
ments are important tools for the management of this interaction.
237
Several techniques have been developed to determine the most stable genotype in a set of repli-
cated trials across years and locations or combinations of both environments (Ortiz and Ekanayake,
2000). Postdictive models are, however, not useful in the identification of which genotypes and
environments contribute to the GxE interaction. Moreover, a breeder may be interested to identify
which genotypes are adapted to specific environments or to predict their performance in a specific
location. The additive main effects and multiplicative interaction (AMMI) model was developed to
provide answers for such questions (Gauch, 1992). AMMI uses the analysis of variance (ANOVA)
to study the main effects of genotypes and environments and principal component analysis (PCA)
for the residual multiplicative interaction. In this regard, Ortiz (1996) assessed the potential of
AMMI analysis for field assessment of Musa genotypes to banana streak virus (BSV) infection in
West Africa. The AMMI1 model revealed that an increase in clonal susceptibility resulted in a more
unstable response to BSV, whereas the AMMI2 model showed that seasonal rather than locational
diversity accounted for most of the interaction patterns. Such results suggested a low level of BSV
strain differentiation in this sub-Saharan Africa region.
12.2 Phenotypic Stability in Banana and Plantain

Plantain and banana breeding programs need to assess thoroughly their materials through multilo-
cation testing (Ortiz, 1998). A sequence of multisite trials will lead to selecting promising clones as
potential new cultivars in the targeted agro-ecozone.
12.2.1 Multienvironment Testing
The first GxE analysis of Musa multilocation trials in West Africa showed that this interaction
affected all traits except fruit circumference (De Cauwer et al., 1995). Moreover, the genotype by
location was significant for bunch weight, number of hands, number of fruits, and fruit weight,
whereas most of the traits were not affected by genotype by cycle interaction in the humid and
degraded forest of Nigeria. These observations suggest that multilocation trials may be more effi-
cient than single-site trials over several years in the humid forest of West Africa.
12.2.2 Stability Analysis

Further stability analysis of bunch weight and yield potential, based on the phenotypic coefficient of
variation (PCV), allowed the identification of high- and stable-yielding tetraploid plantain-banana
hybrids for West African humid forests (Ortiz et al., 1997). Selections from the cross [‘Obino l’Ewai’
× ‘Calcutta 4’] were among the highest yielders in this set of multilocation trials, which confirmed
the potential of this cross to generate diverse but stable high-yielding hybrids for further advanced
testing and subsequent cultivar release(s) in the “plantain belt” of West Africa. Likewise, de Cauwer
and Ortiz (1999) indicated that the cooking banana cultivar ‘Cardaba’ had the most stable yield
potential in the West African humid and degraded forest as revealed by the AMMI biplots of the
GxE analysis.
Stability and AMMI analyses provided a means for identifying Musa cultivars and exper-
imental hybrids with stable bunch weight across environments in sub-Saharan Africa (Ortiz,
1998). These analyses also facilitated the interpretation of multilocation testing results, which
suggested that the yield potential of a Musa clone should be assessed in the ratoon cycle because
plantain and banana are perennial crops. Furthermore, Tenkouano et al. (2002) showed that there
was a higher genetic expression for most yield components during the second crop cycle in all
the environments, which confirms that the selection should be better in ratoon than in the first
crop cycle.
Selection of high-yielding clones with specific adaptation should be in environments showing
the respective stress to select for. Baiyeri et al. (2000b) suggest that farm gate and postharvest
Genotype by Environment Interaction and Musa Improvement 239
processing traits should be taken into account when selecting for broad or specific adaptation.
Selection indices considering both groups of traits could allow Musa breeders to perform multitrait
selection and enhance breeding efficiency. Further research showed that simultaneous use of dif-
ferent stability statistics may protect Musa breeders from wrongly identifying presumably stable
genotypes (Baiyeri et al., 1999a). For example, Musa genotypes selected by AMMl and the regres-
sion coefficient were also classified as stable by PCV and Kang’s statistic for simultaneous selection
for high and stable yield.
12.3 Effect of Genotype by Environment

Interactions on Musa Breeding
With reduced budgets allocated for agricultural research, site rationalization had become an impor-
tant issue to consider when carrying out multilocation testing of promising selections. Experimental
results from West Africa showed that multilocation testing is more profitable than single-site evalu-
ation over several years in a Musa breeding station. Ortiz and de Cauwer (1998) also suggested,
based on correlated responses across environments for yield potential, that a selection site in the
humid forest could be dropped when selections in one site may be well adapted to the other location
in the same agro-ecozone. Their results further indicated that the relatively poor performance of
most Musa genotypes in dry environments reinforced the importance of early testing across a wide
range of environments. In this way, selections with broad or specific adaptation may be identified
for further release to targeted farmers.
12.3.1 Genotype by Environment Interactions and Trait Heritability and Repeatability

The “easy to breed” traits are those that can be scored easily and have a constant phenotypic expres-
sion in all environments: high heritability and low environmental influence. Traits with low (or
nonsignificant) GxE, although they may be affected by the environment, are more important for
assessing their potential across environments.
Components of variance are estimated to calculate broad-sense heritability (the ratio between the
genetic and phenotypic variances) and repeatability (or the ratio between the genetic and the GxE
plus the environment variances). Table 12.1 lists heritability and repeatability estimates for growth,
bunch, and fruit traits in Musa germplasm. Plant height and bunch weight appear to be significantly
influenced by the GxE and thus showed lower heritability than traits such as pseudostem girth,
number of fruits, and fruit sizes, which are highly heritable and repeatable across environment, and
thereby likely to be more responsive to selection than the former.
12.3.2 Indirect and Multitrait Selection, Selection Index, and Ideotype Breeding

Indirect selection may be useful for selection of traits that are difficult to score or that show low heri-
tability. Intraclass correlations (Ortiz, 1997b) suggest that selection for large fruit may lead to heavy
fruits in plantain-banana hybrids, thereby resulting in plants bearing heavy bunches. However, sig-
nificant correlations between plant height and bunch weight in polyploidy hybrids indicate that
selection of dwarf cultivars with heavy bunches may be difficult. Further research by Tenkouano
et al. (2002) showed that associations between bunch weight and yield components were higher
than between bunch weight and phenological traits. In the former cases, the genetic correlation was
similar to the phenotypic correlation, indicating that GxE effect on the relationship between bunch
weight and yield components was neutral. This result confirms that yield components could serve as
indirect selection criteria for bunch weight in Musa.
Breeding efforts for genetic improvement of banana and plantain have gradually shifted from indi-
vidual trait selection to simultaneous improvement of several traits following an ideotype concept in
Table 12.1
Broad-Sense Heritability (H2) and Repeatability (R) Estimates for Growth, Bunch, and
Fruit Traits in Triploid Musa Germplasm after Trials across Crop and Ratoon Cycles in a
Single West African Humid Forest Location
Plantain (75) and Banana Plantain (75) and Plantain (52) and
(17) Cultivarsa Banana (18) Cultivarsb Banana (51) Cultivarsc
Trait H2 R H2 R H2 R
Days to flowering – – 0.80 1.50 – –
Plant height 0.84 2.16 0.91 1.52 0.75 1.47
Plant girth 0.72 1.29 0.89 1.26 0.86 3.08
Leaf number – – 0.86 2.30 – –
Leaf length/width ratio at flowering – – 0.69 1.06 – –
Tallest sucker height at flowering 0.61 0.45 0.89 2.36 0.82 2.31
Tallest sucker height at harvest 0.75 1.06 0.78 1.51 0.82 2.20
Days to harvest – – 0.80 1.43 – –
Days to fruit filling – – 0.76 1.58 – –
Bunch weight 0.42 0.87 0.66 0.98 0.68 1.05
Fruit weight 0.86 2.18 0.89 4.11 – –
Hand number 0.89 4.13 0.93 6.61 0.78 1.70
Fruit number 0.96 13.06 0.94 8.40 0.80 1.99
Fruit per hand 0.94 0.96 7.86 12.83 – – 0.82 0.92 2.26 5.49
Fruit length – – 0.95 9.67 0.91 0.93 4.87 6.42
Fruit girth – – 0.82 2.14 0.86 0.90 3.48 4.24
a Baiyeri, K.P. and R. Ortiz, 2000, Agronomic evaluation of plantain and other triploid banana in Africa, Acta Hort., 540,
125–135.
b Ortiz, R., D. Vuylsteke, R.S.B. Ferris, J.U. Okoro, A.N. Guessan, O.B. Hemeng, et al., 1997, Developing new plantain
varieties for Africa, Plant Var. Seeds, 10, 39–57.
c Ortiz, R., 1997, Morphological variation in Musa germplasm, Genet. Res. Crop Evol., 44, 393–404.
Musa. There are, however, a few common pathways determining yield potential among plantain land
races, making it difficult to define a common ideotype for plantain breeding (Ortiz and Langie, 1997).
This finding suggests that plantains may possess different genes controlling similar pathways, or dif-
ferent traits contributing to yield potential. Likewise, defined ideotypes may differ for each landrace,
according to the production system. Plantain breeders should therefore consider those relationships that
are affected by both genotype and production system when selecting for improved hybrid germplasm.
12.3.3 Genotype by Crop Management Interactions

Careful management of organic matter is essential to achieve sustained perennial productivity of
plantain under large-scale field production conditions. Agro-forestry systems such as alley crop-
ping and the management of regrowth of natural bush fallow species in plantain fields have been
investigated for their capacity to maintain productivity over long periods of cultivation without
degradation of the resource base (Ortiz and Langie, 1997). Alley cropping generally leads to better
performance in plantains than sole cropping (Baiyeri et al., 1999b, 2000b, 2004).
The performance of improved genotypes relative to cropping systems should be also assessed
because on-site cultural practices used in breeding trials may differ considerably from typical farmer
practice and could influence genotype selection as well as the size of testing plots (Ortiz, 1995). For
example, early selection of sucker for the ratoon crop and other crop management options that will
enhance healthy growth of the plant crop will sustain high yield in Musa genotypes (Baiyeri et al.,
2000). Baiyeri et al. (2004) warn on generalizing Musa cultivar recommendation across cropping
systems because they found a significant genotype by cropping cycle interaction for yield and post-
harvest traits in multilocation trials in Nigeria (Baiyeri et al., 1999b).
12.3.4 Black Sigatoka Host-Plant Resistance Interactions with the Environment

The aim of any resistance breeding program is to develop superior high-yielding genotypes with
durable resistance (Craenen and Ortiz, 2003). Host-plant resistance breeding needs therefore meth-
ods that are reliable for discriminating between resistant and susceptible genotypes across target
environments and strains of the pathogen. Moreover, epidemiological studies aimed at the identifi-
cation of different pathotypes with variable degrees of virulence on well-known standard cultivars
(or differentials) help in monitoring the appearance of more virulent strains and to establish a resis-
tance breeding strategy able to maximize the probability of generating durable resistance.
Although field-screening methods are relatively simple, they are time consuming and affected by
environmental factors such as weather and soil, which may affect symptom expression. Hence, it is
usual to include genotypes of known host response as checks (from susceptible to highly resistant)
and to gather a large dataset to validate results. Theory from population genetics indicates that the
smaller the selection intensity(s), the lower the response to selection; that is, increases in frequency
of favorable alleles will be at maximum when there are no escapes during screening (or maximum
selection efficiency). Host-plant resistance breeding becomes inefficient (and it may be worthless)
with increasing escape rates due to the screening method.
Genotype by environment interaction influences black Sigatoka reaction in plantain and banana,
which explains the low repeatability of host-plant resistance traits to this fungal pathogen (Craenen
and Ortiz, 1997). GxE was, however, not important when the analyses were based on the marker
genotype bs1, which is regarded as the major gene in the host response (slow disease development) to
black Sigatoka (Ortiz and Vuylsteke, 1994). This finding confirms that Mendelian genetic markers
are seldom affected by the environment.
Genotype by location effects are more important that the nonsignificant genotype by year inter-
action (Ortiz et al., 1997). These differences between locations may be attributed to the bias of
different surveyors. Nonetheless, results from multilocation trials in sub-Saharan Africa showed
that all partially resistant hybrids had a homeostatic resistant host response to black Sigatoka. Their
slow disease development may lead to durable resistance to black Sigatoka because such host-plant
response slows the progress of an epidemic without inhibiting its initiation.
12.3.5 Genotype by Environment Interactions on Root and Reproductive Traits

12.3.5.1 Roots
Root phenotypic plasticity refers to the ability of roots to adapt their structure to changes in the
environment. Soil temperature, moisture level, partial pressure of carbon dioxide and oxygen, and
nutrient availability are among the most important edaphic factors influencing root development.
Likewise, air temperature, day length, light intensity, and partial pressure of carbon dioxide affect
the provision of nutrients and growth regulators from the shoot to the root system and the shoot–root
ratio of a plant. Results from trials in two contrasting Nigerian agro-ecozones showed that location
significantly affects most aerial growth, corm, and root traits (except for the leaf number), total cord
root length of the mother plant and mat, and average cord root diameter of the mother plant (Blomme
et al., 2006). The genotype by location interaction was also significant for all above traits.
12.3.5.2 Reproductive Traits
The environment influences seed set in female-fertile plantain cultivars (Jenny et al., 1993). Ortiz and
Vuylsteke (1995) indicated that seed production in plantain, as well as embryo and seed germination
success of plantain-banana hybrids, followed a seasonal variation pattern in which production of

desired tetraploids hybrids (after triploid-diploid crosses) are highest when hand pollinations are
done under high solar radiation and low relative humidity. Such weather factors seem to be the most
convenient for relatively high production of 2n eggs as compared to haploid gametes. There was
also a seasonal variation in 2n pollen production in some diploid banana cultivars that was posi-
tively correlated with solar radiation (Ortiz, 1997c), which confirms that high solar radiation could
enhance 2n gametes production in Musa.
Environmental conditions may also influence the quantity and quality of pollen produced in
fertile Musa clones (Dumpe and Ortiz, 1996). Ortiz et al. (1998) indicated that solar radiation was
also significantly associated with pollen stainability in Musa. Their results further confirm that this
weather factor as well as temperature, total pan evaporation, rainfall, and minimum relative humid-
ity correlate significantly with 2n pollen production. Hence, Musa breeders should identify the best
time of the year to maximize both fertility and 2n gamete production to enhance the synthesis of
polyploid Musa hybrids through sexual polyploidization.
12.4 Genotype x Environment Interactions

on Reaching the End User
Plant breeding programs aim to develop and deploy improved cultivars that consistently display
distinct phenotypic superiority in cultivation or utilization when compared to existing cultivars
across their cropping range in farmers’ fields. The fundamental determinants of the performance
of a cultivar (P) are the biological potential embedded in the genetic makeup of the cultivar (G), the
quality of the environmental context in which it is grown (E), and the responsiveness of the cultivar
to changes in the environment (GxE) so that P = G + E + GxE.
Typically, superior genotypes are identified and selected in experimental fields that only margin-
ally represent the range of target environments, but the breeders’ reward depends on the suitability
of such superior cultivars to the biophysical and socioeconomical circumstances of the farmers.
GxE interactions may cause discrepancies between expected and observed performance of bananas
and plantains both spatially and temporally as has been observed in Nigeria (Baiyeri, 1998), Uganda
(Baiyeri et al., 2008), and outside Africa (Orjeda, 2000).
Ideally, therefore, each cultivar should be targeted to the environment that maximizes the expres-
sion of its biological potential. Causes of spatial variation include differences in climate (rainfall
pattern and temperature), soil quality (biophysical characteristics), and cultural practices. The same
factors can change over time and explain temporal variations. Understandably, plant breeders can-
not replicate each target environment at their breeding stations even if the target environments were
not subject to stochastic variations. Nor is it logistically possible for breeders to carry out validation
tests of selected lines in the whole range of targeted environments. However, it is conceivable that
data on long-term climatic characteristics, soil quality, and other factors can be used to define broad
target zones within which representative sites could be chosen for additional evaluation of most
promising lines selected in research stations.
Perhaps equally or more important than the biophysical context, it is the cultural and economic con-
text of the farming communities that dictates cultivar choice for adoption (Tenkouano et al., 2009).
12.4.1 Market Potential: Beyond the Farm Gate

The concept of market potential (Bressani, 1976) has been adapted to banana and plantain by Baiyeri
et al. (1999b), who used it to discuss the likelihood of adoption of a new cultivar by a farming com-
munity as a function of the value of the products derived from the harvested yield. The utilizable
fraction of the yield is a function of pulp weight of the harvested fruit, dry matter content of the
pulp, and shelf life of the fruit. Dry matter content determines the biological value of the crop, that
is, the harvested fraction that is available for processing into food products (Sanchez et al., 1968).
Ripening patterns—that is, shelf life—dictate processing options and the array of food products
that can be derived from the crop, and this may determine the economic value of the crop (Dadzie
and Orchard, 1996; Ferris et. al., 1996).
Ripening is associated with progressive hydrolysis of starch into simple sugars, which induces
changes in pulp firmness and peel color (Stover and Simmonds, 1987). Three ripening stages, eas-
ily distinguished by the color of the peel and the consistency of the fruit, can be broadly defined.
The first stage encompasses the period from complete greenness (unripe fruits) to yellowing of the
fruit tip. The fruit is very firm and can be subjected to transport over relatively long distances and
processed into dried/solid products, such as flour, that can be subsequently used in various prepa-
rations. The second stage goes from the end of the first stage to about 60% yellowing of the fruit.
This is usually the preferred stage of consumption of dessert bananas with sufficient hydrolysis of
starch into sugars while retaining a soft/firm consistency. Some solid food products can be derived
at this stage, but options for dried products become limited or uneconomical while those for liquid
products, such as soft drinks, become appealing.
The third stage begins with the end of the second stage, with increased browning of the peel
and increased conversion of the sugars from hydrolyzed starch into alcohol, through fermentation.
Virtually no solid product can be derived at this stage without addition of solidifiers from other
crops for traditional preparations, but options for fermentation and distillation become attractive.
In some West African countries, the market price of ripe fruits is higher than that of the green fruit
(Ahenkora et al., 1996), but the increased number of food processing options with green fruits com-
pared to ripe fruits would normally make fruits in earlier stages of ripening more marketable than
those in later stages.
Therefore, the duration of each stage for a cultivar, not just the total shelf life, is an important
determinant of what the cultivar can be processed into, hence its market potential. Taking this
further, it is known that cultural preferences exist for traditional food preparations. Therefore, it is
likely that a given food preparation option may have higher social value in some environments but
not in others. Consequently, a high-yielding cultivar that has a good segmentation of its shelf life so
that it can be used for a given food preparation preferred by populations in a particular area should
be targeted to that area for release. On the breeding side, knowledge of the biological characteristics
associated with a given food preparation can be used to understand the underlying genetic causes
of these characteristics and use this information for developing new cultivars that would fit the par-
ticular food preparation, a process that can be termed “end-usage breeding.”
The extension of this concept to industrial products is straightforward, with a broadening of the
target clients from traditional farmers producing for their own consumption or local village markets
to specialty crop growers feeding industrial processors. Thus, a market potential index can be esti-
mated from biological yield components, the number of days in each ripening stage, and the value
of corresponding food products across target environments, using the following formula:
M = Y ∑ijsiajk(i)
where M is the estimated market potential index of the cultivar, Y is the dry pulp yield of the culti-
var, si is the number of days spent in the ith ripening stage by the cultivar, and ajk(i) is a subjective
index value attributed to the jth product in the kth target environment (Figure 12.1).
12.4.2 Defining Best Bets for Dissemination

Before attempting to introduce new cultivars to farmers, it is important to establish their likely
performance when grown under smallholder-managed environments. This can be done by bringing
farmers’ voices into the cultivar development process, notably through farmer-participatory variety
evaluation, whereby breeders and farmers jointly conduct and evaluate trials in order to identify
Socio-environmental context (E) and product

Ripening stages Products value in each context (a)
S1 S2 S3 E1 E2 ........ EJ ........ EP Mean/P
P1 a11 a12 ........ a1j ........ a1p a1.

P2 a21 a22 ........ a2j ........ a2p a2.
: : : : : :
: : : : : :
: : : : : :
Pi ai1 ai2 ........ aij ........ aip ai.
: : : : : :
: : : : : :
: : : : : :
Pn an1 an2 ........ anj ........ anp an.
M = YΣsiajk(i) Mean/E a.1 a.2 ........ a.j ........ a.p
Breeding Targeting new cultivars
Figure 12.1 Illustration of market potential as a breeding and targeting tool: Harvested fruits undergo a
ripening process made of distinct stages with genetically governed duration (S). There is a range of processed
food products (P) obtainable from each cultivar and the value of such products (a) depends on the cultural and
economic context (E). Thus, besides the yield (Y), the adoption potential of a cultivar depends on the relative
value components of shelf life and the value of the processed products. A new cultivar that is good for a pro-
cessed product that has high value in a given environmental context should be targeted to that environment.
Likewise, knowledge of the biological characteristics associated with a given food preparation can be used to
develop niche-specific cultivars.
those cultivars most attractive to the farmers, which are often location specific. This allows farmers
to cast their cultivar choices without exposing the household to any risk (Ceccarelli and Grando,
2004; Sperling et al., 2001) and facilitates the subsequent deployment of the chosen cultivars on a
large scale in matching regions, constituting a cost-efficient means of enhancing crop productivity
and farmers’ incomes. Regardless of the method chosen for capturing farmers’ voices, cultivars can
be classified into four broad categories, depending on the extent of congruence between agronomic
performance and farmers’ assessments (Figure 12.2).
12.4.3 Nondisruptive Dissemination
Resource-poor farmers in many regions of the world, particularly in Africa, have adopted cropping
strategies based on intraspecific (cultivar mixtures) and interspecific (various forms of intercrop-
ping) to maximize land and labor use efficiency and to minimize risks of crop failure. Cultivar
mixtures are common in subsistence farming systems, offering growers diversity of diet, stability
of income, and reduced losses to pests (Smithson and Lenne, 1996; Selatsa et al., 2009). Thus, the
chances for adoption of new cultivars from breeding programs would partly depend on the extent
that traditional cropping and consumption practices are not drastically disrupted.
One of the lessons learned over the years by the breeding programs of the International Institute
of Tropical Agriculture (IITA) was that as much as farmers seemed attracted to new cultivars,
Let go varieties
Best Bet
(Associate with
Varieties
other options)
Farmers’ preference ranking

SUBSISTENCE INNOVATIVE
Need further Seek alternative uses to

improvement diversify options
HOLD BACK SPECIALTY
Agronomic ranking
Figure 12.2 Schematic representation of the outcome of farmer participation in cultivar selection. High
congruence between agronomic performance ranking and farmers’ preference ranking indicates that the cul-
tivars are best bets for further evaluation under farmers’ conditions (top right quadrant) or should be held back
and subjected to further improvement (bottom left quadrant); in the lack of congruence between agronomic
performance and farmers’ preference, one group of cultivars can be promoted if they lend themselves to
economically attractive ways of processing unfamiliar to the grower, provided the new processing options
are included in the dissemination process (bottom right quadrant). This group can then easily move into the
group of best bets. The last group of cultivars are those not performing well agronomically but are nonetheless
attractive to farmers (top left quadrant), which the farmers should be allowed to have.
owing notably to their higher yields under disease pressure where the traditional cultivars succumb,
they also insisted on keeping their traditional cultivars. This suggested that the disease-resistant
cultivars should be introduced into the farmers’ cropping system through association with their own
landraces and other crops.
Nondisruptive dissemination of new cultivars through mixtures promotes plant diversity. This
has the potential of increasing the productivity and stability of the mixture, but it depends on the
success of each individual in the mixture composition, which in turns depends on the extent of
competition among individuals in the mixture. The performance of plants in communities departs
significantly from their performance in isolation, a discrepancy that is usually attributed to negative
or positive competitive interactions, but the relative importance of competition and facilitation may
vary inversely along gradients of abiotic stress (Callaway et al., 2002). Diversity is considered to be
a stabilizing factor for an ecosystem that operates like insurance, allowing for greater resilience of
the ecosystem in the face of stressful conditions (Kahmen et al., 2005).
There may be different options for cultivar mixtures, but the simplest approach could be one
that achieves equal representation of new and old cultivars. Binary mixtures can achieve a 1:1 old-
to-new cultivar ratio or 50% landrace substitution level. This approach was used for large-scale
on-farm testing and dissemination of improved cultivars in Nigeria, using a checkerboard design
whereby each hybrid plant was surrounded by four plants of a landrace and vice versa (Tenkouano
and Swennen, 2004; Tenkouano et al., 2009). The mixture was effective in reducing black Sigatoka
effects on the landrace, whereby the index of nonspotted leaves (INSL) increased from 43.2% to
51–56% when the landrace was mixed with the hybrids, and the bunch weight of the landrace
increased from 4.9 kg when grown solely to 7.1–8.1 kg when grown in mixture with the hybrids
(Table 12.2). Similar results were observed in Cameroon, with the mixture increasing the INSL of
the local variety ‘Essong’ from 24.3% when grown solely to 30.8–37.7% when mixed with hybrids.
Table 12.2
Improved Hybrids Act as “Bio-Pesticides” to Enhance Landrace
Performance and Preserve Diversity (50% Substitution) in Cultivar
Mixtures in Nigeria and Cameroon
Bunch Weight Index of Nonspotted Leaves
Clone Treatment (kg) (%)
Nigeria
Agbagba Agbagba sole 4.9 ± 0.2 43.2 ± 3.1
Agbagba Agbagba + BITA3 8.1 ± 0.6 55.7 ± 2.1
Agbagba Agbagba + PITA14 7.1 ± 0.4 51.4 ± 3.6
Agbagba Agbagba + PITA17 7.3 ± 0.3 52.8 ± 2.7
BITA3 Agbagba + BITA3 16.7 ± 1.1 73.8 ± 2.8
PITA14 Agbagba + PITA14 12.7 ± 0.6 89.7 ± 2.0
PITA17 Agbagba + PITA17 12.7 ± 0.7 75.4 ± 3.0
Cameroon
Essong Essong sole 9.6 ± 1.9 24.3 ± 0.5
Essong Essong + BITA3 9.1 ± 2.6 30.8 ± 3.7
Essong Essong + PITA14 11.2 ± 0.8 37.7 ± 3.6
Essong Essong + PITA21 10.6 ± 3.0 39.6 ± 7.5
BITA3 Essong + BITA3 11.5 ± 1.4 68.7 ± 1.2
PITA14 Essong + PITA14 9.0 ± 1.2 72.0 ± 0.6
PITA21 Essong + PITA21 6.6 ± 0.7 66.6 ± 1.8
Note: The cultivars ‘Agbagba’ and ‘Essong’ are among the most preferred plantains in Nigeria and
Cameroon, respectively. They were grown in mixture with the improved hybrids ‘BITA3,’
‘PITA14,’ and ‘PITA17’ (Nigeria) or ‘PITA21’ (Cameroon).
Likewise, increased bunch weight of the local cultivar was observed with mixtures, although not
statistically different from the bunch weight obtained under sole cropping (Table 12.2).
It is noteworthy that although the performance of the landrace is significantly enhanced, this
is still less than that of the hybrids. Thus, cultivar mixtures appear to enhance the performance of
the landraces without altering that of the hybrids. This mechanism not only helped to increase the
yield of the landraces (preferred for their culinary properties), but it also preserved genetic diversity
while exposing farmers to new, high-yielding hybrids. Hence, cultivar mixtures can be considered
a nondisruptive mechanism for introducing new hybrids into farmers’ fields, where the landraces
can satisfy traditional consumption needs while hybrids can be processed for commercial purposes
(there is a wide variety of options).
The epidemiological factors that are responsible for the apparent improvement of the perfor-
mance of the landraces have not been fully investigated, but there are several theoretical possibili-
ties, such as inoculum trapping by resistant cultivars resulting in reduced disease spread to plants
of the susceptible landraces. Neighbors can beneficially change the situation of a plant, most com-
monly when the neighbors contribute to alleviation of environmental conditions that are unfavor-
able to the survival, growth, and reproduction of the plant (Bertness and Hacker, 1994). Thus, the
mixture approach should be based on a careful analysis of the blending ability of the cultivars
proposed for dissemination. The blending ability can be experimentally calculated using formu-
las analogous to those developed for analysis of genetic combining ability (Gallandt et al., 2001;
Springer et al., 2001).
Regardless of the theoretical basis of the beneficial effects of mixtures, mixtures may be a
better option than chemical control. Chemical control is done either with protectant or systemic
fungicides. Protectant fungicides are applied topically to the leaves and are effective pre-infec-
tionally but they have adverse effects on leaf physiology depending on the amount of oil emul-
sions and weather conditions. Systemic fungicides are effective post-infectionally but have been
reported to cause development of resistance without loss of fitness in M. fijiensis populations
(Marin et al., 2003).
Growing susceptible cultivars is not recommended, unless there are environmentally acceptable
means of controlling the biological organisms to which the cultivars are susceptible. Biological
principles dictate that the causal organisms thrive to maintain a balance between their growth that
is detrimental to the host and the need to circumvent extinction of the host, which is synonymous of
extinction for the organisms unless alternate hosts are available. Similarly, exclusive deployment of
cultivars that are highly resistant could impose extreme pressure on the biological threat, forcing it
to mutate to more virulent forms in order to ensure its survival.
Under such circumstances, it is advisable to develop a strategy that maintains an acceptable
balance between threatened and threatening organisms. Thus, growers of resistant cultivars should
provide a biological refuge consisting of susceptible cultivars to the threatening organisms, similar
to the practice for deployment of transgenic plants with Bt resistance to insects (Gopalaswamy et al.,
2003). The cultivar mixture approach to the dissemination of new cultivars of banana and plantain
adheres to this principle.
References
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13 Quality Improvement
of Cultivated Musa
Edson Perito Amorim, Sebastião de Oliveira e Silva,
Vanusia Batista de Oliveira Amorim, and Michael Pillay
Contents
13.1 Introduction........................................................................................................................... 251
13.2 Nutritional Value................................................................................................................... 252
13.2.1 Nutrition in Banana................................................................................................... 252
13.2.2 Identification of Genotypes with Functional Food Attributes................................... 253
13.3 Breeding Objectives for Quality Improvement.....................................................................260
13.3.1 Biofortification...........................................................................................................260
13.3.2 Crop Improvement..................................................................................................... 263
13.3.3 Genetics.....................................................................................................................264
13.3.4 GxE Interaction.........................................................................................................264
13.3.5 Breeding for High Yield and Micronutrient Density.................................................264
13.3.6 Strategies for Agronomic Superiority........................................................................ 265
13.4 Conclusions............................................................................................................................ 265
References....................................................................................................................................... 265
13.1 Introduction
The world’s production of banana and plantain is estimated at about 125 million tons per year,
placing the crop first in the world’s ranking of fruit production, followed by grape, orange, mango,
and pineapple (FAO, 2010). Bananas are grown in more than 130 countries in the tropical and sub-
tropical zones in five continents (Jones, 2000). The world’s largest producers are India, Uganda,
the Philippines, China, and Brazil. Besides its importance as a food crop, banana cultivation has a
strong socioeconomic significance, since it is the only source of income for many small-scale grow-
ers (Mattos et al., 2010; Bakry et al., 2009).
Banana is a fruit that is rich in natural antioxidants such as vitamin C and vitamin E (Someya
et al., 2002; Amorim et al., 2009a). The antioxidant properties of many fruits are associated with
flavonoids and β-carotenes. Flavonoids are found in the pulp and peel of banana while β-carotenes
are present only in the pulp of some banana varieties (Vijayakumar et al., 2008). β-carotenes are
precursors of vitamin A. In low-income regions of the world, such as parts of Asia, Africa, and
Latin America, high levels of vitamin A deficiency lead to serious health problems, especially
in children (Bloem et al., 2005). Vitamin A is associated with cell differentiation, vision, bone
growth, reproduction, and integration of the immune system and is considered to play a role in
the prevention of cancer, cardiovascular disease, cataracts, and macular diseases as well as neu-
rologic, inflammatory, and immune disorders (Arora et al., 2008a, b). Micronutrient deficiencies
of iron and zinc also result in serious health problems such as mental and physical retardation,
reduced resistance to infections, and hypogonadism (Whittaker, 1998). Despite various efforts to
251
improve the vitamin and micronutrients intake of people the past 50 years, over 2 billion people,
about one third of the world’s population, suffer from vitamin A, Zn, and/or Fe deficiencies (FAO,
1997).
To maintain a healthy status, it is believed that the human body requires more than 20 minerals
and about 40 nutrients, especially vitamins and essential amino acids that can be easily obtained
from a healthy diet. However, the vast majority of the world’s population has little or no access to
sufficient quantity, quality, and variety of foods, resulting in deficiencies in minerals and essential
nutrients (Pfeiffer and McClafferty, 2007). García-Alonso et al. (2004) suggested that there is a
direct relation between diets rich in fruits and vegetables and a reduced risk of cardiovascular dis-
eases and some types of cancer. In this context, the biofortification of banana by conventional breed-
ing combined with the use of biotechnological tools has the potential to increase the concentrations
of micronutrients (Fe, Zn) and vitamin A in new cultivars, with the aim of improving the health
status of populations in both rural and urban areas of developing countries. Bananas are already
used in special diets where ease of digestibility, low fat, minerals, and vitamins are required. These
special diets are used for babies, the elderly, and patients with stomach problems, gout, and arthritis
(Nakasone and Paull, 1999).
This chapter deals with some aspects of banana as a food crop, emphasizing the nutritional value
and breeding strategies for the development of biofortified cultivars. Findings of different research
groups working on the identification, measurement, and use of genotypes with functional properties
are presented.
13.2 Nutritional Value
The nutritional value of any food crop depends on several variables such as growth stage, climatic
conditions, soil quality, and of particular importance, the genotype (Mozafar, 1994; Lee and Kader,
2000). Therefore assessments of the same micronutrients in a crop can differ significantly, depend-
ing on growth conditions and method of evaluation. This observation is also valid for banana.
13.2.1 Nutrition in Banana
The pulp of ripe banana consists essentially of sugar and is therefore easily digestible. The fruit is
composed of approximately 70% water, 27% carbohydrate, 0.3% fat, and 1.2% protein (Robinson,
1996). In addition, each gram contains approximately one calorie of energy. Twelve vitamins are
found in the fruit, which is considered a good source of the vitamins A, B1, B2, and C (Robinson,
1996). Banana and plantain differ in moisture content, with water content of banana averaging
75%, while plantains contain around 65% of water (www.answers.com/topic/banana-and-plantain).
Therefore the conversion of starch to sugars is quicker in bananas. Plantains are generally cooked
before being eaten, although they are eaten as a fresh fruit when ripe. The total lipid content is low
both in banana and plantain (<0.2%) and the energy the fruits provide is therefore not related to fat.
Similarly, the concentrations of amino acids are usually low (http://www.nal.usda.gov).
There are no significant amounts of antinutritional compounds in banana (Adeniji et al., 2007).
This contrasts sharply with other staple crops such as cassava that contains cyanogenic glycosides
(Koch et al., 1992; White et al., 1994) and potato that has high concentrations of glycoalkaloids
(Hellenas et al., 1995), which are toxins. Banana contains high concentrations of serotonin and
dopamine, two neurotransmitters that act directly in controlling the release of some hormones and
in regulating the circadian rhythms of sleep and appetite (Adao et al., 2005).
Recently, banana has been labeled as an ideal vehicle for the development of vaccines, in view of
the easy digestion, pleasant taste, and great acceptability of banana by children. Studies are under-
way to develop different vaccines in banana (Sala et al., 2003; Arntzen et al., 2004; Sunil Kumar
et al., 2005).
Quality Improvement of Cultivated Musa 253
The quantity of different nutrients in bananas is presented in Table 13.1 and includes protein,
sugar, minerals, vitamins, lipids, and amino acids, obtained from the U.S. Department of Agriculture
(UDA) based on the evaluation of banana samples, particularly of the subgroup Cavendish (AAA).
13.2.2 Identification of Genotypes with Functional Food Attributes

Studies on the identification and use of banana genotypes with functional properties have been
conducted around the world by different research institutions, including the following: Embrapa
Cassava and Tropical Fruits (Brazil), Catholic University of Leuven (Belgium), Bioversity (France),
University of Queensland (Australia), Tohoku University (Japan), University of Kerala (India),
Agricultural Research Service (United States), and the International Institute of Tropical Agriculture
(IITA), among others. This chapter presents the research results of the identification, assessments,
and quantification of functional compounds in different banana genotypes. Particular attention was
given to the presence of the following compounds: carotenoids and their fractions, vitamin C, total
polyphenols, flavonoids, and antioxidant activity.
Sharrock and Lutsy (1999) assessed the contents of carbohydrates, protein, fats, vitamins, and
minerals in banana and plantain, and compared the nutritional value of banana with those of sweet
potato, potato, cassava, and apple. The vitamin C content of banana and plantain was similar to that
found in other species (18.4 mg 100 g–1). Vitamin A content was higher in plantain (1127 UI/IU)
than in banana (81 IU) and exceeded that found in potato, cassava, and apple.
Kanazawa and Sakakibara (2000) estimated the dopamine content, a strong natural antioxidant
in banana (subgroup Cavendish). The amount varied from 80 to 560 mg 100 g–1 in the peel and from
2.5 to 10 mg 100 g–1 in the flesh. The antioxidant activity of dopamine was higher than of butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), flavonoids, catechin, and glutamine, and
was similar to that of other antioxidants, such as gallocatechin gallate and vitamin C. According to
the authors, dopamine is easily absorbed by the body and plays an important role in the control of
Parkinson’s disease. Someya et al. (2002) also found higher levels of the antioxidant gallocatechin
in the peel (158 mg/100 g dry wt) than in the pulp (29.6 mg/100 g dry wt) in Cavendish banana.
Together, these results suggest that bananas should be considered as a good source of antioxidants.
Englberger et al. (2003a, 2003b, 2003c) identified banana genotypes, commonly known as
the Fe’i bananas (section Australimusa), with much higher concentrations of β-carotene than in
Cavendish cultivars, which account for approximately 41% of the world’s total banana production.
There was a wide variability for β-carotene in the Fe’i bananas, with concentrations ranging from
56 µg 100 g–1 to 6360 µg 100 g–1 (Table 13.2). The genotypes from the islands of Micronesia (Karat
and Uht en Yap, Musa troglodytarum) contained 275 times more carotenoids than Cavendish. These
genotypes, with upward-growing bunches and fruits with variable and dark coloration, are promis-
ing sources of alleles for improving the nutritional quality of other bananas in breeding programs.
Adeniji et al. (2006) determined micronutrients in plantain and banana hybrids in Nigeria (‘PITA
14,’ ‘PITA 17,’ ‘BITA 3,’ ‘FHIA 17,’ ‘FHIA 23,’ and ‘Agbagba,’ used as a control). The cultivars
‘Agbagba’ and ‘FHIA 17’ had high Fe (36.5 µg g–1 and 16.13 µg g–1) and zinc contents (12.5 µg g–1
and 12.49 µg g–1), respectively. The carotenoid contents of the cultivars were similar (mean of 3.6
µg g–1) with the highest values recorded in ‘PITA 17’ (4.80 µg g–1). The authors are of the opinion
that it is possible to develop new banana hybrids with high yield and high micronutrient concentra-
tions. Such varieties may be potentially useful in reducing health problems related to deficiency of
vitamin A and/or other micronutrients.
Wall (2006) determined the mineral composition and vitamins C and A content in the banana
varieties ‘Prata,’ ‘Santa Catarina,’ and ‘Williams’ (Cavendish) and in papaya cultivars in Hawaii.
The variety ‘Prata’ contained 1.5 times more carotenoids than ‘Williams’ and the mean vitamin C
content was 12.7 mg 100 g–1 for ‘Prata’ and 4.45 mg 100 g–1 for ‘Williams.’ In addition, the P, Ca,
Mg, Mn, and Zn contents of ‘Prata’ were higher than of Williams, indicating a better nutritional
quality of the former.
Table 13.1
Nutritional Values for Banana and Plantain (per 100 g Raw Edible Portion)
Value per 100 g
Nutrient Units Banana Plantain
Water G 74.91 65.28
Energy Kcal 89 122
Energy kJ 371 510
Protein G 1.09 1.30
Total lipid (fat) G 0.33 0.37
Ash G 0.82 1.17
Carbohydrate, by difference G 22.84 31.89
Fiber, total dietary G 2.6 2.3
Sugars, total G 12.23 15.00
Sucrose G 2.39 –
Glucose (dextrose) G 4.98 –
Fructose G 4.85 –
Lactose G 0.00 –
Maltose G 0.01 –
Galactose G 0.00 –
Starch G 5.38 –
Minerals
Calcium, Ca Mg 5 3
Iron, Fe Mg 0.26 0.60
Magnesium, Mg Mg 27 37
Phosphorus, P Mg 22 34
Potassium, K Mg 358 499
Sodium, Na Mg 1 4
Zinc, Zn Mg 0.15 0.14
Copper, Cu Mg 0.078 0.081
Manganese, Mn Mg 0.270 –
Fluoride, F mcg 2.2 –
Selenium, Se mcg 1.0 1.5
Vitamins
Vitamin C, total ascorbic acid Mg 8.7 18.4
Thiamin Mg 0.031 0.052
Riboflavin Mg 0.073 0.054
Niacin Mg 0.665 0.686
Pantothenic acid Mg 0.334 0.260
Vitamin B-6 Mg 0.367 0.299
Folate, total mcg 20 22
Folic acid mcg 0 0
Folate, food mcg 20 22
Folate, DFE mcg_DFE 20 22
Choline, total Mg 9.8 13.5
Betaine Mg 0.1 –
Vitamin B-12 mcg 0.00 56
Vitamin B-12, added mcg 0.00 0
Vitamin A, RAE mcg_RAE 3 56
Retinol mcg 0 0
(continued)
Table 13.1 (Continued)
Value per 100 g
Carotene, beta mcg 26 457
Carotene, alpha mcg 25 438
Cryptoxanthin, beta mcg 0 0
Vitamin A IU 64 1127
Lycopene mcg 0 0
Lutein + zeaxanthin mcg 22 30
Vitamin E (alpha-tocopherol) Mg 0.10 0.14
Vitamin E, added Mg 0.00 0.00
Tocopherol, beta Mg 0.00 –
Tocopherol, gamma Mg 0.02 –
Tocopherol, delta Mg 0.01 –
Vitamin D (D2 + D3) mcg 0.0 0.0
Vitamin D IU 0 0
Vitamin K (phylloquinone) mcg 0.5 0.7
Lipids
Fatty acids, total saturated G 0.112 0.143
4:0 G 0.000 0.000
6:0 G 0.000 0.000
8:0 G 0.000 0.000
10:0 G 0.001 0.001
12:0 G 0.002 0.002
14:0 G 0.002 0.002
16:0 G 0.102 0.096
18:0 G 0.005 0.005
Fatty acids, total monounsaturated G 0.032 0.032
16:1 undifferentiated G 0.010 0.009
20:1 G 0.000 0.000
Fatty acids, total polyunsaturated G 0.073 0.069
18:4 G 0.000 0.000
20:5 n-3 (EPA) G 0.000 0.000
22:5 n-3 (DPA) G 0.000 0.000
22:6 n-3 (DHA) G 0.000 0.000
Cholesterol Mg 0 0
Phytosterols Mg 16 –
Amino Acids
Tryptophan G 0.009 0.015
Threonine G 0.028 0.034
Isoleucine G 0.028 0.036
Leucine G 0.068 0.059
Lysine G 0.050 0.060
(continued)
Value per 100 g
Methionine G 0.008 0.017
Cystine G 0.009 0.020
Phenylalanine G 0.049 0.044
Tyrosine G 0.009 0.032
Valine G 0.047 0.046
Arginine G 0.049 0.108
Histidine G 0.077 0.064
Alanine G 0.040 0.051
Aspartic acid G 0.124 0.108
Glutamic acid G 0.152 0.116
Glycine G 0.038 0.045
Proline G 0.028 0.050
Serine G 0.040 0.041
Other
Alcohol, ethyl G 0.0 0.0
Caffeine Mg 0 0
Theobromine Mg 0 0
Source: USDA National Nutrient Database for Standard Reference, Release 22, 2009, http://www.nal.usda.gov. With
permission.
Davey et al. (2007, 2009) reported that the genetic variation for carotenoids is strongly related to
differences in fruit maturity and that orange-colored varieties (plantains of the AAB group) usually
had higher carotenoid concentrations than triploid AAA fruits. The authors found variability for
carotenoid content within fruit, within hand, and within plant, as well as between plants. It was sug-
gested that sampling methods for correct carotenoid analyses should take two fruits in the central
region of the bunch.
Arora et al. (2008a, b) quantified the content of carotenoids, β-carotene, and antioxidant enzymes
in some banana genotypes in India. ‘Red Banana’ was found to have the highest carotenoid concen-
trations (241.91 ug 100 g–1 in the peel and 117.2 ug 100 g–1 in the flesh), followed by ‘Karpooravalli’
(1786.0 μg g−1 dry wt in peel and 544.85 μg g−1 dry wt in flesh). The catalase enzyme activity in
the peel of these varieties ranged from 5.66 to 35.57 nmol min−1 mg−1 protein. Both the peel and
flesh of these varieties are rich sources of bioactive compounds such as carotenoids, enzymes, and
antioxidant carbohydrates.
Davey et al. (2009) assessed the genetic variability for provitamin A carotenoids (pVACs), lutein,
and micronutrients (Fe and Zn) in 171 different banana cultivars, including diploid (genome AA
and AB), triploid (genome AAA, AAB, and ABB), and tetraploid accessions of unknown ploidy.
The results indicated a substantial variation in the pVAC concentration between genotypes, genomic
constitution, and ploidy, although the frequency of genotypes with high pVAC content was low.
The cultivars with highest pVAC values were ‘Bantol Red’ (unknown ploidy), ‘Pusit’ (unknown
ploidy), ‘Iholena Lele’ (AAB), ‘Henderneyargh’ (AAS), ‘Katimor’ (AAB), and ‘Chek Porng Mean’
(unknown ploidy), with values of 1500–2800 μg t-BCE 100 g−1 dry wt.
González-Montelongo et al. (2010) quantified the antioxidant activity in banana and observed
that the peel contains a large amount of dopamine and L-dopamine, two catecholamines with con-
siderable antioxidant activity.
Table 13.2
Carotenoid Content of Selected Cultivars of Ripe Micronesian Banana (µg
100 g–1 Edible Portion)
Sci. Name Local Name Source Color of Raw Flesh β-Carotenoids
Mt Uht en yap Pohnpei Orange 6360
Mt Uht en yap Pohnpei Orange 5860
Ms Usr wac Kosrae Orange 2082
Ms Uht ipali Pohnpei Orange 1181
Mt Uht karat Pohnpei Yellow-orange 918
Mt Uht karat Pohnpei Yellow-orange 578
Ms Usr wac essie Kosrae Yellow-orange 686
Ms Usr wac essie Kosrae Yellow-orange 309
Ms Usr taiwang Kosrae Yellow 662
Ms Usr taiwang Kosrae Yellow 571
Ms Usr kuria Kosrae Yellow 653
Ms Usr kuria Kosrae Yellow 218
Ms Usr macao Kosrae Yellow-orange 589
Ms Uht akatan Pohnpei Yellow 515
Ms Uht akatan Pohnpei Yellow 227
Ms Usr in yeir Kosrae Yellow 421
Ms Usr in yeir Kosrae Yellow 360
Ms Uht enkerinis Pohnpei Yellow 310
Ms Marechg Chuuk Yellow 189
Ms Usr fiji/uhten fijih Fiji White 56
Note: Ms: Musa spp.; Mt: Musa troglodytarum.

Source: Englberger, L., J. Schierle, G.C. Marks, and M.H. Fitzgerald, 2003, Micronesian banana, taro, and
other foods: Newly recognized sources of provitamin A and others carotenoids, J. Food Composition
Anal., 16, 3–19. With permission.
Prebreeding initiated at Embrapa to identify accessions with functional properties has identified 80
accessions that have been characterized for the presence of carotenoids, flavonoids, polyphenols, vita-
min C, and antioxidant activity. The active germplasm bank (AGB) of banana maintained by Embrapa
currently contains 276 accessions of different genomic groups (Figure 13.1). Amorim et al. (2007)
estimated the amount of functional foods of 11 genotypes. The mean content of total polyphenols was
46.68 mg 100 g–1, and ranged from 21.58 mg 100 g–1 in the triploid ‘Ambei’ to 120.97 mg 100 g–1 for
the diploid ‘Khai.’ For vitamin C, the mean content was 38.46 mg 100 g–1, ranging from 20.76 mg 100
g–1 for ‘Ambei’ to 54.20 mg 100 g–1 for diploid ‘Lidi.’ The mean total carotenoid content was 4.39 µg
g–1 and the highest mean was observed for diploid ‘Khai’ with 9.02 µg g–1 (Figure 13.2).
Cohen et al. (2009a, b) quantified the flavonoid and polyphenol concentrations in 26 diploid
banana accessions at Embrapa (Table 13.3). The mean total polyphenol content was 37.06 mg 100
g–1, ranging from 12.84 mg 100 g–1 for ‘Thu Mambee’ to 120.97 mg 100 g–1 for ‘Khai.’ The mean fla-
vonoid content was 2.58 mg 100 g–1, ranging from 1.29 mg 100 g–1 for ‘NBA 14’ to 4.68 mg 100 g–1 for
‘Pipit’ (Figure 13.3). Cohen et al. (2009a, b) also determined the antioxidant activity of three banana
accessions rich in vitamin C, carotenoids, and polyphenols (Table 13.4). The antioxidant activity of
tetraploid ‘Teparod’ (ABBB) was considerably higher (119.19 µM trolox g–1) than those of ‘Khai’
(6.32 µM trolox g–1) and ‘F3P4’ (6.44 µM trolox g–1). Mattos et al. (2010) characterized 26 banana
accessions of the Embrapa AGB for agronomic, physical, and physical-chemical characteristics
and by microsatellite molecular markers. Wide variability was observed for most agronomic traits,
particularly for fruit number and bunch weight. In the physical and physical-chemical assessments
2% 3%
6%
8% 26%
AA
BB
AAA
4%
AAB
29% ABB
22% AAAA
Others
AAAB
Figure 13.1 Frequency of banana genomic groups in the germplasm collection of Embrapa Cassava and
Tropical Fruits, Cruz das Almas, Bahia, Brazil, 2010.
PT (mg/100g) VitC (mg/100g) CT (ug/g)
140 10
120 8
100
80 6
60 4
40
20 2
0 0
n
a
n
an
ish
tu
1
t
di
i
ai
gi
bu
be
ka
ai
io
-0
Pi
Kh
N
Li
nd
u
nm
na
Ba
m
03
al
Tu
M
ve
A
ai
28
Ke
Kh
Ca
P.
Figure 13.2 Polyphenols (PT), vitamin C (Vit C), and carotenoids in 12 banana genotypes, including
diploids (AA) and triploids (AAA) of the germplasm collection of Embrapa Cassava and Tropical Fruits, Cruz
das Almas, Bahia, Brazil, 2010.
Table 13.3
Banana Genotypes of Germplasm Collection of the Embrapa Cassava and Tropical Fruits
and Their Respective Origins (Cruz das Almas, Bahia, Brazil, 2010)
Accessions Ploidy Origin Accessions Ploidy Origin
2803–01 AA Brazil Mambee Thu AA New Guinea
Berlin AA Indonesia Modok Gier AA New Guinea
F3P4 AA Ecuador NBA 14 AA New Guinea
Jambi AA Indonesia Ouro AA Brazil
Jaran AA Indonesia P. Kenmain AA New Guinea
Jari Buaya AA Honduras Pa Phatalung AA Thailand
Khai AA Thailand Pipit AA Indonesia
Khai Nai On AA Thailand P. Nangka AA Thailand
Khi Maeo AA Thailand Pitu AA Indonesia
Lidi AA Honduras SN2 AA New Guinea
M-48 AA Ecuador Sowmuk AA New Guinea
M-61 AA Ecuador Tongat AA Honduras
Malbut AA New Guinea Tuugia AA Hawaii
1.63
Tuugia 31.04
2.07
Tongat 38.27
1.34
Sowmuk 13.13
2.48
SN2 25.12
2.10
Pitu 40.61
3.15
P. Nangka 23.89
4.68
Pipit 61.48
2.54
Pa Phatalung 32.61
3.61
P.Kenmain 44.73
2.16
Ouro 33.32
1.29
NBA 14 31.17
1.73
Modok Gier 19.87
2.04
Mambee Thu 12.84
2.09
Malbut 26.90
4.56
M-61 37.38
4.07
M-48 41.18
2.44
Lidi 35.48
2.86
Khi Maeo 54.81
2.81
Khai Nai On 42.67
2.17
Khai 120.97
2.76
Jari Buaya 32.39
2.46
Jaran 28.75
1.82
Jambi 24.82
4.02
F3P4 43.41
1.30
Berlin 28.09
2.87
2803-01 38.51
Flavonols (mg/100g) Poliphenols (mg/100g)
Figure 13.3 Flavonols and polyphenols in 26 diploids of the Embrapa Cassava and Tropical Fruits and
their respective origins, Cruz das Almas, Bahia, Brazil, 2010.
Table 13.4
Functional Compounds in Banana Diploids of the Embrapa Cassava and Tropical Fruits
and Their Respective Origins (Cruz das Almas, Bahia, Brazil, 2010)
Genotype Ploidy Origin Vit. C CT PET FLAV AAT
Teparod ABBB Indonesia 76.82a 1.44b 257.80a 6.63a 119.19
Khai AA Thailand 34.51b 9.02a 120.97b 2.17b 6.32
F3P4 AA Equador 17.85c 2.52b 43.41c 4.02a 6.44
Note: Vit. C: vitamin C (mg 100 g–1); CT: carotenoids (µg g–1); PET: polyphenols (mg 100 g–1); FLAV: flavonols (mg 100
g–1); AAT: antioxidant activities (µM trolox g–1). Averages followed by the same letter in the columns belong to the
same group by the Tukey test at 5% probability.
of the fruits, accessions with high concentrations of carotenoids (diploid ‘Jaran’), polyphenols (trip-
loid ‘Caipira’ and tetraploid ‘Teparod’) and vitamin C (diploid ‘Tuu gia’ and an unknown triploid
AAA) were identified (Table 13.5). Thirteen microsatellite primers were used to assess diversity
detecting a mean of 7.23 alleles. Using the mean genetic divergence as cut-off point, three groups
were formed: G1 with the diploids ‘Jaran,’ ‘028003-01,’ and ‘M-48’; G2 containing the diploids
‘Malbut’ and ‘Ido 110’; and G3 with 21 tri-and tetraploid accessions, including ‘Tuu gia’ a diploid.
The triploids with the B genome, ‘Thap Maeo,’ ‘Walha,’ ‘Pacha Nadan,’ and ‘Champa Madras’
were grouped in G2 (Figure 13.4). Segregating populations for carotenoids and other functional
compounds are being developed at Embrapa with the aim of analyzing the genetic control of these
characteristics and identifying markers linked to the genes responsible for metabolic pathways.
13.3 Breeding Objectives for Quality Improvement

The goal of conventional banana breeding is to develop hybrids with high yield, short stature, short
cycle, and resistance to abiotic (water stress, cold, and so forth) and biotic factors (such as pests, yel-
low and black Sigatoka, Panama disease). The specific features of breeding for biofortification are
presented and discussed in this section.
13.3.1 Biofortification
Biofortification is a new approach that combines conventional breeding with modern tools of biotech-
nology with the aim of increasing the micronutrient concentrations in staple crops (Zimmermann
and Hurrell, 2002). Biofortification represents a great opportunity to improve the health status
of low-income populations in rural and urban areas of developing countries (Bouis et al., 2003).
According to the World Health Organization (WHO), the micronutrients considered critical to
human health are iron (Fe), zinc (Zn), and vitamin A. To minimize the deficiency of these elements,
biofortification needs to become a multidisciplinary endeavor with public health being one of the
targets of agricultural research.
Research aimed at biofortification has been carried out primarily with cassava (Manihot
esculenta), rice (Oryza sativa), maize (Zea mays), sweet potato (Ipomoea batatas), and com-
mon bean (Phaseolus vulgaris) (Welch, 2003). These crops were designated as “Phase I crops”
for the HarvestPlus Challenge Program coordinated by the Consultative Group for International
Agricultural Research (CGIAR), with the participation of more than 70 scientists from 46 research
institutions worldwide, including Embrapa. Other crops designated as “Phase II crops” included
banana (Musa spp.), barley (Hordeum vulgare), cowpea (Vigna unguiculata), lentil (Lens culinaris),
pearl millet (Pennisetum glaucum), pea (Cajanus cajan), potato (Solanum tuberosum), sorghum
(Sorghum bicolor), and yam (Dioscorea spp.).
Quality Improvement of Cultivated Musa
Table 13.5
Average of the 10 Agronomical and Physical-Chemical Characteristics Evaluated in 26 Banana Accessions (Cruz das Almas, Bahia, Brazil,
2010)
Accessions Ploidy ALP DPC NPC NFR PSC SIF CTN FLA PLF VIT
Jaran AA 2.83b 15.73d 8.00b 148.00a 3.20d 2.35a 8.23a 8.23a 28.76m 17.61k
2803–01 AA 1.76d 9.75f 5.00c 67.00b 3.30d 0.71e 3.53e 3.53e 38.51h 31.52f
Malbut AA 2.55c 15.00d 6.00c 64.00b 2.93d 2.12a 6.88b 6.88b 26.90n 20.42j
Idu-110 AA 2.33c 9.67f 7.00b 87.00b 3.30d 0.71e 2.86f 2.86f 40.96g 20.10j
Tuugia AA 2.53c 12.67e 6.00c 59.00b 2.09d 0.71e 1.41g 1.41g 31.051 51.10c
M-48 AA 2.75b 14.67d 6.00c 84.00b 4.57d 0.71e 3.52e 3.52e 41.18g 9.03n
Pipit AAA 2.21c 12.00e 5.00c 92.00b 3.80d 1.14d 2.95f 2.95f 61.48e 15.721
Caru Roxo AAA 3.33a 21.00b 5.00c 64.00b 7.00c 1.58c 5.91c 5.91c 33.32j 24.63i
Wasolay AAA 2.64c 13.33d 5.00c 51.00b 3.03d 0.88e 3.15e 3.15e 17.51p 14.721
Markatooa AAA 2.45c 17.50c 5.00c 63.00b 4.75d 1.73c 2.29f 2.29f 16.23q 14.411
Bakar AAA 3.33a 18.00c 6.00c 79.00b 9.80c 1.22d 3.99e 3.99e 79.14c 29.43g
AAA Desc. AAA 2.90b 16.50d 6.00c 57.00b 6.20c 1.55c 2.45f 2.45f 35.48i 54.20b
Nam AAA 2.23c 16.50d 6.00c 87.00b 4.33d 1.87b 2.77f 2.77f 31.86k 44.67d
Towoolle AAA 2.20c 14.50d 4.00c 42.00b 2.85d 1.40c 2.33f 2.33f 12.84s 10.87m
Caipira AAA 2.46c 17.17c 7.00b 132.00a 9.67c 0.71e 1.05g 1.05g 146.31b 11.48m
Thap Maeo AAB 3.43a 20.60b 10.00a 158.00a 15.03b 0.71e 3.78e 3.78e 15.71q 37.21e
Walha AAB 1.44d 14.67b 4.00c 30.00b 1.71d 1.90b 2.52f 2.52f 43.41f 17.85k
P. Nadan AAB 3.47a 18.00c 7.00b 88.00b 8.47c 1.58c 5.83c 5.83c 64.90d 26.85h
C. Madras ABB 3.49a 21.50b 7.00b 94.00b 12.90b 0.71e 3.34e 3.34e 27.41n 12.45m
Ambrosia AAAA 3.54a 24.42a 9.00a 154.00a 21.26a 0.71e 1.39g 1.39g 27.52n 11.60m
Calipso AAAA 3.15a 24.56a 8.00b 138.00a 18.62a 0.71e 1.40g 1.40g 27.12n 9.49n
Tropical AAAB 2.76b 20.40b 6.00c 92.00b 9.96c 0.71e 0.98g 0.98g 14.83r 14.681
Maravilha AAAB 2.66c 20.60b 5.00c 53.00b 6.74c 1.58c 1.89g 1.89g 16.03q 9.66n
Porp AAAB 2.85b 20.25b 5.00c 51.00b 5.95c 1.22d 2.34f 2.34f 14.77r 13.651
O. da Mata AAAB 3.22a 20.67b 5.00c 78.00b 6.75c 1.29c 4.70d 4.70d 24.56o 19.45j
Teparod ABBB 2.93b 18.00c 6.00c 37.00b 3.87d 0.71e 1.44g 1.44g 257.80a 76.83a
(continued)
261
262
Average of the 10 Agronomical and Physical-Chemical Characteristics Evaluated in 26 Banana Accessions (Cruz das Almas, Bahia, Brazil,
2010)
Accessions Ploidy ALP DPC NPC NFR PSC SIF CTN FLA PLF VIT
F (Trat.) 12.30* 22.05* 4.59* 5.97* 14.93* 22.80* 40.79* 40.79* 34877.64* 1161.36*
CV (%) 9.77 9.12 21.53 32.76 33.77 17.07 12.72 12.72 0.86 2.90
Mean 2.79 17.76 6.00 83.00 7.78 1.08 3.19 3.19 45.31 23.82
Note: PH: Plant height (cm); PD: pseudostem diameter (cm); NS: number of suckers; NH: number of hands; NF: number of fruits, BW: bunch weight (kg); YSF: yellow Sigatoka during
flowering; CTN: carotenoids (µg.g–1); FLA: flavonoids (mg 100 g–1); PLF: polyphenols (mg 100 g–1); VIT: vitamin C (mg 100 g–1). * significant at 5%; ns: not significant. Averages fol-
lowed by the same letter in the columns belong to the same group by the Scott and Knott (1974) test at 5% probability.
Banana Breeding: Progress and Challenges

Jaran
2803-01
M-48
Malbut
Idu-110
Tuugia
AAADesc
ChampaMadras
Calipso
Towoolle
ThapMaeo
Walha
Nam
PachaNadan
OurodaMata
Ambrosia
Maravilha
Porp
Pipit
Wasolay
Tropical
Markatooa
Bakar
CaruRoxo
Caipira
Teparod
0.20 0.33 0.45 0.57 0.70
Figure 13.4 Genetic diversity between six banana accessions from the Germplasm Bank at Embrapa
Cassava and Tropical Fruits integrating agronomical, physical, and physical-chemical data from fruits and
molecular data using the Gower (Gower, 1971) algorithm. Cruz das Almas, Bahia, Brazil, 2010.
The success of biofortification depends, in part, on target concentrations of micronutrients in

crops that should be set by nutritionists who understand the complexity associated with the expres-
sion of these elements and who can quantify their impact on human health. Information on the
retention time of nutrients after storage, appropriate processing methods and forms of food prepa-
ration, as well as the daily requirements need to be taken into consideration when breeding new
biofortified cultivars (Nestel et al., 2006).
Another aspect to be considered is the appearance of the food, in the form of grains, tubers,
or fruits. Foods that are rich in provitamin A tend to have an orange color (Krinsky, 1998;
Russell, 1998). Food color plays an important role for the consumer, who often chooses a par-
ticular food due to its appearance. In general, housewives prefer, for example, white wheat,
maize, and cassava flour as well as white or cream-colored banana fruit. On the other hand,
micronutrient-rich biofortified cultivars, except in the case of carotenoids, are visually not dif-
ferent from traditional varieties. This may be an obstacle, since it is not possible to identify the
biofortified cultivar visually. Before the adoption of a biofortified product, farmers and consum-
ers must be convinced of the benefits of this new variety. Therefore, studies that quantify the
benefits of consuming this modified food are required for marketing programs about the benefits
of biofortified foods.
13.3.2 Crop Improvement
In conventional breeding, the target characteristics are of direct benefits to farmers, and include
pest resistance, drought tolerance, high productivity, and so forth. Breeding for improved nutritional
status, however, may in many cases not benefit the growers directly. Consequently this breeding
objective has been largely ignored by breeders when selecting promising genotypes.
Breeding for improved nutritional qualities has primarily focused on the exploration and quan-
tification of natural variability for different micronutrients (Bouis et al., 2003). At the same time
(or during subsequent selections), agronomic evaluations should be performed. If there is suffi-
cient variability, breeders can exploit the additive effects, heterosis, and transgressive segregation to
increase micronutrient concentrations.
The genetic variability can be used in: (1) identification of parents for hybridization, (2) genetic
studies, (3) development of molecular markers, and (4) direct use of the genotypes. The next step
involves the development and evaluation of new biofortified cultivars/hybrids, based on genetic
studies and development of markers for assisted selection. The quantification of environmental
effects (genotype by environment [GxE] interaction) on the expression of micronutrient contents
must be performed under field conditions in different environments and years.
13.3.3 Genetics
Knowledge of the heritability and expected genetic gain (Gs = iσph2) where Gs is the genetic gain from
selection, i is a constant based on selection intensity, σp is the standard deviation of the phenotypic
variance, and h2 is the heritability is crucial for the choice of adequate breeding methods and appropri-
ate strategies to quantify the GxE interaction. Research results of different crops have indicated that
Fe and Zn concentrations are controlled by two to five genes, and that heritability is low (Philip and
Maloo, 1996; Maloo et al., 1998; Long et al., 2004; Cichy et al., 2005). Moreover, these two micronu-
trients are strongly correlated, which facilitates simultaneous improvement of these traits.
Provitamin A is apparently controlled by few genes (~2), and heritability is high (Egesel et al.,
2003; Gruneberg et al., 2005). For both minerals (Fe and Zn) and provitamin A, the additive gene
effects and general combining ability are high. Transgressive segregation is also found for provita-
min A.
13.3.4 GxE Interaction
The expression of a particular trait and the extent of GxE interaction in different environments
determine the method of selection, breeding, and evaluation, and can influence results in terms of
heritability, genetic variation, and even the genetic gain.
Research results indicate that the expression of provitamin A is relatively stable, independent of
the environment (Egesel et al., 2003; Menkir and Maziya-Dixon, 2004).
The expression of Zn and Fe is more variable due to environmental variations. In the case of Fe,
the environmental influence on the trait expression is particularly high.
Micronutrient-rich germplasm should be selected using more stringent criteria, due to the varia-
tion observed for these elements between plants of the same genotype. The use of controls, a greater
number of replications, and statistical designs that minimize the effects of soil variation (microenvi-
ronments) are therefore recommended. Large plots are more suitable to sample the genetic variation
and allow a comparison of results between different experiments. In addition, results of experiments
installed at different weather stations may also vary, and these temporal and spatial variations ham-
per the selection of germplasm for higher micronutrient contents.
13.3.5 Breeding for High Yield and Micronutrient Density

Breeding for characteristics that are not productivity related often results in a low progress rate for
yield traits. When resources are not limiting, breeding can substantially increase both σp and selec-
tion intensity i (Gs = iσph2) and provide genetic gain for productivity without affecting the selection
of micronutrient-rich genotypes.
Another strategy to increase the genetic gain over breeding generations is to breed and test
micronutrient-rich genotypes in controlled environments (greenhouse) to simulate the climate and
soil conditions for which these new genotypes will be recommended. By this practice the heritabil-
ity and correlation between selection and environmental variation can be increased and a greater
number of plants can be selected in little space and in less time. In addition, molecular markers can
be used for assisted selection. Promising results of this strategy have been reported in the literature
(Guzmán-Maldonado et al., 2003, Wong et al., 2004).
13.3.6 Strategies for Agronomic Superiority

The increase in productivity of a crop may be due to: (1) genetic gain in yield potential, (2) genetic
gain in tolerance or resistance to biotic and abiotic factors, (3) genetic gains through management
practices, and (4) synergistic effects of all the above factors. Indirect selection for tolerance to biotic
and abiotic factors has led to yield gains in banana. The development of hybrids resistant to black
Sigatoka and Panama disease in the breeding program of Embrapa Cassava and Tropical Fruits can
be cited as an example (Amorim et al., 2009b).
Pleiotropic effects associated with high micronutrient contents and yield performance have been
reported in the literature. Cakmak et al. (1990) identified a strong relationship between Zn content
in wheat seed and seedling vigor, resulting in greater uniformity of the plantation and, consequently,
higher productivity. Higher economic returns through tiered pricing for higher-quality fruits that
address specific market niches can motivate the adoption of biofortified banana cultivars by farm-
ers. Additionally, federal programs that encourage the consumption of biofortified banana cultivars
in public schools have led to an increased demand for banana.
13.4 Conclusions
Biofortification of banana, especially in terms of provitamin A, Fe, and Zn is possible and can
potentially minimize health problems caused by the deficiency of micronutrients in the world’s low-
income populations. The accessibility of the fruit and the facts that it is a staple food of millions of
people, especially in Africa, suggests that banana can play a fundamental role as a health-promoting
food. There is enough genetic variability for micronutrients, making genetic improvement and bio-
fortification of banana possible.
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14 Postharvest Processed
Products from Banana
Cherukatu Kalathil Narayana and Michael Pillay
Contents
14.1 Introduction........................................................................................................................... 269
14.2 Banana/Plantain Flour........................................................................................................... 270
14.3 Banana Chips/Crisps............................................................................................................. 272
14.4 Banana Puree/Pulp................................................................................................................ 274
14.5 Banana Powder...................................................................................................................... 274
14.6 Banana Figs and Dehydrated Banana.................................................................................... 274
14.7 Banana Flakes........................................................................................................................ 275
14.8 Banana Jam............................................................................................................................ 275
14.9 Banana Beverages.................................................................................................................. 276
14.9.1 Banana Juice............................................................................................................ 276
14.9.2 Banana Beer and Wine............................................................................................ 276
14.9.3 Banana Alcohol........................................................................................................ 277
14.9.4 Banana Vinegar........................................................................................................ 277
14.10 Banana Sauce...................................................................................................................... 277
14.11 Other Banana-Based Products............................................................................................ 278
14.11.1 Banana Fiber.......................................................................................................... 278
14.11.2 Banana Starch........................................................................................................ 278
14.12 Conclusion........................................................................................................................... 279
References....................................................................................................................................... 279
14.1 Introduction
Bananas and plantains are staple food crops grown in over 130 countries in the tropics and subtrop-
ics. They are considered to be one of the most important sources of energy in the diet of millions
of people in Africa, the Caribbean, Latin America, Asia, and the Pacific (Jones, 2000). Most of
the bananas produced in many countries such as India, Uganda, Brazil, and China are consumed
locally, with only a small percentage being exported (Pillay and Tripathi, 2007). Bananas and
plantains are consumed in various ways in different countries. Traditionally dessert bananas are
consumed raw in the ripe stage while plantains and cooking bananas are cooked as part of the
staple diet, or for processing into longer lasting products such as flour (Wainwright and Burdon,
1991; Dadzie, 1995). The need for processed banana products has not been high in many countries
since fresh bananas are readily available throughout the year (Sole, 1996, 2005). The development
of processed products from bananas has been slow compared with that of other crops. Processing
of bananas has been stimulated by the high volumes of bananas that are lost after harvest, espe-
cially those that are rejected for the export market in the main banana-growing countries of South
America. The climatic conditions of the tropics that favor banana production also play a major
role in increasing postharvest losses. The optimum storage temperature for green bananas and
269
plantains is 13–14°C, at which temperature ripe bananas can be stored for 1 to 2 weeks (Chia
and Huggins, 2003). Storage facilities are not available in many banana-producing countries to
guarantee a longer shelf life (Wills et al., 1989). The exact quantity of postharvest banana losses
in different countries is not known but in general losses of fruits and vegetables in the tropics are
reported to be about 40–50% (Mejia, 2003). One report estimated that 40% of bananas produced
in Brazil are lost after harvest (Agrianual, 2003). Postharvest banana losses vary from country to
country according to market chains and modes of consumption (Tchango Tchango et al., 1999).
The marketing chain in many developing countries is not well organized, and farmers incur losses
in many ways, including: the bulky nature of bananas makes transportation and handling costs
from the rural to urban areas very high and unaffordable for many small-scale farmers, small
bunches are considered below market size and are rejected, the shelf life of both green and ripe
banana is limited, and there is a lack of appropriate technology, as well as insufficient or scarce
access to information (Pekke et al., 2004; Mukhtar, 2009). The shelf life of green banana is short-
ened further by black Sigatoka (Mycosphaerella fijiensis), a widespread disease of the crop (Chillet
et al., 2008).
Dehydration is one of the oldest methods used to preserve agricultural products (Adams, 2004).
Drying prevents or reduces the growth of microorganisms responsible for the decay of food.
Advances in dehydration techniques and development of novel drying methods have in recent years
enabled the preparation of a wide range of dehydrated products and convenience foods from fruits
and vegetables meeting the quality, stability, and functional requirements coupled with economy
(Jayaraman and Das Gupta, 1992).
Dehydration is traditionally used in West Africa (Dadzie and Wainwright, 1995) and to a lim-
ited extent in East Africa (Aked and Kyamuhagire, 1996) to preserve unripe banana. In addition to
drying and preparation of banana flour, banana is now being processed into several products that
are being consumed and traded throughout the world. For example, banana “figs” produced by
sun drying of ripe bananas are one of the oldest processed food products. Other popular processed
banana products are dehydrated flakes and banana powder. Brazil, Honduras, and Mexico were
the main countries exporting dried, desiccated, or evaporated bananas (Sole, 1996). Banana puree
or mashed bananas are at present the banana product with the highest volume processed in the
world. The Philippines have emerged as one of the important countries exporting large quantities
of processed banana products with an exported volume of 16,964 tons of banana chips/crackers
valued at US$18.7 million in 1998 (hvcc.da.gov.ph/pdf/banana_phil_prodn_market.pdf, accessed
14 May 2010). The Philippines also exported 1,474 tons of banana catsup/ketchup valued at US$1.3
million in the same year. The biggest importers are the United States, capturing almost half of the
total export volume, followed by Canada, Saudi Arabia, and the United Arab Emirates. Over 2.2
tons of banana flour, meal, and powder valued at US$14,771 were exported solely to Japan in 1998
(Narayana and Sathiamoorthy, 2002). Banana is favorable for industrial processing due to its rich
content of soluble solids, minerals, and low acidity (Carvalho et al., 2009a, b). The major processed
banana products are discussed fully in Sole (2005). Two products from banana that appear to be
ubiquitous in many banana-producing countries are banana flour and banana chips.
14.2 Banana/Plantain Flour

One strategy to increase the utilization of banana and plantain includes the production of flour from
ripe and unripe fruit and to incorporate the flour into various innovative products such as slowly
digestible cookies (Aparicio-Saguilan et al., 2007), high-fiber bread (Juarez-Garcia et al., 2006),
and edible films (Sothornvit and Pitak, 2007). Many methods to prepare flour from unripe banana
and plantain have been reported (Oyesile, 1987; Ukhum and Ukpebor 1991; Rodriguez-Ambriz
et al., 2008). The flour from unripe banana possesses thickening and cooking properties that are
almost identical to that of starch (Suntharalingam and Ravidran, 1993). As a technology to preserve
banana for later use, the production of banana flour is widespread in Africa and in countries such as
Postharvest Processed Products from Banana 271
Bolivia. Large-scale production of banana flour is carried out by peeling and slicing green fruits and
exposure to sulfur dioxide gas or dipping in sulfurous acid, and then drying to a moisture content
of 8% in a countercurrent tunnel dryer for 7–8 hours with an inlet temperature of 75°C and outlet
temperature of 45°C, and finally milling (Sole, 2005). The full three-quarter-maturity stage of fruits
contains the correct proportions of starch and sugar that produces good quality flour. If immature
fruits are used, the resultant flour tastes bitter and astringent due to a high tannin content. It was
shown that the different dehydration methods used to produce flour significantly affect its proximate
composition and physical characteristics (Pacheco-Delahaye et al., 2008).
In the traditional method, the banana/plantain is peeled, cut into small pieces, and sun dried for
a few days. Nowadays, new miniature solar dryers that are more hygienic have been developed in
many low-income countries. The dried pulp is then ground into a powder in a mortar and pestle.
Flour produced in this manner is usually brown due to the effect of enzymes such as polyphenol oxi-
dase and peroxidase or other nonenzymatic reactions (Cano et al., 1997). It is known that bananas
undergo rapid browning as a result of tissue disruption and exposure to oxygen during peeling
and slicing operations (Cano et al., 1997). Enzymatic browning of banana can cause undesirable
changes in quality during handling, processing, and storing of the fruit. Various methods have
been used to try and reduce the browning of processed banana, including: the addition of sodium
bisulphate (Tonaki et al., 1973; Garcia et al., 1985), blanching to inactivate the enzymes (Ngalani,
1989; Cano et al., 1990; Giami, 1991), and mild heat treatment with addition of sodium bisulphate,
citric acid, and potassium sorbate. Cano et al. (1997) showed that microwave and steam blanch-
ing significantly reduced the enzymes that cause browning in banana, with blanching being more
effective. However, blanching had a significant negative effect on the proximate analysis, mineral
content, and pasting properties of whole flour prepared from plantain and banana hybrids (Adeniji
and Tenkouano, 2008). Steaming has been used to reduce enzymatic discoloration before process-
ing (Hanson, 1976; Suntharalingam and Ravidran, 1993). Steaming made peeling easier, reduced
discoloration, improved rehydration, and reduced cooking loss in the East African Highland banana
(Muyonga, 2000). Color changes in foods during the drying process are affected by a number of
variables such as temperature, moisture content, degree of ripeness, wet-bulb temperature, and air
flow (Baini and Langrish, 2009). Bananas were dried in a kiln at dry-bulb temperature of 50–100°C.
The bananas were dried continuously and intermittently for 72 h. For continuous drying it was found
that browning increased and reached equilibrium. The rate of browning was found to decrease with
drying time and moisture content (Baini and Langrish, 2009). The rate of browning for overripe
and ripe bananas was higher than those of unripe banana, suggesting that the sugar content may
affect browning.
Peeling of banana for flour production is difficult and time consuming. Haslinda et al. (2009)
prepared flour from both peeled and unpeeled banana. They observed that flour prepared from
unpeeled banana (‘Awak,’ AAB) had enhanced nutrition values with higher concentrations of miner-
als, dietary fiber, and total phenolics. This is supported by the work of Izonfuo and Omuaru (1988),
who reported that plantain peel is richer in minerals such as potassium, calcium, magnesium, phos-
phorus, copper, and iron when compared with the pulp and that the concentrations of potassium,
calcium and iron increase as the fruit ripens. In addition, flour fortified with peel showed higher
antioxidant activity and had better pasting properties than flour without peel. Choo (2007) showed
that noodles containing 30% banana (‘Awak’) flour and oat β-glucan showed very high antioxidant
properties and very low glycemic index (GI) and high rate of carbohydrate digestibility. These
noodles compared well with control samples in a sensory test. Banana peel and pulp flour was also
found to be useful for controlling starch hydrolysis when used in the preparation of yellow noodles
(Ramli et al., 2009). Acid treatment of unripe banana flour produced a fiber-rich product that may
be important for the development of food and medical products (Aguirez-Cruz et al., 2008).
Banana flour has usually been obtained from unripe banana and plantain. Flour from ripe fruits
has the potential to offer new products for industrial and domestic uses (Abbas et al., 2009). Flour
from ripe banana containing a small quantity of sugar can be incorporated into food products
requiring solubility, sweetness, and high energy content.
Studies have shown that ripe banana flours from different cultivars (‘Cavendish,’ AAA, and Musa
acuminata ‘Pisang Berangan,’ AAA, can be differentiated from each other (Abbas et al., 2009).
Banana flour is used as an adjuvant in several food preparations and baby food formulations.
Banana is an important weaning food in Uganda (Bukusuba et al., 2008). Soyamusa, a baby food
made from plantain flour (60%), full-fat soybean flour (32%), sucrose (8%), and fortified with 0.15%
of multivitamins and 0.85% calcium carbonate, has been made and used in Nigeria (Ogazi et al.,
1991; Ogazi, 1996). Banana flour can be blended with other cereal flours for making chapattis
(Indian flat bread), biscuits, instant porridge, bread, papadams, and extruded foods. Pekke et al.
(2004) reported that the inclusion of flours from millet, cassava, and soybean improved the accept-
ability of banana flour products. The acceptability of porridge made from a combination of banana-
millet-soybean flour in the ratio 7:2:1 was rated highly in Uganda. Bukusuba et al. (2008) showed
that pregelatinization and extrusion cooking can significantly raise the energy content of banana
with significant impact on its viscosity and reduction of its bulkiness. This has implications for
weaning foods in Uganda since high rates of malnutrition were reported in banana-consuming areas
in the country. In western Nigeria, plantain flour is mixed with an appropriate quantity of boiling
water to prepare thick dough that is eaten with vegetable soup (Ogazi, 1998).
Banana flour has been used as an additive in the preparation of bread and whole maize meal. It
was shown that 15% plantain flour substitution could be adopted in the bread-making processes,
without affecting the quality of the bread that is made exclusively with wheat flour (Olaoye et al.,
2006). Similarly it was found that different formulations of banana flour, especially from Cavendish,
when added to whole-maize meal imparted improvements in formulated products in terms of mouth
feel and binding properties with a significant difference in the color of the base product (Daramola
and Osanyinlusi, 2006).
In the 1980s dietary fiber (DF) was identified as an important component of a healthy diet. The
interest in foods rich in DF has increased in recent decades and there is a large market for fiber-rich
products and ingredients (Juarez-Garcia et al., 2006). Unripe banana represents a good source of
indigestible carbohydrate, due to the starch content of the pulp and high cellulose, hemicelluloses,
and lignin levels. Bread made with banana flour had significantly higher quantities of resistant
starch, dietary fiber, and indigestible fractions than control bread made primarily from wheat flour.
Bakery products with banana flour have a low glycemic index and could be used as a dietary aid for
people with low caloric requirements (Juarez-Garcia et al., 2006).
Banana flour is produced for export in Ecuador, Colombia, Canada, and Switzerland, gener-
ally in small volumes. Confoco S.A. Trobana in Ecuador produces green banana flour for export
(Sole, 1996). Commercial banana flour production is not yet common in Asia (Abbas et al., 2009),
although this industry is gaining popularity in major banana-growing countries in Africa (Emaga
et al., 2008).
14.3 Banana Chips/Crisps

The production of banana and plantain chips is a widespread activity in many banana-growing
countries. Chips are the most popular snack in many fast food outlets in Bangladesh (Molla et al.,
2009) and are the most popular plantain product in Nigeria (Onyejegbu and Olorunda, 1995). Chips
are an important ingredient in breakfast cereals. The chips are either fried in oil or dried to be pro-
cessed into flour. Chips can also be coated with sweeteners and then fried. Chips can be preserved
for a long time (months, possibly years) with adequate packaging and storage facilities. Several
studies have been conducted to determine the best Musa cultivars for chip making (Adeniji and
Tenkouano, 2007; Molla et al., 2009).
In India, the most popular processed banana product is ‘Nendran’ chips, which has grown from a
household art to a well-established cottage industry in the states of Kerala and Tamil Nadu. In India,
banana chips or crisps are made primarily from the cultivar ‘Nendran’ (AAB) while ‘Saba’ (ABB) is
mainly used in the Philippines. The Philippines also export large quantities of banana chips to the
United States and Canada. In Africa, plantains are used for chip production in West Africa while
other varieties are used in East Africa. Banana chips/crisps are made by deep-frying raw banana
slices of 1.75 to 2.0 mm thickness in a suitable cooking medium and salting them. Coconut oil is the
most preferred medium in India, while cottonseed oil or corn oil is used in other countries. Many
variations of banana chips exist. Chips are coated with cane sugar and refried to produce sugar-
coated banana chips. Another variation is that chips are coated with a mixture of banana puree
and sugar before frying the second time. The stage of maturity and use of antioxidants play a very
important role in the quality and storability of banana chips.
Plantains are commonly used to prepare chips in Nigeria and Cameroon (Onyejegbu and
Olorunda, 1995). Chips made from plantains absorb less frying oil than those produced from cook-
ing and dessert banana (Lemaire et al., 1997). Hydrocolloids such as alginate, carboxyl methyl cel-
lulose, and pectin were shown to decrease the amount of oil absorption in banana chips (Singthong
and Thongkaew, 2009). Plantains and certain cooking banana also have the advantage of not turn-
ing brown during chip preparation and antioxidizing treatments are not necessary (Lemaire et al.,
1997). Badia (1985) reported that the degree of ripeness of plantain is important in chip preparation
and that browning occurs if the sugar content is higher than 1% in the fruits. Varieties used for chip
preparation have to be selected carefully since genetic differences exist among cultivars in pigment
composition and/or pulp browning potential (Tourjee et al., 1998). This was also shown for plantain
and plantain-derived hybrids in Nigeria (Adeniji and Tenkouano, 2007). Studies on the effects of
systems for contact of banana slices (solid phase) and palm oil during deep frying of banana chips
showed that shaking homogenizes the temperature of the oil and enhances mass energy transfer.
Exchange of matter and heat took place during the first 3 minutes of cooking. Diaz et al. (1996)
observed that chip quality can be improved if the frying temperature is maintained at 167°C. At
this temperature, water loss was less than 5 g/100 g of raw material and gain in lipids was less than
16 g/100 g. The optimum cooking time was 200 s with a minimum ratio of energy consumption to
evaporation of water.
Suvittawat and Babpraserth (1996) tried to identify the best cultivar for banana chip production
in Thailand using nine banana cultivars with different genome compositions. The most preferred
cultivar was the tetraploid ‘Khai’ (unknown genomes). At room temperature (25–30°C) and super-
market temperature (20–25°C) the chips could be stored for up to 1 or 3 months, respectively. Shere
et al. (1993) studied the effect of maturity stage on the quality of fried banana chips by using the
cultivar ‘Basrai,’ AAA, Cavendish subgroup. A maturity stage of 100 days after flowering was not
ideal for chip preparation because of the high sugar content that caramelized at high temperatures.
Bananas harvested after 85 days from flowering produced better chips.
The shelf life of banana and plantain chips depends to a large extent on the type of packaging.
Visual appearance, especially color, is the major quality criterion for determining the commercial
quality and cost of banana chips (Anand et al., 1982). Chips must be packed in moisture-proof bags
to prevent loss of crispness (Stover and Simmonds, 1987). Chips packed in polyethylene bags become
rancid with time, due to oxidation and changes in color and taste of the product (Ogazi, 1996).
Addition of antioxidants like propylene glycol, propyl gallate, and citric acid has been recommended
for reducing the rancidity (Badia, 1985; Ogazi, 1996; Narayana and Mustaffa, 2000). The inclusion
of different absorbents inside the polyethylene bag can prolong the shelf life of banana chips by 3–5
days (Goswami and Barua, 1996). These researchers found that inclusion of the ethylene absorbent
(KMnO4) together with the carbon dioxide absorber [Ca (OH)2] showed the best results not only in
terms of shelf life but in physicochemical properties and incidence of fungal rots. The viability of
such a technical method may not be useful to small-scale producers of banana chips.
In Nigeria, cellophane has been recommended as the packing material for plantain chips (Ogazi,
1996). A number of packaging techniques have been outlined by González-Aguilar et al. (2010).
Some of these techniques may be suitable for banana products and need further investigation.
Banana chips could play an important role in intervention programs to combat micronutrient
deficiencies by virtue of their iron, zinc, and total carotenoid content (Adeniji and Tenkouano,
2007). Chips are a ready-to-eat food and are a favorite among children and adults alike. It was
already shown that chips made from some banana varieties could contribute substantially to the
recommended daily allowance (RDA) of retinol, iron, and zinc of both children and adults (Adeniji
and Tenkouano, 2007). Unpublished data (Fungo and Pillay) showed that banana accessions from
Papua New Guinea have very high levels of ß-carotene with values ranging from 205 µg/100 g to
2594 µg/100 g. It would be interesting to produce chips from such varieties and assess the effect of
processing on their micronutrients.
14.4 Banana Puree/Pulp

Banana puree is obtained by pulping peeled, ripe bananas and then preserving the pulp by one of
three methods: aseptic canning, acidification followed by normal canning, or quick-freezing (www.
vuatkerala.org, accessed 22 April 2010).
Most of the world’s banana puree is processed by aseptic canning. Peeled, ripe fruits are conveyed
to a pump that forces them through a plate with 8 mm holes, then on to a homogenizer, followed by a
centrifugal deaerator and into a receiving tank with 100 kPa vacuum, where removal of air helps in
preventing discoloration. The sterilized puree is then packed aseptically into steam-sterilized cans,
which are closed in a steam atmosphere (Sole, 1996, 2005). Pretreatments of plantain by blanching
in hot water, steam, microwave treatment, or calcium chloride modifies the texture and the orga-
noleptic characters. Calcium chloride and microwave pretreatments hardened frozen banana pulp.
Microwave pretreatment seemed to be the most suitable for frozen banana (Giami, 1991). Garcia et
al. (1987) conducted a study to reduce the time required to lower the pH of banana puree by lactic
acid fermentation. The best production conditions were found to be 37°C and 10% (w/w) of inocu-
lum, under which conditions the pH decreased from 4.8 to 4.3 in approximately 8 hours.
14.5 Banana Powder

Banana powder is made from fully ripe banana pulp. Passage through a colloidal mill converts the
pulp into a fine paste. The color of the final product is improved by adding approximately 1–2%
potassium metabisulfite solution. In addition, the pulp is spray or drum dried and the solids are
recovered. In the latter method, the moisture content of the pulp is reduced to 8–12% and then it is
further decreased to 2% by drying at 60°C in a tunnel or cabinet dryer. The dry powder is highly
hygroscopic and is packed in bag-in-box packages. The bags are made from laminated material
with moisture barrier layers. This product has a high market value as it is used in the confection-
ary industry, making of ice creams, and weaning and baby foods. The edible pulp of the cultivar
‘Bhimkol,’ ABB, was shown to be a nutritious baby food (Barthakur and Arnold, 1990). The fruit
was found to be very rich in macro- and micronutrients, and 100% of a 6-month-old infant’s RDA
for K, Mn, Zn, and Se could be satisfied with 100 g of fresh pulp. It appears that this cultivar may
be useful for the production of banana powder.
14.6 Banana Figs and Dehydrated Banana

Fruit softening is characteristic of fruit ripening, and mature preclimacteric bananas lose their firm-
ness rapidly, even after 4 days of storage (Yang et al., 2008).
The production of banana figs represents another way in which the shelf life of ripe banana can
be prolonged. Banana figs are dried or dehydrated banana with a sticky fig-like consistency and very
sweet taste. Banana figs were initially produced in the tropics by sun drying, but hot air circulation
in tunnel or cabinet dryers is now used. The product is made from fully ripe fruits. Varieties with
a high total soluble solids (TSS) content and a sugar content of about 19.5% are highly suitable for
making figs. Dried banana is an important food product in Ethiopia (Osman and Asrat, 1985). The
dried bananas are produced by treating batches of sliced banana for 5 minutes in a boiling 50%
sugar solution and then drying them in the sun in glass-covered black boxes.
In Brazil, ‘Nanica,’ AAA, Cavendish subgroup, bananas are dried to produce banana-passa.
Banana-passa is obtained by the natural or artificial drying of ripe bananas until they reach a
moisture content of 20–25% wet weight (Cano-Chauca et al., 2004). Banana-passa is a commercial
product of Brazil and is exported in limited quantities to the United States, Germany, France, and
the United Kingdom. The commercialized product is often rejected due to microbiological contami-
nation and physical degradation of the product. Therefore various drying procedures were studied
to produce the best banana-passa (Nogueira and Park, 1992; Cano-Chauca et al., 2004; Leite et
al., 2007). The finding of Nogueira and Park (1992) suggested a temperature of 60°C or 70°C and
airflow of 1.5 m/s. The best drying time to reach a final moisture content of 23–25% depends on
temperature, drying speed, and relative humidity (Cano-Chauca et al., 2004). Large-scale produc-
tion of banana-passa is likely to become a profitable enterprise since it requires low initial invest-
ment. The export market for this product is largely unexplored.
In India, osmotic dehydration of ‘Karpuravalli,’ ABB, Pisang Awak, banana in 60–70% sugar
syrup for 12 hours followed by drying in a hot air oven at 50°C produced high-quality figs with
excellent color and taste (Narayana and Sathiamoorthy, 2002). Wrapping the figs in cellophane
paper and packaging in polymeric containers gave an extended storage life of 6 months under ambi-
ent conditions.
In a study in Africa, undertaken to develop an organoleptically acceptable and shelf-stable dry
plantain product using an osmotic drying technique, Ukkuru and George (1996) showed that an
ideal plantain product can be obtained when plantains with sugar concentration of 70°Br were
immersed in an osmotic solution for 60 min and heated to a temperature of 60°C.
Moisture content is an important quality attribute that directly influences the storability of
fruits and vegetables (Romano et al., 2008). It was found that ripe banana halves dehydrated
to a moisture content of about 30%, followed by bleaching and/or sulfating, are quite astrin-
gent (Ramirez-Martinez et al., 1977). Therefore accurate methods for determining the mois-
ture content of dried banana are essential. In this regard, Romano et al. (2008) developed an
approach for monitoring the moisture content of banana with laser-light backscattering imag-
ing. Significant relationships were obtained between changes in backscattering area and mois-
ture content especially at temperatures of approximately 53°C. This technology will be useful
in rapidly evaluating the moisture content of dried banana and preventing spoilage of dried
banana products.
Ecuador is one of the main producers of banana figs, but substantial amounts are now produced
in India, Brazil, Philippines, Costa Rica, and other countries (Sole, 2005). The main importers of
the product are the European countries, especially France and Germany.
14.7 Banana Flakes

Banana flakes are made by drying puree in large chrome-plated drum dryers. The puree, extracted
in a similar way to that of banana puree, is fed directly to the dryers. The water evaporates as the
drum turns, forming a film of dried material on the drum surface. Carefully adjusted knives remove
the dry film as a continuous sheet, which passes to an air-conditioned room where it is broken into
flakes. It is then sifted and packaged in bag-in-box containers (Sole, 1996). Confoco S.A. is the
world’s largest supplier of banana flakes, produced in their factories in Ecuador (Sole, 2005).
14.8 Banana Jam

Several varieties of banana are suitable for preparing jam. Banana jam is prepared by cooking ripe
fruit pulp with an equal quantity of sugar and with pectin and acid in right proportions until the
product sets well. This product has a good commercial value and is used in several mixed-fruit jams
(Narayana and Mustaffa, 2000).
14.9 Banana Beverages

14.9.1 Banana Juice
Banana has a widely appreciated flavor and aroma and will be able to compete favorably in the
fruit juice market either as banana juices or mixed with other juices (Viquez et al., 1981). Generally
juices are extracted from fruits by crushing or grinding. Banana pulp is too pectinaceous to yield
juice by simple pressing or centrifugation (Viquez et al., 1981). This method produces a sticky,
lumpy mass with no juice (Surendranathan et al., 2003). Therefore, various methods have been uti-
lized to extract banana juices. Earlier methods for juice extraction involved the use of calcium oxide
and sulfuric acid. Through pectolytic enzyme clarification a clear transparent juice to an extent of
75–80% of pulp weight could be obtained with a TSS content of 23–26°Br depending on the cultivar
(Narayana and Sathiamoorthy, 2002). The clarified juice could be dispensed as a “ready-to-serve”
(RTS) beverage by adjusting the TSS to 18–20°Br and acidity to 0.3% with sugar, citric acid, and
water. After chilling it forms an excellent thirst-quenching and nutritious drink (Narayana et al.,
2003). The RTS can also be carbonated using carbon dioxide gas at an appropriate level. Sole (1989)
presents, in his patent, a method to recover concentrated clarified banana juice that will probably
be used more as a “filler” ingredient in several other fruit juice blends rather than being served as
a sole beverage. In fact, the first such product launched by Chiquita Brands International Inc. was
an orange-banana juice. However, it has grown to become an “anytime drink,” slightly eroding the
market for carbonated beverages (Sole, 1996, 2005).
Koffi et al. (1991) reported that two different combinations of pectinase, cellulase, and hemicel-
lulase were more effective in reducing viscosity and improving the filterability of both green and
ripe banana purees when producing banana juice. The addition of potassium metabisulfate at 100
mg/L was more effective in producing a light-colored juice with a stable color. By adding cellulase
(0.06%) and pectinase (0.05%) at 45°C for 2 hours, a 73% clear juice yield can be obtained. On the
other hand, amylase was not effective in obtaining juice from banana (Pheantaveerat and Anprung,
1993; Sim and Bates, 1994).
In East Africa, banana juice is extracted with traditional technology. The banana pulp is mixed
with grasses, especially Imperata cylindrica, and worked manually until the juice appears. The
extracted juice is a clear, creamy liquid with a strong banana taste and aroma with a sugar content
of 18–28% soluble solids (Aked and Kyamuhagive, 1996). This method is laborious and time con-
suming, and mechanical methods will be required if banana juice has to be marketed as a profitable
fresh drink. However, the banana juice is often fermented to prepare different types of “banana
beer,” which is widely consumed in many countries in Africa (Stover and Simmonds, 1987).
14.9.2 Banana Beer and Wine

Fermenting banana juice for making banana beer and wine is an age-old traditional practice. In
Central and East Africa, the juice from the ripe “beer banana” fruits are consumed either fresh or
fermented to a low alcohol content having a short shelf-life (Sharrock, 1996). These ‘Mbide’ variet-
ies (AAA) are used principally for making banana beer, which is a typical feature of the banana-
based highland cropping systems in Burundi, Kenya, Rwanda, Tanzania, Uganda, and Zaire (now
Democratic Republic of Congo). Making banana beer in East Africa consists of four main stages:
(1) forced maturation of green banana, (2) pressing and mixing, (3) recovery of filtered juice and
fermentation, and (4) racking, filtering, and cooling (Davies, 1995).
Various researchers have reported on methods for producing banana wine from different banana
varieties in different continents (Adeniji, 1995; Narayana et al., 2002a; Akubor et al., 2003; Carreno
and Aristizabal, 2003). The method of Narayana et al. (2002a) involved mashing ripe pulp with about
10% water and after adjusting the TSS to 26°Br, the pulp was sterilized and inoculated with wine
yeast, Saccharomyces cerevisiae var. ellipsoideus, and incubated at 24–26°C for 7 days with inter-
mittent aeration. After a week, the fermented juice is filtered and kept under anaerobic conditions
with a water seal for secondary fermentation. After a further 2 weeks the secondary fermentation is
terminated. The wine is racked or centrifuged, bottled, and pasteurized at 50°C for 20 minutes. The
pasteurized wine is aged, giving the wine a characteristic flavor and aroma. Akingbala et al. (1994)
reported that banana and mango juice can be fermented to produce wine using Saccharomyces cerevi-
siae. Pasteurization of the banana-based alcoholic beverages increases the ester and alcohol content.
Banana has been used as an adjunct in the production of regular beer (Carvalho et al., 2009b).
The wort of regular beer was adjusted with different concentrations of banana juice. The results
showed that ethanol production increased by approximately 0.4 g/g ethanol yield, suggesting that
banana could be used in brewing methods for the development of new products. This provides a new
opportunity for banana growers and a different outlet for excess banana.
14.9.3 Banana Alcohol

In Uganda and Sudan, banana beer is distilled to produce banana alcohol, or “Waragi,” that is sold
commercially. Homemade banana alcohol, although considered illegal, is widely produced in many
countries in Africa and many small-scale rural farmers depend on this for their income. Commercial
or medicinal alcohol from banana has also been produced in several countries. Iizuka et al. (1985)
reported a 100% transformation with glucoamylase and yeast at 70°C; pectic enzymes were added
to reduce the viscosity of the crushed material. Large quantities of Cavendish (AAA) bananas in the
Philippines are rejected for export and are used for preparation of alcohol. Juice extracted from the
ripe fruits was found to contain approximately 126 g /L total sugars. A technique for preparation
of the inoculum, immobilization of yeast, and fermentation of juice to alcohol has been described
by Del Rosario and Pamatong (1985). Tewari et al. (1986) produced alcohol from banana peels by
subjecting the material to saccharification treatments using either sulfuric acid, steam under pres-
sure, or cellulase. The fermentation was carried out with Saccharomyces cerevisiae var. ellipsoi-
deus. The alcohol yield was approximately 150 ml/kg of peel. Commercial alcohol was produced
at the National Research Centre for Banana, Tiruchirapalli, from banana peel of ripe banana fruits
through saccharification followed by fermentation and distillation. The yield of alcohol was around
200 ml/kg peel (Sree Prabha et al., 2002).
14.9.4 Banana Vinegar

Vinegar is produced in Panama from banana wastes by subjecting it to alcoholic and acetic fermen-
tation (Ortega and Blanco, 1986). A simple technique for processing rejects or overripe ‘Robusta’
(AAA), Cavendish subgroup, banana into vinegar was developed by Suresh and Ethiraj (1991).
The pulp was crushed with an equal volume of water, and pectinase was added at 40 mg/kg. This
method produced a clear juice that was seeded with a strain of Saccharomyces cerevisiae var.
ellipsoideus to initiate fermentation. A good quality alcoholic base for producing vinegar contain-
ing 5–6% acetic acid was obtained. Simmonds (1966) reported that vinegar has been prepared by
fermenting a mash of banana pulp and peel.
14.10 Banana Sauce

In the Philippines, bananas are used to produce ketchup on a commercial scale. Banana sauce resem-
bles tomato ketchup in appearance but not in flavor. A recipe for the preparation of banana sauce
was developed at the National Research Centre for Banana, Tiruchirapalli, where the ‘Monthan’
(ABB) cultivar was blanched, peeled, mashed, and cooked with spices and vinegar to yield banana
sauce with favorable acceptability. The product is stable for almost a year (Narayana et al., 2002b).
14.11 Other Banana-Based Products

The technology for several other value-added products based on banana flour such as biscuits, papads,
health drink, baby food, sweet chutney, and cakes have been developed at the National Research
Center for Banana, Tiruchirapalli, India (Narayana and Sathiamoorthy, 2002; Narayana et al., 2002c;
Ramajayam et al., 2003). Dried, ground, and roasted green bananas are also used as a coffee substi-
tute in some places. Starch can be extracted from the pseudostems of banana and plantain. Banana
starch has been used for producing glue used in manufacturing of cartons for export of fresh banana
(Sharrock, 1996). Maini and Sethi (2000) reported that 5% edible starch can be obtained from 20–25
tons of banana pseudostem from 1,000 plants. Dried banana and banana/cassava pancakes called
Kabalagala are consumed in Uganda (Aked and Kyamuhagive, 1996). Ogazi (1996) reported that in
Nigeria unripe plantain peel and pulp were pounded to obtain a meal and eaten with any of the popular
soups. Various delicious banana products (savory and sweet dishes) were prepared by Manimegalai et
al. (1999), the Annual Report of the National Research Centre (2002), and the Annual Report of NATP
(2002) using raw and ripe plantain, pseudostems, and flowers from banana.
Green pseudostems, corm, and sun-dried ripe banana peels provide feed for cattle and sheep.
Ripe banana rejects, supplemented with protein, vitamins, and minerals are also used for fatten-
ing hogs. Good silage can be made from equal parts of chopped green bananas and grass or from
chopped green banana mixed with 1.5% molasses. In the Philippines meal made from dehydrated
rejected bananas could form up to 14% of total broiler rations without any adverse effects. Peeled
leaf sheaths are used fresh or after drying as packaging material for flowers, betel leaves, fruits, and
other items. The leaf sheaths are stripped into shreds, dried, and used for tying packages and mak-
ing garlands in Southern India.
14.11.1 Banana Fiber

Bananas and plantains are frequently used as a source of fiber. Banana fiber is extracted from
dried petioles and leaf sheaths that make up the pseudostem. Dependent on the cultivar and
method of extraction, a fresh banana plant yields about 0.6–1.0% fiber (Uma et al., 2002).
Banana fiber is gaining popularity because it can be used in the pulp and paper industry for
manufacturing of certain papers, particularly where great strength is required. The paper is
used for, among other things, making tea bags and bank notes. The fiber has numerous other
uses, including textile manufacture; making rope, string, and thread; and for production of
handicrafts such as baskets, toys, table mats, wall hangings, and lamp shades (Uma et al.,
2002). Mechanical devices for efficient extraction of fiber from banana pseudostem have been
developed by the Central Institute for Research on Cotton Technology, Mumbai (Nachene and
Uma, 2003). Although fiber can be extracted from all cultivars, variability for fiber yield exists
according to genomic groups.
The various uses of banana fiber are explained by Uma et al. (2005) and can be summarized
as follows: (1) as a natural absorbent for spillages including oil; (2) as a base material for bioreme-
diation and recycling; (3) as a natural water purifier; (4) as a base material for the pulp and paper
industry; (5) as a medium for mushroom production; (6) for handicrafts and textiles; (7) for printing
currency; and (8) as a cottage industry for handicrafts and source of income.
14.11.2 Banana Starch

The production of starch from rejected bananas in the major banana-producing countries could
be an important source of income and employment (Zhang et al., 2005). Rejected bananas are
apparently dumped in rivers in countries such as Costa Rica, causing serious pollution problems.
Bananas are rich in starch and when green the pulp contains about 70–80% starch on a dry weight
basis (Guilbot and Mercier, 1985; Waliszewski et al., 2003). The starch content of banana is com-
parable with that found in the endosperm of corn and the flesh of white potato. There is growing
interest in processing banana starch because of its importance for food and other industrial purposes
(Waliszewski et al., 2003). A number of studies have been conducted on the isolation and charac-
terization of starch from different banana varieties (Lii et al., 1982; Bello-Perez et al., 1999, 2000a,
2000b; Nunez-Santiago et al., 2004). These studies showed that banana starch has potential in both
its digestion and functional properties to be used in processed foods and become a commercially
viable starch product. These studies also suggested that banana starch has low stability to freezing
treatments, and its use in frozen products is not recommended (Bello-Perez et al., 1999). Other stud-
ies (Siriwong et al., 2003) showed that the stability of banana starch paste over a range of pH makes
it suitable for food applications that require high amylase and high retrogradation after processing.
Banana starch has potential applications in food systems that require high-temperature processing,
such as jellies, sausages, and bakery and canned products (Aurore et al., 2009). Chemical modifica-
tions of banana starch can produce improvements to its chemical properties. For example, phos-
phorylated and hydroxypropylated banana starches showed improvement in clarity and phosphated
starches have shown the best freeze–thaw ability. Debranching of native banana starch from the
cultivar ‘Nandigobe’ (EA-AAA) and retrogradation under different storage temperatures and starch
concentrations allowed the production of a high-quality resistant starch with prebiotic properties
with health applications (Lehmann et al., 2002).
Further details about banana starch are available in a number of reviews, including one by Zhang
et al. (2005).
14.12 Conclusion
Postharvest losses of most crops, including banana, are relatively high especially in developing
countries. Advances in biotechnology are now being applied to food commodities to increase the
production of new products. The development of processing industries will see an increase in
the production of processed products (chips, flour, dried pulp, jam, and beverages) from banana.
Although there are many innovative laboratory studies of processed banana products, the indus-
trial application of these products is still not exploited (Aurore et al., 2009). But there is marked
increase in the number of studies that are attempting to utilize banana in various forms. In addition
to exploring various ways of reducing the large postharvest losses incurred in banana production,
these studies are developing new products from banana. The discovery that banana flour has a low
glycemic index has the potential of increasing the utilization of banana flour, especially for control-
ling diabetes. The fact that alcohol can be produced from banana also suggests that banana could be
used for the production of biofuel.
The large genetic diversity of banana is an asset for any program attempting to explore the vari-
ous uses of this genus. Better use of bananas and plantains could be achieved by investigating their
suitability for different types of processing (Aurore et al., 2009). Bananas represent a great potential
raw material for food and nonfood processing industries.
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15 Propagation Methods in Musa
Michael Pillay, Christopher A. Cullis,
David Talengera, and Leena Tripathi
Contents
15.1 Introduction........................................................................................................................... 285
15.2 Embryo Culture in Musa....................................................................................................... 286
15.2.1 Background................................................................................................................ 286
15.2.2 Factors Affecting Success in Embryo Culture.......................................................... 287
15.2.2.1 Embryo Culture Media Composition.......................................................... 287
15.2.2.2 Parental and Seasonal Effects..................................................................... 287
15.2.2.3 Effects of Seed Quality............................................................................... 287
15.2.2.4 Seed Treatments.......................................................................................... 287
15.2.2.5 Culture Physical Environment.................................................................... 288
15.2.2.6 Culturing Techniques.................................................................................. 288
15.3 Micropropagation.................................................................................................................. 288
15.4 Low-Cost Multiplication Methods for Banana...................................................................... 289
15.5 Molecular Characterization of Somaclonal Variation in Musa............................................. 297
15.5.1 Background................................................................................................................ 297
15.5.2 Molecular Techniques for Detecting Genomic Changes in Somaclonal Variants.... 297
15.5.2.1 Cytogenetics and Flow Cytometry............................................................. 297
15.5.2.2 Random Amplified Polymorphic DNA (RAPD)........................................ 298
15.5.2.3 Selective Amplification of Microsatellite Polymorphic Loci..................... 298
15.5.2.4 Representation Difference Analysis (RDA)............................................... 298
15.5.2.5 Transposable Element Activation............................................................... 299
15.6 Conclusions............................................................................................................................ 299
References.......................................................................................................................................300
15.1 Introduction
Bananas (Musa spp.) are propagated vegetatively using suckers or corms. The rate of multiplica-
tion of suckers is slow and variety dependent. Suckers may be infected with diseases and pests
and are transferred to new fields. With the exception of some wild diploids, bananas do not set
seeds. Although the seeds from such diploids have very high germination rates, seeds obtained
from crossing programs have very low rates of germination (Pillay and Tripathi, 2007). Embryo
culture is widely used to increase germination rates in breeding programs. The potential of in vitro
propagation, via shoot-tip cultures, has gained worldwide popularity for rapid mass multiplication
of planting materials. Since the first report of banana in vitro clonal propagation in the 1960s, the
tissue-culture technology in banana has undergone significant improvement and is now used widely
in banana production worldwide. Small-scale farmers are, however, unable to afford the cost of tis-
sue-culture-derived planting material. Low-cost multiplication techniques have been developed to
285
rapidly produce planting materials. Tissue culture is universally associated with somaclonal varia-
tion. The genome of banana has also been shown to vary during tissue culture. Variation has been
observed at the cytogenetic level as well as in specific DNA sequences. As with other plants, these
changes in DNA sequence are limited to a subset of the genome and may also include the activation
of some transposable element families. A series of genomic scanning techniques has been applied
to characterize the DNA variation, and two of these techniques have identified molecular markers
that could be useful in identifying one of the common off-types in banana, namely the dwarf phe-
notype. This chapter examines embryo culture, in vitro multiplication, low-cost multiplication, and
somaclonal variation in Musa.
15.2 Embryo Culture in Musa

15.2.1 Background
Embryo culture was probably the first tissue-culture technique to be applied to plant improvement
(Drew, 1997). Embryo culture has been used to rescue hybrid plants from wide crosses, which
often fail to produce mature viable seeds (Hu and Wang, 1986). In these cases the immature
embryonic tissue is removed from the developing seeds and cultured in the laboratory to produce
the hybrid plants. Immature embryos represent the most regenerable tissue for many species,
including banana. Embryo culture enables the breeder to access a much wider range of genes that
can be used for genetic improvement of crop plants. Wide crosses and embryo culture have been
valuable tools, especially for the transfer of disease resistance genes from wild relatives into crop
plants (Drew, 1997).
Embryo culture was the first application of a tissue-culture technique in banana nearly 40
years ago (Cox et al., 1960). The principal applications of embryo culture are rescuing embryos
after interspecific hybridization, clonal propagation of recalcitrant species, and overcoming seed
dormancy and seed sterility. Interspecific crosses are often attempted to transfer desired traits
such as disease resistance, stress tolerance, or high yield from wild species into important crop
species (Liang et al., 1978). However, embryo abortion often occurs due to breakdown of the
endosperm or embryo-endosperm incompatibility (Hu and Wang, 1986; Elmsweller and Uhring,
1962). In vitro culture of hybrid embryos often successfully bypasses postzygotic incompatibili-
ties (Drew, 1997).
Seed set in triploid banana cultivars is very low due to high levels of sterility (Ortiz and Vuylsteke,
1995). This makes crossbreeding of plantain and banana difficult. Nonetheless, several triploid plan-
tain and banana cultivars produce seeds after hand pollination with diploid parents (Ssebuliba et
al., 2006). Axenic in vitro germination of hybrid seed has been a cornerstone of banana breed-
ing programs. Indeed, embryo culture increases rates of seed germination by a factor of 10 or
more (Vuylsteke et al., 1990). At the International Institute of Tropical Agriculture (IITA), in vitro
embryo culture is being used to create interspecific mapping populations and to rescue hybrid mate-
rials from breeding programs.
In vitro technique of banana embryo culture involves surface sterilizing of banana seeds, asep-
tically cracking them to extract the embryos, and culturing the embryos on appropriate nutrient
medium and growth conditions to induce germination. Since the early 1970s (Menendez and
Shepherd, 1975), embryo culture has been relevant in banana conventional breeding work where
poor fertility due to low seed-set rates has been aggravated by poor seed germination. Seed germi-
nation as low as 1% in plantains (Swennen et al., 1992) and 1.4% in East African Highland bananas
(AAA-EA) (Talengera et al., 1996) has been recorded from direct seed sowing. However, embryo
culture has improved embryo germination to 22% in East African Highland bananas (EAHB,
AAA-EA) (Ssebuliba et al., 2006) and up to 60% in plantains AAB and ‘Popoulou’ (ABB) (Bakry
and Horry, 1992).
Propagation Methods in Musa 287
15.2.2 Factors Affecting Success in Embryo Culture

In vitro banana embryo germination rates vary considerably and are ascribed to several factors.
15.2.2.1 Embryo Culture Media Composition

Embryo culture media are based on the low nutrient formulations of Knudson (1950) or MS
(Murashige and Skoog, 1962) at half strength (Afele and De Langhe, 1991; Vuylsteke et al., 1990)
and the mineral rich salts of MS at full strength (Bakry and Horry, 1992; Vuylsteke and Swennen,
1992; Ssebuliba et al., 2005) and without complex natural supplements. Modifications of the culture
media have been employed to improve embryo germination. Addition of growth regulators such as
BAP (benzylaminopurine) at 1–2 mg/L and IAA (indoleacetic acid) at 0.4–1 mg/L to the medium
(Bakry and Horry, 1992; Vuylsteke and Swennen, 1992; Jenny et al., 1994) gave varied germina-
tion rates and adverse responses. Callus formation was observed on East African Highland banana
embryos when the above-growth regulators were used in the medium. Rescue of immature embryos
from young fruits has been suggested but the low seed fertility in bananas limits this operation.
Instead, white to cream mushroom-shaped banana embryos are routinely obtained from seeds
extracted from mature and ripened fruits. Such embryos are mature and do not require complicated
nutrient medium (Pierik, 1987).
15.2.2.2 Parental and Seasonal Effects

In vitro embryo germination rates vary considerably from cross to cross, genomic composition,
and ploidy of parents. In addition, factors that influence fertility and seed quality positively
affect embryo germination. Generally, 2x × 4x crosses gave higher germination rates than 3x ×
2x crosses. Besides, the presence and proportion of the B genome in the parents is associated
with higher seed quality and embryo germination. Seasonal effects on embryo germination
have been reported, with high germination rates obtained in seeds extracted from bunches pol-
linated during the wet seasons (Vuylsteke et al., 1990). Embryo germination is strongly associ-
ated with the seed quality and number of extracted embryos (Bakry and Horry, 1992; Ssebuliba
et al., 2005).
15.2.2.3 Effects of Seed Quality

Aborted and empty seeds with soft coats and those lacking endosperm are frequently obtained
from crosses and must be discarded. These seeds can be identified by squeezing them between the
thumb and finger or by a water gravity test in which they would float. Caution is needed with seeds
derided from crosses involving EAHB as they do not have a compact endosperm such as the seeds
derived from BB, AAB, and ABB genotypes, and the majority will float. Seed abnormality has
been attributed to failure of fertilization, failure of embryo sac development, and zygote develop-
ment (Shepherd, 1954; Vuylsteke et al., 1990). The proportion of such imperfect seeds vary with
the parents used in the crossing scheme and ranges between 10 and 70% in ‘Popoulou’ (ABB) and
plantains (AAB) (Vuylsteke et al., 1990; Bakry and Horry, 1992) and 41% in AAA–EA pollinated
bunches (Ssebuliba et al., 2005).
15.2.2.4 Seed Treatments
Soaking the seeds for 5 days in water doubled the embryo germination in Musa balbisiana (Afele
and De Langhe, 1991) compared to nontreated seeds. Three days of seed soak in plantain diploids
raised germination rates remarkably from 2 and 30% (untreated) to 45 and 75% (treated) (Talengera,
unpublished). However, seed soaking increases contamination with fungi, yeasts, and bacteria that
can be controlled by a harsh disinfectant such as 1% (w/v) silver nitrate instead of sodium hypochlo-
rite (0.75% w/v). The effectiveness of sterilization can be improved further by sterilizing the seeds
for 15 minutes before soaking and prior to embryo extraction. Disinfection is made easier by wrap-
ping the seeds in cotton gauze and immersing them in the disinfectant.
15.2.2.5 Culture Physical Environment

Banana embryos are cultured at 26–32°C, reflecting the tropical nature of the crop. Although con-
tinuous light was used by Afele and De Langhe (1991), embryos are generally incubated in dark-
ness. Upon germination, the seedlings are exposed to light for further growth, as is required in
normal in vitro micropropagation work.
15.2.2.6 Culturing Techniques
Germination of cultured embryos of hybrids occurs within 1 to 2 weeks compared to the 6-week
period required in Musa balbisiana (Afele and De Langhe, 1991). Late germination usually pro-
duces normal seedlings. After germination, seedlings are cultured for 2 months before they are
ready for potting. Where several plants of a seedling are required, embryo culture has been com-
bined with shoot-tip culture technique (Vuylsteke, 1998). This involves cutting back the seedling
near the base and culturing them onto medium supplemented with cytokinins such as BAP. To
reduce the potential for somaclonal variation due to high cytokinin levels, the BAP concentration
can be scaled down to 2.5 mg/L.
For in vitro rooting, microshoots are separated and cultured onto root-inducing media supple-
mented with 0.225 mg/L BAP and 0.186 mg/L NAA (naphthalene acetic acid) (Vuylsteke, 1998).
However, phytohormone-free medium can be used to root the shoots (Talengera et al., 1994). Rooted
shoots are potted for a month or two acclimatized in shade before the plantlets are ready for field
establishment. Rouging out of off types arising from aneuploidy is required at this stage.
15.3 Micropropagation
Micropropagation has been defined as in vitro regeneration of plants from cells or protoplasts, tis-
sues, or organs (Beversdorp, 1990) on specially formulated nutrient media. Under the correct condi-
tions, an entire plant can be regenerated from a single cell. Plant-tissue culture has been in existence
for more than 30 years. Tissue culture is seen as an important technology for developing countries
for the production of disease-free, high-quality planting material and the rapid production of many
uniform plants. Micropropagation is the production of multiple copies of a single plant using tissue-
culture techniques. Micropropagation increases the number of planting materials to facilitate dis-
tribution and large-scale planting. In this way, thousands of copies of a plant can be produced in a
short time. Micropropagated plants are observed to establish more quickly, grow more vigorously,
have a shorter and more uniform production cycle, and produce higher yields than conventional
propagules. Often, the tissue used is the meristem from which new leaves and stems are produced.
Each growing tip on a plant can be excised and grown into a complete new plant.
Micropropagation of plants has many advantages over conventional methods of vegetative propa-
gation, which suffer from several limitations (Nehra and Kartha, 1994). Tissue-culture systems allow
propagating plant material with high multiplication rates in an aseptic environment. For example,
an in situ banana plant can produce about 10 shoots per year whereas in vitro meristem culture
can produce about 125–144 shoots within 8 weeks. Through repeated subculturing of proliferating
shoots, an open-ended system can be maintained (Tripathi et al., 2003). Propagation from existing
meristems yields plants that are genetically identical with the donor plants (Hu and Wang, 1983).
Tissue-culture propagation is capable of producing disease-free planting materials. Several
major diseases of bananas are known to be transmitted through vegetative planting materials.
In addition, some pests, such as nematodes and weevils, and some pathogens, such as those
causing Fusarium wilt, moko, and bacterial wilt, are also transmitted through the soil and root
tissues. Micropropagation of banana can eliminate virus diseases. The use of tissue-culture and
disease-free planting materials has rehabilitated the banana industry in many countries (Molina,
2002). In Taiwan, the epidemic of Panama wilt, Fusarium race 4, on the popular export cultivar
Cavendish had been successfully managed with the massive use of tissue culture, coupled with
selection and use of somaclonal-variation-derived resistant varieties. In India and China, the
use of tissue-culture planting materials is a very effective control tactic in reducing the inci-
dence of banana bunchy top virus (BBTV). In the commercial export banana production in the
Philippines, tissue-culture-planted farms are known to have fewer problems with nematodes.
Fewer nematicide treatments are required than those of the traditional sucker-planted farms and
perennially maintained plantations.
The micropropagation technique for cultivated Musa is now well established (Cronauer and
Krikorian, 1984; Banerjee and De Langhe, 1985; Vuylsteke and De Langhe, 1985). Use of this
technique may be limited by the risk of somaclonal variation, a widespread occurrence in some
in vitro cultures (Vuylsteke et al., 1988). Shoot-tip culture is simple, easy, and applicable to a wide
range of Musa genotypes (Vuylsteke, 1989). An efficient regeneration protocol, which seems to be
independent of ploidy level and genomic background, was developed for Musa species using apical
meristems (Tripathi et al., 2003). The selected species represented major groups of Musa, includ-
ing fertile diploid bananas (AA and BB genomes), the sterile triploid plantains (AAB), Cavendish
bananas (AAA), and tetraploid hybrids (AAAA and AAAB).
Propagation of banana through encapsulated shoot tips has also been reported (Ganapathi et al.,
1992). An efficient, simple, and rapid regeneration system was also established for bananas using sec-
tions of corm containing intercalary meristematic tissues as explants (Tripathi and Tripathi, 2008).
Several workers have reported that cellular differentiation and organogenesis in tissue culture are
controlled by concentrations of cytokinin and auxin (George, 1993). George (1993) suggested that
organogenesis in monocotyledonous plants was promoted on media supplemented with cytokinin/
weak auxin combinations. The effects of cytokinin/auxin interactions on in vitro shoot proliferation
of bananas were investigated (Arinaitwe et al., 1999). Higher proliferation rates were observed in
cytokinin/auxin combinations in which a weak auxin IBA (indole-3-butyric acid) was included.
Micropropagation has played a key role in plantain and banana improvement programs world-
wide (Vuylsteke et al., 1997). Planting material derived from micropropagation performs equal to
or superior to conventional material (Smith and Drew, 1990; Vuylsteke, 1998). Micropropagated
banana and plantain establish faster, grow more vigorously, are taller, have a shorter and more
uniform production cycle, and yield higher than conventional propagules (Vuylsteke and Ortiz,
1996). Bananas propagated from apical meristem in Kenya have been shown to have increased
vigor and suffer lower yield loss from weevils, nematodes, and fungal diseases (ISAAA, 2006).
Micropropagation was considered an appropriate option to provide sufficient quality and quantity
of such materials. With proper management and field hygiene, yield losses caused by pests and
diseases at the farm level have been reduced substantially. Tissue-culture technology has made
it possible for farmers to have large quantities of superior, clean planting materials that are early
maturing, with bigger bunch weights and higher annual yield per unit of land. Moreover, uniformity
in orchard establishment and simultaneous plantation development can make marketing easier to
coordinate with the possibility of transforming banana growing from merely subsistence to a com-
mercial enterprise. Tissue-culture banana production is more remunerative as an enterprise than
traditional banana production.
15.4 Low-Cost Multiplication Methods for Banana

Cultivated banana and plantain are triploid and seedless. Most cultivars are female sterile and only
produce seeds during artificial hybridization. Seeds are only used during the breeding process for
the genetic improvement of banana. On the contrary, wild diploids are fertile and produce copious
amount of seeds that are capable of germination after dispersal. Bananas are clonally propagated.
Their survival in nature and geographical dispersal is not possible without intervention by humans.
Large plantations purchase new planting material from tissue-culture laboratories. The high cost
of tissue-culture planting material (averaging about $US1.00 per plant) is unaffordable by most
small-scale farmers in most countries. Bananas in general have poor suckering ability (Robinson,
1996). The difficulty in obtaining healthy planting material is one of the most important constraints
for large-scale banana production or expansion of existing plantations in many countries. Several
types of planting materials that vary in their degree of suitability include: the maiden sucker, water
sucker, sword suckers, butt, peeper, and bits that can be used for the establishment of new banana
plantations (Ndubizu and Obiefuna, 1982; Baiyeri and Ndubizu, 1994). The major concern of veg-
etative planting material of this nature is its tendency to perpetuate pests (weevils, nematodes),
wilts (Fusarium, Xanthomonas), and viral diseases (banana bunchy top, BSV) from existing to
newly established fields. The movement of unhealthy planting material is probably the biggest fac-
tor in transmitting diseases and pests not only from one country to another but from one continent
to another.
An alternative to tissue culture for producing large numbers of planting material in a relatively
short time will benefit farmers who consider the cost of tissue-cultured plants prohibitive. In addi-
tion, most farmers in the developing countries where banana is grown are not trained in handling
tissue-culture plantlets. For example, a plant arising from tissue culture produces a large num-
ber of suckers in the very early growth stage due to the residual effect of the growth hormones.
These suckers have to be rouged out carefully without damaging the mother plant. The underground
banana stem (rhizome) has numerous axillary buds at the base of the leaf sheaths which provide a
source of a large number of plantlets if they are allowed to develop (Barker, 1959). One of the first
experiments in the vegetative multiplication of bananas involved splitting of the rhizome and plant-
ing the pieces under nursery conditions (La multiplication, 1955). Navarre (1957) reported that over
180 plants were obtained from one rhizome by initiating callus formation. The technique involved
digging, cleaning, and injuring of the rhizome to initiate callus formation. The callus tissue gave
rise to new plants that were removed and rooted. This method was also adopted by Hamilton (1965)
who obtained over 150 plants from a single rhizome in 5 to 7 months (Hamilton, 1965). Barker
(1959) used the stripping technique in which the outer leaf sheaths of the pseudostem were removed
to expose the axillary buds. Soil was then mounded around the buds to initiate sucker development.
The process was repeated when a sucker reached 1 m in height. This method was capable of pro-
ducing about 20 plants from a single parent plant. In a series of experiments, Loor (1978, cited in
Menendez and Loor, 1979) tried to improve vegetative multiplication of bananas. One experiment
identified the most suitable kind of corm to use while another experiment defined the type of wound
that produced the best results. A third experiment looked at the action of hormones and cytokinins
in stimulating the growth of adventitious buds.
In vitro banana cell culture research was first studied in Jamaica by Cook (Menendez and Loor,
1979). Later in vitro work was carried out in Honduras (Berg and Bustamante, 1974), Taiwan
(Su-Shien and Shii, 1974; Ma and Shii, 1972, 1974), and the Philippines (de Guzman et al., 1976).
Micropropagation using meristem/tissue culture can rapidly multiply and produce disease-free
planting materials. Modern methods, especially micropropagation using meristem/tissue culture,
are very efficient in rapid multiplication and production of healthy, vigorous, and disease-free plant-
ing materials. The method, however, requires sophisticated techniques, skill, and care to handle
(Vuylsteke and Talengera, 1998). Tissue culture as a method of generating planting materials is
being established in many developing countries. Though effective and fast, tissue culture (in vitro
multiplication) is not an option for the majority of traditional banana producers.
A number of robust, low-cost means of multiplying bananas are now available (Pillay
and Tripathi, 2007). They include false decapitation (Figure 15.1a, b), complete decapita-
tion (Figure 15.2a, b), excised corm (Figure 15.3a, b), split corm (Figure 15.4a, b), and a bud
manipulation technique (Figure 15.5a–i). These techniques, known as macropropagation, are
easy affordable alternatives for tissue culture for large-scale sucker production at the farm level.
Macropropagation methods can generate from 16 to 50 or more plantlets from one sword-sucker
corm utilizing sawdust as a plantlet initiation medium. Other media such as rice hulls could also
be used (Baiyeri and Aba, 2005).
(a)
(b)
Figure 15.1 False decapitation method. (a) The actively growing region (meristem) of the plant is destroyed
by cutting a small hole (arrow in picture) in the pseudostem (the trunk) with a sharp knife. New plantlets will
appear around the plant in about 1 month. (b) The suckers are detached carefully once they reach a height of
20 to 30 cm and have three to four leaves and are then planted directly to the field. (Reprinted from Pillay, M.
and L. Tripathi, 2007, Breeding major food staples, M.S. Kang and P.M. Priyadarshan, eds., 393–428. New
York. With permission from John Wiley.)
False decapitation destroys the actively growing region (meristem) of the mother plant by carefully
cutting a hole through the pseudostem base with a sharp instrument such as a knife (Figure 15.1a).
New plantlets arise from axillary buds at the base of the plant (Figure 15.1b). Suckers with a height
of 30–40 cm can be detached carefully with a sharp instrument and planted directly in the field.
Complete decapitation differs from the earlier method in that the pseudostem is completely cut down
(a)
(b)
Figure 15.2 Complete decapitation method: (a) The pseudostem is completely cut down. The meristem is
killed and the corm left to sprout (within a month). Young plants are detached and planted directly. (b) New
suckers formed from mat. (Reprinted from Pillay, M. and L. Tripathi, 2007, Breeding major food staples, M.S.
Kang and P.M. Priyadarshan, eds., 393–428. New York. With permission from John Wiley.)
and the meristematic tissue is removed or destroyed (Figure 15.2a). The destruction of the apical
meristem stimulates the growth of the axillary buds (Figure 15.2b), which are detached and planted
in a field. Best results with both false and complete decapitation occurs when there is no competing
ratoon sucker in the same mat. A sucker in the same mat retards the growth of the axillary buds,
perhaps by exerting apical dominance. In the excised corm technique, the large visible buds are
(a)
(b)
Figure 15.3 Excised corm method: (a) Buds are cut out in mini sets (about 100 g each). (b) Each bud
is grown in a separate polyethylene bag and transferred to the field when three to four leaves have been
attained. (Reprinted from Pillay, M. and L. Tripathi, 2007, Breeding major food staples, M.S. Kang and P.M.
Priyadarshan, eds., 393–428. New York. With permission from John Wiley.)
carefully removed together with some of the surrounding corm tissue and sown in suitable-sized
polythene bags (Figure 15.3a, b). Enough water is applied to keep the soil moist. The split-corm
technique, as the name suggests, involves splitting of the corm into two or more fragments. The
pieces are planted, preferably in sterile soil with the axillary buds facing the soil (Figure 15.4a, b).
Sprouted plantlets are detached and potted before transplanting to the field. The technique that has
the highest potential of producing the largest number of suckers is the bud manipulation technique.
This is a modification of the technique of Barker (1959). Medium-sized corms are obtained from
healthy mats (Figure 15.5a). The sucker is pared with a sharp knife to remove roots, old dead tissue,
(a)
(b)
Figure 15.4 Slit corm method. (a) The corm is dug out of the soil, pared, and split into two or more frag-
ments depending on its size. (b) Plant split corm face down in polyethylene bag. Detach sprouted plantlets and
pot in appropriate substrate. (Reprinted from Pillay, M. and L. Tripathi, 2007, Breeding major food staples,
M.S. Kang and P.M. Priyadarshan, eds., 393–428. New York. With permission from John Wiley.)
and pseudostem remains (Figure 15.5b). Removal of the outer corm tissue gets rid of superficial
weevil and nematode eggs. The pseudostem is then cut about 20 cm above the collar (Figure 15.5c).
The leaf sheaths are removed individually in order to expose the axillary buds (Figure 15.5d). The
apical meristem is destroyed. A well-prepared corm in the bud manipulation technique is repre-
sented in Figure 15.5e. The bud is carefully placed in sawdust or an appropriate medium in a plastic
(a)
(b)
(c)
(d)
Figure 15.5 Bud manipulation method. (a)

Medium-sized sucker obtained from healthy
mats. (b) Sucker is pared. (c) The pseudostem is
cut about 20 cm above the collar. (d) The sheaths
are removed individually in order to expose the
buds at the v end of each sheath. (e) The inner-
most section (meristematic doom) is cut to create
a small depression. The corm is placed in sawdust
in the propagation chamber. (continued)
(e)

(f) (g)

(h) (i)
Figure 15.5 (Continued) Bud manipulation method. (f) Corms in a propagation chamber, regular water-
ing is required. Presence of condensed water droplets on the inside of the plastic material indicates sufficient
humidity in the chamber. First sprouting commences from the second week of operation. (g) Manipulation
of primary bud sprouts. Sawdust is removed to expose the bulbous shoot bases. The shoots are cut off just
above the collar and meristem excised to induce production of secondary bud sprouts. They are recovered
with sawdust. Secondary bud sprouts are observed 2 to 4 weeks after primary bud manipulation. (h) A
clump of plantlets regenerated from bud sprouts. A range of 16 to 30 plantlets is obtainable between 10 to
18 weeks. At this stage plantlets are detached and put in plastic materials with nutrient-rich porous topsoil.
(i) Plantlets just detached from the mother corm ready to be taken to the root initiation chamber from where
they are weaned after developing small whitish roots. (Reprinted from Pillay, M. and L. Tripathi, 2007,
Breeding major food staples, M.S. Kang and P.M. Priyadarshan, eds., 393–428. New York. With permission
from John Wiley.)
growth chamber as shown in Figure 15.5f. The axillary buds begin to develop after approximately
3 weeks (Figure 15.5g). This technique is capable of producing 16–30 plantlets in 10–18 weeks
(Figure 15.5h). The plantlets are detached, rooted in soil in a low-light chamber, and then hardened
under direct sunlight (Figure 15.5i).
Although macropropagation is genotype specific, it certainly provides a low-cost method of pro-
ducing new planting material. The method has been used to rapidly multiply and supply farmers
with planting material in emergency situations such as the recent threat of Xanthomonas wilt in
eastern Africa.
15.5 Molecular Characterization of
Somaclonal Variation in Musa
15.5.1 Background
The term “somaclonal variation” was coined to describe the variation observed in plants when they
were taken through one or more cycles of culture and regeneration (Larkin and Scowcroft, 1981).
This observation was unexpected, as the process of tissue culture and regeneration was not origi-
nally considered to be mutagenic. The characteristics of the somaclonal variants have led to varying
hypotheses concerning their origin. The appearance of a very limited range of aberrant phenotypes
is inconsistent with the notion that the mutagenic effect is simply a random process within the
genome. Across many plant species, flower and leaf abnormalities are very common. Therefore
there may be a common mechanism that targets a specific compartment of the genome for variation
while cells are being propagated through tissue culture. Additionally, the observation that some of
the somaclonal variants can revert during growth has led to the suggestion that the process is “epige-
netic” (Kaeppler et al., 2000); that is, it is not based in DNA sequence variation but in modifications
such as cytosine methylation that can be reversed.
In banana, the set of phenotypic aberrations, known as off-types, is limited but can arise at a
variable frequency, which can be as high as 90% (Cote et al., 1993; Israeli et al., 1995; Reuveni
et al., 1996). Among the most common variants are ‘Dwarf,’ ‘Giant,’ ‘Massada,’ and chloro-
tic variegation. The ‘Massada’ variant is mosaic variegation on the leaves while the remaining
names are descriptive of the phenotype. This limited set of variants coupled with the high rates of
appearance indicate that a search for molecular markers associated with this phenomenon would
be successful.
15.5.2 Molecular Techniques for Detecting Genomic Changes in Somaclonal Variants

Since the somaclonal variants appear to be heritable, and some somaclonal variants are also mei-
otically transmissible, genome scanning techniques have been applied to banana to identify DNA
variations associated with somaclonal variation. Cytogenetics and flow cytometry methods (Roux
et al., 2004; Gimenez et al., 2001), random amplified polymorphic DNA (RAPD) (Damasco et al.,
1996; Walther et al., 1997; Sheidai et al., 2008), the selective amplification of microsatellite poly-
morphic loci (SAMPL) (Gimenez et al., 2005), and representation difference analysis (RDA) (Oh
et al., 2007) have all been used to characterize normal and off-type bananas. In addition, there is
evidence that retrotransposons are also activated during culture (Khayat et al., 2004).
15.5.2.1 Cytogenetics and Flow Cytometry

Genetic instability in the DNA content of embryogenic cell suspension cultures of triploid clones
has been identified using flow cytometry and chromosome counting (Roux et al., 2004). They dem-
onstrated that the ploidy level of Musa embryogenic cell suspension cultures can be determined
by flow cytometry. The ploidy level of such cells can vary over a short time in culture but the
contribution of such ploidy alterations is debatable since the abnormal ploidy levels coincide with
lowered regeneration ability. However, a somaclonal variant of banana, ‘CIEN BTA-03,’ resistant
to yellow Sigatoka disease (Gimenez et al., 2001), was shown to be a tetraploid clone through cyto-
genetics and flow cytometry.
15.5.2.2 Random Amplified Polymorphic DNA (RAPD)

A number of studies have used RAPD comparisons between normal and off-type bananas (Sahijram
et al., 2003; Lakshmanan et al., 2007; Sheidai et al., 2008). These studies have screened large num-
bers of RAPD primers to assess variation among parental identified off-types from micropropaga-
tion. In one study, 66 primers were used in an initial screening of approximately equal numbers of
normal and dwarf plants (Damasco et al., 1996). Nineteen of the 66 primers identified polymor-
phisms between normal and dwarf plants, and one primer, OPJ-04 (5´-CCGAACACGG-3´), could
be used to identify a specific polymorphism between normal and dwarf plants. The polymorphism
was in the form of an approximately 1.6 kb band, which was consistently present in all normal but
absent in all dwarf plants. This RAPD band was converted into a sequence characterized amplified
region (SCAR) marker for use in detection of dwarf plants in regenerated bananas (Ramage et al.,
2004). Since the test is one of a band present in the DNA from normal plants and not from dwarf off-
types, a control for all the amplifications needed to be included in order to distinguish between the
absence of a PCR product due to the plant being a dwarf or simply a problem with the PCR amplifi-
cation. None of the other polymorphisms that were observed between normal and dwarf phenotypes
were specifically associated with the dwarf phenotype. In another study, Sheidai et al. (2008) used
30 RAPD primers to study somaclonal variation among the parental as well as regenerated plants.
A total of 289 bands were scored, of which 147 were polymorphic. There were no associations with
any of the polymorphisms and particular phenotypes. In contrast to these two studies, Lakshmanan
et al. (2007) screened a large number of random primers but found no changes in the banding pat-
terns of tissue-cultured plants and those of the mother plant. Since Sheidai et al. (2008) found that
the level of variation was associated with the culture conditions, this last set of data (Lakshmanan et
al., 2007) supports previous studies that the appearance of genomic variation is dependent on both
the variety and the culture conditions.
15.5.2.3 Selective Amplification of Microsatellite Polymorphic Loci

The selective amplification of microsatellite polymorphic loci (SAMPL) technique is a modification
of the amplified fragment length polymorphism (AFLP) procedure (Vos et al., 1995). In the case
of SAMPL, the primers are compound microsatellite sequences combined with AFLP oligonucle-
otides. High numbers of monomorphic bands were amplified in all cultivars and somaclones but
some markers were present in only a single cultivar or somaclone (Giminez et al., 2005).
15.5.2.4 Representation Difference Analysis (RDA)

RDA is a technique whereby all the fragments that are in common between two genomes are
removed, leaving only those that are different between the genomes (Lisitsyn et al., 1993). These
subtractions were performed using the tall (off-type) and dwarf forms of the cultivar ‘Curare enano’
(Oh et al., 2007). The series of different products that were isolated were sequenced. Using the
available sequence, primers were designed and used in a PCR-based system to monitor changes
in genomic integrity of in vitro–produced banana plants. A subset of the primers identified dwarf
from normal plants. The most informative primer pair amplified a band in the normal or giant
phenotypes, but failed to amplify a band in the dwarf phenotypes. This is identical to the SCAR
marker described earlier, although there was no similarity between the two sequences. A sequence
analysis over an extended region containing the marker identified numerous other small DNA varia-
tions between the phenotypes that are consistent with the notion that specific genomic regions are
targeted when banana cells are propagated through tissue culture.
An extensive analysis of this characterized region also identified some differences in the methy-
lation status of the DNAs from normal and dwarf phenotypes, with the level of methylation being
greater in the dwarf than the normal phenotype. More studies on the levels of methylation covering
a greater part of the banana genome are necessary to determine the role of methylation in soma-
clonal variation in banana. Are the methylation differences a cause or a consequence of the mecha-
nisms that are responsible for the genomic restructuring?
15.5.2.5 Transposable Element Activation

Retrotransposons have been identified as being activated by stresses in plants (Grandbastien, 1998),
including the stresses imposed by tissue culture. Following tissue-culture cycles, retrotransposon
activation was observed in banana (Khayat et al., 2004). As with many of the other observations
concerning the genomic changes associated with passage through tissue culture, there were no
directly identifiable changes in the retrotransposon patterns with a particular phenotype. The activa-
tion of previously quiescent retrotransposons by stresses is one hallmark of some epigenetic changes
where the instability is mediated by modification of the heterochromatin.
15.6 Conclusions
Despite efforts to improve the rate of embryo rescue in banana, the current in vitro technique does
not allow total germination of all embryos. Failure to achieve total germination even after ridding
the embryo of a suspected inhibitor from the endosperm and seed coat suggests the presence of
embryo-related dormancy. There is need to explore dormancy-breaking treatments on the excised
embryos. Attempts to improve embryo germination must consider other factors such as seasonal
effects on seed fertility. Genomic composition and ploidy of the hybrids should also be considered
to improve the rate of embryo germination.
The main advantage of tissue-culture technology lies in the production of high-quality and uni-
form planting material that can be multiplied on a year-round basis under disease-free conditions
anywhere irrespective of the season and weather. However, the technology is capital, labor, and
energy intensive. Although labor is cheap in many developing countries, the resources of trained
personnel and equipment are often not readily available. In addition, energy, particularly electricity,
and clean water are costly. In many African countries there are difficulties in adoption of tissue-
culture material because of the high cost of the material in comparison to conventional planting
material. Hence, it is necessary to have low-cost options for weaning, hardening of micropropagated
plants, and finally growing them in the field. Low-cost tissue-culture technology is the adoption of
practices and use of equipment to reduce the unit cost of the micropropagule and plant production.
Low-cost options should lower the cost of production without compromising the quality of the
micropropagules and plants. Alternative low-cost macropropagation techniques for producing large
numbers of planting material in a relatively short time will benefit farmers who consider the cost of
tissue-cultured plants prohibitive.
The data for the genomic changes in banana in response to passage through tissue culture is
consistent with that observed for other plants and includes cytological abnormalities, frequent quali-
tative and quantitative phenotypic mutation, sequence change, and gene activation and silencing.
Clearly there are a number of different mechanisms in play during the occurrence of somaclonal
variation in banana. The changes in ploidy level as well as possible chromosome rearrangements
are directly observable. The physical changes within the genome that have been identified through
the application of a series of molecular screens of the genome include polymorphisms identified
through RAPD and RFLP analysis, the characterization of difference products by RDA, and the
activation of transposable elements. Finally, some variation in the methylation status of regions of
the genome has also been identified. The primary events that are responsible for the appearance
of the variants have not yet been definitively identified. However, two different methods of analy-
sis have both identified changes that identify dwarf from normal banana plants. In both cases, a
DNA fragment is absent from the dwarf but is present in the normal plants. The observation that a
reproducible genomic event is associated with one of the phenotypes is again consistent with all the
other plant data that limits the genomic variation in response to tissue culture to a small subset of
the genome. The evidence for the involvement of epigenetic alterations in banana somaclonal varia-
tion is minimal at present, but future research will determine the relative importance of epigenetic
versus sequence or chromosome variation in the appearance of somaclones. In some respects the
appearance of somaclonal variation resembles tumorigenesis in animals. Here again, the genome is
subject to many different restructuring processes and it has been difficult to directly assign the root
event responsible for the tumor progression. In the case of somaclonal variation, the same problem
exists: Which of the many variations that have been observed are the specific causes of the altered
phenotype and which are simply associated with the restructuring that accompanies the stresses
imposed by the growth environment of cells in culture?
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16 Hybrid Distribution to Farmers
Adoption and Challenges
Abdou Tenkouano, Michael Pillay, and Ousmane Coulibaly
Contents
16.1 Introduction........................................................................................................................... 305
16.2 Access to Improved Cultivars and Quality Planting Materials.............................................306
16.2.1 Seed Systems: Promotion of Rapid Plant Propagation Techniques...........................306
16.2.2 Farmer-to-Farmer Diffusion of Hybrids in Nigeria...................................................307
16.2.2.1 Delivery Package........................................................................................307
16.2.2.2 Delivery Process.........................................................................................308
16.2.2.3 Preliminary Assessment of Impact.............................................................309
16.3 Understanding and Managing Diversity................................................................................ 310
16.4 Banana Trade Prospects for Africa....................................................................................... 310
16.4.1 Trends in Commercial Banana Production in Sub-Saharan Africa.......................... 310
16.4.2 Strategic Implications................................................................................................ 312
16.5 International Distribution of Hybrids.................................................................................... 312
16.6 Conclusion............................................................................................................................. 313
References....................................................................................................................................... 319
16.1 Introduction
The adoption of new banana cultivars by large-scale producers is a relatively straightforward pro-
cess when the commercial incentives for adoption are high or when there are no other alternatives.
This was evidenced by the replacement of ‘Gros Michel’ by the Cavendish varieties. Therefore, this
chapter will consider hybrid distribution to smallholder farmers who are responsible for the bulk of
banana and plantain production in developing countries, usually under complex cultural and tech-
nological circumstances.
Productivity gains achievable from improved agricultural technologies have not been fully
exploited by farmers in developing countries. There is a big gap between potential and achieved
yields on farmers’ fields, mainly due to various factors such as poor extension services, institu-
tional and cultural constraints, and farmers’ firm adoption of traditional practices and their limited
financial ability and willingness to increase input levels in farming (Ghatak and Ingersent, 1984;
Ali and Byerlee, 1991; Xu and Jeffrey, 1998; Pingali and Heisey, 1999; Kalirajan and Shand, 2001;
Alene and Manyong, 2006a, 2006b). Although measures have been taken to achieve a high rate
of adoption of new cultivars, little or no emphasis has been given to the adoption of other types of
complementary information, which taken together represent the “best practice” technological pack-
age (Kalirajan, 1991; Pingali and Heisey, 1999; Kalirajan and Shand, 2001; Alene and Manyong,
2006a, 2006b). These general principles strongly apply in the case of banana and plantains.
305
One of the alternative measures taken to achieve a high adoption rate of new cultivars—espe-
cially in areas where formal seed systems do not exist, are ineffective, and/or are inefficient—has
been the farmer-to-farmer seed diffusion. The basic premise behind such farmer-led technology
diffusion is the rich theory of diffusion of innovation, which is described as “the process by which
an innovation is communicated through certain channels over time among the members of a social
system” (Rogers, 1995). The most striking feature of the diffusion theory is that, for most members
of a social system, the innovation decision depends heavily on the innovation decisions of the other
members of the system. Farmer-to-farmer technology transfer thus builds upon farmers’ traditional
seed-transfer methods and is based on the observation that farmers prefer that their fellow farmers
are the primary sources of information even when they have alternative sources (Feder and Slade,
1985; Rogers, 1995). This must be considered when designing technology dissemination programs,
particularly for improved seeds (Grisley, 1994; Kormawa et al., 2004).
However, when farmers have access to modern inputs such as improved seeds, they often lack
the knowledge and agronomic and crop-management technologies that are crucial for bridging the
yield gap (Pingali and Heisey, 1999). This is often the case with farmer-to-farmer technology diffu-
sion, because crop management information is either not integrated into the farmer-to-farmer seed
transfer scheme or is not effectively communicated.
Promoting improved seeds without the complementary management technologies through
farmer-to-farmer transfer means that potential yields remain unexploited. For example, the yield
achieved on farmers’ fields depends not only on the amount of fertilizer applied but also on when
and how it is applied—not only on whether improved cultivars are adopted but also on whether
the recommended cropping patterns and systems are followed. In this situation, farmer-to-farmer
seed transfer will promote partial adoption and results in underutilization of yield potential of new
cultivars by adopters.
It is thus important to conceive seed transfer programs within the context of a technology
package, particularly for bananas that have a crop cycle of 12–18 months from planting to harvest.
This chapter focuses on the direct components of seed systems, including access to improved
cultivars and quality planting materials, which are discussed with emphasis on technical and
institutional issues.
16.2 Access to Improved Cultivars and Quality Planting Materials

We have discussed the concept of breeding and targeting new cultivars in Chapter 9 and Chapter
12. In essence, we indicated that the prospects for adoption of new cultivars increase as the new
cultivars are (1) adapted to biophysical and socioeconomic characteristics of the target area, (2)
chosen with inclusive participation of the farmers in the selection process, and (3) introduced in a
nondisruptive approach that preserves the existing cultivars. Once these prerequisites are met, it is
important to devise a user-friendly approach for supply of planting materials.
16.2.1 Seed Systems: Promotion of Rapid Plant Propagation Techniques

A major constraint to expansion of banana and plantain cultivation is the scarcity of healthy plant-
ing materials. Farmers usually depend on natural regeneration of plants for the supply of planting
materials. This is a very slow process that often produces small numbers of planting materials that
are usually contaminated by various soil-borne pathogens such as nematodes. Transplanting of the
contaminated materials often spreads nematode problems and shortens the lifetime of plantations
to only one or two cycles of cultivation, beyond which most plants fall and become unproductive. In
prelude to the wide-scale distribution of hybrids, the International Institute of Tropical Agriculture
(IITA) has been looking at alternative means of producing planting materials, since it is unlikely
that the average farmer will develop the capacity to multiply plants by micropropagation (tissue cul-
ture). The alternative methods (low-cost multiplication) can be classified into two categories: field
Hybrid Distribution to Farmers 307
techniques based on complete or partial decapitation and detached corm techniques practiced away
from the field (Tenkouano et al., 2006). These techniques are discussed in detail in Chapter 15.
These approaches for multiplying planting material have been adopted by farmers on a com-
mercial scale not only in Cameroon and Nigeria but also in Ghana. The method was widely used
across eastern (Uganda, Tanzania) and southern (Malawi, Mozambique) Africa for mass-testing
and delivery of new hybrids. This low-cost method was also used to rapidly multiply material for
distribution to farmers in East Africa due to the ravages of Xanthomonas wilt (Crop Crisis Control
Project, 2007).
16.2.2 Farmer-to-Farmer Diffusion of Hybrids in Nigeria

Breeding programs at the International Institute of Tropical Agriculture (IITA), the Fundación
Hondureña de Investigación Agricóla (FHIA) in Honduras, and the Centre Africain de Recherches
sur Bananiers et Plantains (CARBAP) in Cameroon have developed disease-resistant hybrids with
appropriate agronomic characteristics. The development of these disease-resistant and high-yielding
plantain and banana hybrids is a significant scientific achievement in recent history in terms of its
potential contribution to food security and poverty reduction in Africa. The major varieties include
‘PITA-14,’ ‘PITA-17,’ ‘PITA-21,’ ‘PITA-22,’ ‘PITA-23,’ ‘PITA-24,’ ‘PITA-25,’ ‘PITA-26,’ ‘BITA-
3,’ ‘BITA-7,’ ‘FHIA-17,’ ‘FHIA-18,’ ‘FHIA-20,’ ‘FHIA-21,’ ‘FHIA-23,’ ‘FHIA-25,’ and ‘CRBP-39’
(Tenkouano and Swennen, 2004).
16.2.2.1 Delivery Package
Multilocation on-farm farmer-participatory trials were set up to evaluate these improved hybrids for
the widespread distribution of those materials fitting the ecological and culinary preparation needs
in different countries across Africa. Several hybrids were identified as promising in many countries,
for example, ‘PITA-2’ and ‘PITA-5’ in Ghana, ‘PITA-17’ and ‘PITA-14’ in Nigeria, ‘BITA-3’ in
Ghana and Uganda. It must be noted that farmers’ preferences for distributed hybrids vary in dif-
ferent countries. Out of four cultivars—‘BITA- 3,’ ‘FHIA-17,’ ‘FHIA- 21,’ and ‘CRBP-39’—farmers
in Cameroon preferred ‘FHIA-21’ followed by ‘FHIA-17’ (M. Pillay and G. Blomme, unpublished
data). In Mozambique, farmers preferred the hybrid ‘SH-3640’ and landrace ‘Grande Naine’ and
showed less preference for ‘FHIA-17,’ ‘FHIA-21,’ and ‘FHIA-23’ (Uazire et al., 2008).
Variety dissemination projects should be designed to have significant economic impact in the
medium term. In Nigeria, for example, on-farm demonstrations were launched in 2000 to promote
an improved plantain and banana package consisting of: (1) planting materials of hybrids, (2) pests
and diseases control methods, (3) agronomic practices, and (4) postharvest utilization. Between
2000 and 2001, over 5,000 farmers obtained planting materials through farmer-to-farmer diffusion
of planting materials, and this had grown to over 20,000 farmers by 2005. Similarly, over 4,000
farmers were targeted in Tanzania, Mozambique, Malawi, and Zambia between 2003 and 2005.
While the growing number of beneficiary farmers over the years certainly shows the effective dis-
semination of hybrid planting materials, little is known about the dissemination of the remaining
components of the improved hybrid delivery package.
Achieving the productivity gains from hybrids also depends critically on the adoption of the rec-
ommended associated technologies. Given that the cropping pattern component of the technology
package is provided in the form of information, its dissemination depends not only on the effective-
ness of the farmer-to-farmer communication but also on the quality of the technical advice provided
during major cropping operation.
16.2.2.2 Delivery Process
Instrumental in the implementation of large-scale variety dissemination is the quality of collabora-
tion among multiple stakeholders, from the farmers to extension institutions, to community-based
organizations, including faith-based and self-help groups (Tenkouano et al., 2010). A careful choice
of partners is essential in implementing large-scale delivery for evaluation of improved hybrids of

banana and plantain to the smallholder farmers. This is because the target regions are usually very
large and cut across ecological and linguistic/cultural landscapes, each with unique socioeconomic
features. For example, the Nigerian plantain belt (NPB) is a region lying between 3° E and 9° E of
longitude and south of 8° N latitude, with a northern boundary running approximately parallel to
the coast of the Gulf of Guinea. This region encompasses the territories of 11 states, namely, Abia,
Akwa-Ibom, Bayelsa, Cross River, Delta, Edo, Imo, Ogun, Ondo, Oyo, and Rivers. Each state has
an agricultural extension network coordinated by erstwhile World Bank–sponsored Agricultural
Development Programmes (ADP). Other field operatives of banana and plantain extension include
the National Horticulture Research Institute (NiHort) and the Plantain and Banana Development
Programme (PBDP), agencies of the federal government of Nigeria. A large number of voluntary
service organizations (VSOs) that operate as nongovernmental organizations (NGOs), sometimes
as community-immersed or community-based organizations (CBOs), and the network of schools
constitute other key components of the landscape.
Four ADPs were chosen by their peers and other stakeholders to serve as relay centers that would
be repositories of the disseminated hybrids. Trials carried out at the relay centers were considered to be
reference trials where hybrids were grown under two cropping regimes: recommended best agronomic
practices and standard farmer practice. Additionally each of these relay centers would also serve as a
source of planting material for further distribution of hybrids selected by the growers. Therefore, IITA
established model plant-propagation units and trained the staff of the relay centers for operating the
propagation facility as a business. The relay centers also served as schools for demonstration of post-
harvest processing options that were components of the delivery package. With their triple function,
the relay centers were also known as Plantain Resource and Training Centers (PRTC), designed to
be self-sustaining. Among the criteria used by peers to designate which ADP would be a PRTC were
geographical position (central enough to cater for several ADPs with minimum distance) and logistical
endowment (including willingness to cofinance the setting up of the facilities and training events for
their own personnel as well as beneficiaries of the dissemination process).
A joint exploratory survey involving the ADPs, NiHORT, PBDP, and IITA was carried out to
identify candidate farmers for the study. Thus, shortlists of five farmer households for each of five
farming communities per state were provided by the extension agencies and site-screened by IITA
on the basis of the suitability of their farms (size and accessibility, biophysical condition) and their
personal credentials (experience with plantain cultivation, leadership status in the community).
One farmer household per community was retained, giving a sample of 55 contact farmer house-
holds (CFH) for the study. The household approach ensured that collective knowledge remains
within the family and not solely in the hands of one person. Thus each CFH served as the social
experimental unit, linking to the communities where they are sited as well as those of neighboring
villages. The entire set of hybrids in the reference trials was also planted at each CFH (mother trials)
to simulate performance under farmers’ conditions.
Each CFH was aggregated with one PRTC in such a way that hybrids selected by the commu-
nities served by the linked CFHs would be the primary targets for mass propagation for further
distribution to new growers in waves of five new growers per crop cycle for each CFH. At this stage,
only those hybrids chosen by farmers from the mother trials would undergo further distribution to
establish baby trials, usually consisting of subsets of the chosen hybrids.
Each farmer receiving planting materials would have to agree to provide two suckers for each
one received so that these could be distributed to more farmers, ensuring a steady farmer-to-farmer
diffusion of the best performing hybrids. Also, each receiving farmer would be subjected to training
in simple field multiplication and sanitation techniques as described earlier.
Finally, regular progress review and planning meetings involving all the stakeholders were held
at least twice a year to exchange experiences and document practices that should be encouraged
(do’s) and those that should not be recommended (don’ts) with respect to both the delivery package
and the delivery process. Table 16.1 illustrates the outcome of one such meeting. These meetings
Table 16.1
Highlights of Project Review and Planning Meeting in Nigeria
Proposed Interventions Specific Tasks
1. Improved hybrids
1.1.Phase I (Old): Hybrids (first batch Select best bets for large-scale distribution.
introduced in 2000) Develop a plan for rapid multiplication of best bets.
Develop a plan for maintenance and sourcing of best bets.
1.2.Phase II (New): Varieties (second Set up large demonstration plots at the four designated PRTCs and at selected
batch introduced in 2005) sites with proven capacity and commitment to handle large demonstrations.
Plots will follow a classical research design, allowing for data collection and
statistical analysis as part of a hybrid release scheme.
Organize field days for public awareness and participatory evaluation of the
hybrids.
Select best bets and undertake rapid multiplication for distribution to contact
farmers.
2. Integrated crop management Superimpose to new hybrids a set of recommended cultural practices to sustain
yield. Hybrid demonstrations plots will be divided into two subplots, a
well-managed subplot [Do’s] and a poorly managed subplot [Don’ts].
Set up additional demonstration plots on pest management (focus on
nematodes) to prolong plantation lifespan.
Make provision for monitoring and preemptive management of pests
(nematodes) and pathogen (Sigatoka, emerging threats) population shifts.
3. Training (capacity building) At least two intensive training sessions on plantain management and sucker
multiplication/sanitation will be encouraged at each PRTC with a well-
coordinated calendar. The training is to be decentralized so that the ADP can
handle it with backstopping from IITA based on needs.
Training curricula, supported by pictorial booklets, will be disaggregated into
component modules that can be handled separately or concomitantly based on
prospective trainees’ interests.
Posters will be developed to capture in a glance key messages and technology
descriptions (hybrid characteristics, multiplication techniques, postharvest
processing).
4. Governance (resource center) Ideally, each PRTC should have a governing or advisory council that will
oversee the operations of the PRTC. The council should be chaired by the
chief executive officer of the host ADP and include one representative from
each of the states in the operating zones, one representative each of
stakeholders in production and processing, one representative of NIHORT,
one representative of PBDP, with IITA participating as an observer.
Address policy issues with respect to seed certification systems, postharvest quality
control (processing, packaging, mycotoxins).
5. Impact assessment of phase I To be carried out by an independent body in order to assess the delivery model
(partnerships, capacity building) and technologies (hybrids, rapid
multiplication, postharvest) of HDP-I.
ensure the collective appropriation of project interventions by all parties involved. With a greater
understanding of the directions the project is heading to and the way this is done, there is a greater
commitment to contributing resources and implementing interventions by all parties.
16.2.2.3 Preliminary Assessment of Impact

Aitchedji et al. (unpublished) attempted to assess preliminary changes associated with the dissemina-
tion project. They concluded that farmers’ capacity to choose and use planting materials and related
production techniques has been improved by training programs over the 2000–2004 period of the
dissemination project. The curricula for training included: information on hybrids, crop management
practices, pests and diseases management technologies, sucker multiplication, and postharvest and
food processing techniques. Also farmer’s awareness has been increased with field days and exchange
of information on hybrids and associated techniques. All participants reported that the capacity-build-
ing curricula were adapted to their needs and opportunities. More than half of the farmers were aware
of and had adopted disease-resistant plantain and banana hybrids at least once by 2005.
Most of the adopters grew both local cultivars and hybrids in sole cropping and/or intercropping.
The main reasons for adoption evoked by farmers were (1) good/high yield, (2) good taste and high
quality, (3) planting material (suckers) availability and accessibility, (4) good sale (high market
demand), (5) resistant to pest and disease (black Sigatoka), and (6) early maturity of plant and fruit.
The main reason reported for mixing hybrids and local cultivars is to control black Sigatoka and to
strengthen resistance of local varieties. Pioneer farmers were key sources of information and sup-
pliers of suckers for other farmers in rural areas.
Farmers’ awareness and knowledge of disease-resistant hybrids through participation in on-
farm trials and/or field days and demonstration plots are important factors in adoption. In addition,
farmers who participated in the project’s training programs adopted the hybrids because of their
participation and benefits gained from their attendance. The project’s collaboration with ADPs for
organization of annual training programs on different aspects of plantain and banana contributed to
the dissemination of hybrids. The attendance to project training programs on plantain and banana
contributed to the adoption of plantain and banana hybrids by small-scale farmers.
The Nigeria experience has since inspired similar large-scale delivery of improved hybrids across
Africa, notably in Cameroon, Ghana, Rwanda, and Uganda. It would be informative to document
the economic and social benefits of these initiatives.
16.3 Understanding and Managing Diversity

Farmers usually grow a number of cultivars that they maintain perennially through successive crops
derived from suckers (shoots). Suckers constitute the planting material of choice for farmers, which
they acquire on an ad hoc basis from neighbors or rural markets, using local names as guides for
purchasing the suckers.
It is difficult to distinguish among cultivars on the basis of vegetative characteristics alone, par-
ticularly in juvenile plants. Even when planting materials are obtained from mature plants, there is
still a high probability of cultivar mixtures, because homonyms might actually be different culti-
vars. This may result in intrafield diversity that may be unintended (since homonymous suckers may
not be identical cultivars) or deliberate (because intrafield diversity may be viewed as risk manage-
ment strategy) as outlined by Smithson and Lenne (1996) and Kahmen et al. (2005).
Managing diversity in farmers’ fields requires a good understanding of the pattern of variation
among popular cultivars, particularly in juvenile plants. This in turn determines to what extent plant
breeding may succeed in releasing new cultivars into existing production schemes, as indicated in
Chapter 9. Selatsa et al. (2009) provide a comprehensive discussion of this matter.
16.4 Banana Trade Prospects for Africa

16.4.1 Trends in Commercial Banana Production in Sub-Saharan Africa
This section is largely drawn from the comprehensive analysis of Arias et al. (2003) of the United
Nations Food and Agriculture Organization (FAO). Since the 1960s, banana production in Africa
has increased at about 2.2% per annum, while the area harvested has grown at a slightly reduced
pace of 1.7%. In the same period land productivity has increased slowly, at less than 1% per annum.
However, yield has not been the same in all countries.
Export-oriented banana producers use a relatively high supply of external inputs. Côte d’Ivoire
and Cameroon are the most important banana-exporting countries, and their markets are in Europe
and, to a lesser extent, neighboring Burkina Faso, Mali, and Senegal. Banana exports from Côte
d’Ivoire and Cameroon, which in 2001 accounted for 98% of all banana exports from Africa, have
increased in the period between 1987 and 2000 at a sustained rate of 10% per annum. This contrasts
sharply with the steady decline in banana exports in the previous period (1960–1986) when exports
decreased by 2% per annum.
In Cameroon, banana exports had declined steadily since the early 1960s from some 140,000 in
1961 to little more than 20,000 in 1987. Exports recovered during the 1970s with the support of the
Organisation Camerounaise de la Banane (OCB) that helped farmers to fight Panama disease.
However, Sigatoka and weather-related problems affected production during the 1980s and
exports declined to a record low in 1987. In 1987 foreign companies began to take a leading role in
the development of banana for export. In that year Del Monte engaged in a joint venture with the
Cameroon Development Corporation (CDC). Today CDC and Del Monte plant some 2,100 ha of
bananas: CDC provides land and labor, while Del Monte provides credit and technical assistance,
and markets the produce. CDC is currently the largest employer after the state, and the government
allows the company tax exemptions for the importation of banana production inputs.
In 1990, the Organisation Camerounaise de la Banane (OCB) was purchased by the Compagnie
Fruitière, the retail company in charge of selling OCB produce in Europe. At present, Compagnie
Fruitière is controlled by Dole. Both Dole and Del Monte made large investments into irrigation,
fruit-handling facilities, and sanitation equipment. Today bananas in Cameroon provide direct
employment to some 10,000 people in rural areas and exports in 2002 reached almost 260,000,
having grown at a rate of 10% per annum since 1988. Of these, an estimated 215,000 went to the
European Union and the rest to Eastern Europe, North Africa, and neighboring African countries.
Bananas in Côte d’Ivoire are, together with pineapples, the most important exported fruits. Bananas
for export are produced by farms of different sizes and under different production techniques, from
small farms using low-input technologies to large and input-intensive plantations. However, smaller
farms, which are usually located in poor soils with steep slopes and limited water availability, are
finding it increasingly difficult to compete and are slowly disappearing. The increase in banana pro-
duction in recent years (5.4% per annum in the period 1987–2001) was accompanied by an increase
in the scale of production, which in turn was caused by the closing down of smaller farms.
Increases in production were accompanied by an expansion of exports in a pattern that resembles
that of Cameroon. Following a period of steady decline from the mid 1970s to the mid 1980s, the
period between 1987 and 2000 saw a sustained growth in exports of 9% per annum, from 82,000 in
1987 to 240,000 t in the year 2000. The increase coincides with a stronger participation of multina-
tional companies and the dissolution by the government of the Cooperative de Producteurs pour la
Commercialization des Fruits et Légumes de la Côte d’Ivoire (COFRUITEL) in 1985, an organiza-
tion that had since 1978 grouped banana producers and was in charge of marketing.
Nowadays banana production for export consists of an integrated system whereby a few large
operators specify the production technology to be applied and market the produce internationally.
The government is no longer involved in production, and its role is limited to monitoring the phy-
tosanitary aspects of the exported fruit. Almost 80% of all exports go to the European Union, and
the rest to North Africa and the Middle East. The bulk of bananas is produced in 65 plantations
covering an area of approximately 5,500 ha and provide employment to about 20,000 people.
Producers are grouped in the Organisation Centrale des Producteurs Exportateurs d’Ananas
et de Banane (OCAB), created in 1991. The largest export companies are the Société pour le
Développement de la Culture de la Banane (SCB), a subsidiary of Dole with a share of about 50%
of all banana exports; Banador, a subsidiary of Chiquita with a share of 25%; and Canavèse, with a
share of 10%.
Currently, major efforts are underway to restore the banana export potential of Guinea—targeting
countries of the Middle East—with support from the Common Fund for Commodities (CFC) of the
World Bank and from private investors from the Arab Peninsula. It is noteworthy that land areas
cultivated with banana and plantain have increased by about 50% over the past 12 years, with a
largely untapped potential in the major production zones of Guinée Maritime (coastal Guinea) and
Guinée Forestière (bordering Sierra Leone, Liberia, and Côte d’Ivoire).
Finally, some export-oriented banana production occurs in the Volta region of Ghana, with a
specific emphasis on fair-trade to target customers in the European Union.
16.4.2 Strategic Implications

Most of banana production is carried out by small-scale farmers in most of the countries in sub-
Saharan Africa, including those that have developed an export-oriented production. However, the
small-scale farmers are hardly part of the export chain. Rather, they supply domestic or regional
markets.
Export-oriented production is carried out by a small number of entrepreneurs operating large-
scale farms with high-input technology and in close partnership with transnational trade companies.
Therefore, it is advised that the development of a fresh produce banana-based economy should fol-
low a dual approach, focusing on:
• Promoting the development of large-scale farms with significant investment support. This
could be done through the promulgation of conducive policies that attract foreign investors
or incite domestic investors towards banana production and commercialization.
• Promoting the organization of small-scale farmers in cooperative-like institutions. These
institutions will benefit from investment support for access to improved production tech-
nologies and serve as brokers for access to international export markets.
It is advised that the domestic and regional markets should also be targeted, particularly in view of
the lower transportation costs, and the availability of numerous value-adding postharvest process-
ing options that could (1) rescue from wastage fruits that are rejected for international export and (2)
subtend the development of an agro-processing industry.
While international trade opportunities should focus on Cavendish varieties (for example,
‘Dwarf Cavendish,’ ‘Giant Cavendish,’ ‘Williams,’ and ‘Grande Naine’), there is also ample scope
for non-Cavendish varieties of the plantain and cooking banana types, particularly for domestic
and regional markets. Only about 10% of world banana production is traded at the international
level. The share of regional trade is not well documented, but it is known that the consumption of
banana and plantain products is well entrenched in the dietary habits of the people, and demand
for the products is high even in countries lacking the climatic conditions for the production of
these crops.
While this section used examples from West and Central Africa, the trends are the same and
the implications similar for other regions of sub-Saharan Africa where the major players currently
are South Africa, Kenya, and Uganda, but other countries like the Democratic Republic of Congo,
Ethiopia, and Mauritius are also potential strongholds for banana production and trade.
16.5 International Distribution of Hybrids

During the past few decades of plantain and banana research, progress has been made in the
area of host-plant resistance to black Sigatoka, through the development of improved hybrids and
populations with Sigatoka resistance (Tenkouano and Swennen, 2004). The development of these
disease-resistant and high-yielding plantain and banana hybrids constitutes a most significant
scientific achievement in recent history in terms of the impact they could have on the alleviation
of hunger in Africa. What is now needed is to introduce these hybrids into farmers’ fields in size-
able quantities.
There are only a handful of active breeding programs across the world, yet bananas are grown
in more than 130 countries. Therefore, international cooperation to facilitate access to improved
varieties has been a commendable achievement of the banana research community through the
International Network for Improvement of Banana and Plantain (INIBAP; see Chapter 9). Thus, an
international transit center (ITC) was set up in a non-banana-producing country (Belgium) to receive,
sanitize (when required), and multiply for distribution genetic stocks from all over the world.
Virtually all agencies engaged in genetic improvement through breeding, mutation, and selection
have deposited specimen of their products at the ITC (Table 16.2). Lacking in this repository are
products from the breeding programs operating in India. Also lacking are cooking banana variet-
ies recently derived from East African Highland bananas by the breeding program jointly operated
by IITA and the National Agricultural Research Organization (NARO) in Uganda and new dessert
banana varieties developed by French Agricultural Research Centre for International Development
(CIRAD) for commercial production in the French Caribbean islands.
There has been no restriction in distributing the available accessions or breeding products
across the world for production or research purposes as long as normal precautionary measures to
ensure that no biological threats (notably, viruses) are accidentally disseminated through infected
plant materials. Through the various mechanisms of INIBAP (now Commodities for Livelihoods,
Bioversity), such as the International Musa Testing Program (IMTP), new varieties were channeled
to an impressive list of recipients across the word (Table 16.3).
16.6 Conclusion
The success of plant breeding programs is measured by the extent to which the breeding products
are adopted and used by the growers, profitably and durably. Beyond the genetic products, perhaps
the more challenging task for the breeders is to understand and help establish the complex battery
of institutional and transactional measures that create a conducive delivery framework.
Access to improved hybrids is extremely important. Therefore, the willingness of breeding pro-
grams to place their products under custody of a reputable international conduit that helps to main-
tain the intellectual property of the contributing programs is commendable. It is expected that the
influx of improved hybrids into the ITC and efflux from the ITC would be asymmetrical, given the
small number of active breeding programs and the large number of institutions needing improved
hybrids for research or commercial use. Intellectual property issues may also be responsible for the
asymmetrical influx/efflux noted in some cases where countries drawing from the ITC have not yet
contributed to the ITC repository, despite having active breeding programs with tangible products.
Once the asymmetry is removed and improved hybrids meet the biological requirements for safe
international exchange, producers throughout the world would have increased access to a pool of
hybrids that would meet their production needs and those of the markets they serve.
Table 16.2
Accessions and Breeding Stocks Available for International Distribution at the
International Transit Center (ITC) of the International Network for Improvement of
Banana and Plantain (INIBAP)
Contributing Breeding Programme Accession or Breeding Stock Designation
Banana board/Imperial College of Tropical I.C. 2 (ITC0019), Bodles Altafort (ITC0366), 2390 (ITC0367), 2390-2
Agriculture (ICTA) (ITC0553), Calypso (ITC0555), TU8 (ITC0581), Buccaneer
(ITC1248), B7925 (ITC1320)
Centre Africain de Recherches sur les CRBP 01 (ITC1337), CRBP 14 (ITC1338), CRBP 15 (ITC1339),
Bananiers et Plantains (CARBAP) CRBP 37 (ITC1342), CRBP 39 (ITC1344)
Centre de Coopération Internationale en IRFA 904 (ITC1266), IRFA 905 (ITC1267), IRFA 908 (ITC1268)
Recherche Agronomique pour le
Développement (CIRAD)
Empresa Brasileira de Pesquisa Agropecuaria Diploide EMBRAPA 205 (ITC1193), Tetraploide EMBRAPA 401
(EMBRAPA) (ITC1194), PC12-05 (ITC1260), PA03-22 (ITC1261), PV03-44
(ITC1262), JV03-15 (ITC1263), PA 12.03 (ITC1302), PV 42-53
(ITC1310), PV 42-81 (ITC1312), PV 42-320 (ITC1313), JV 42-41
(ITC1314)
Fundación Hondureña de Investigación SH-3142 (ITC0425), Balbisiana tetraploide (ITC0454), FHIA-21
Agricólà (FHIA) (ITC0503), FHIA-01 (ITC0504), FHIA-02 (ITC0505), FHIA-03
(ITC0506), FHIA-04 (ITC0968), FHIA-17 (ITC1264), FHIA-23
(ITC1265), SH-3640 (ITC1307), FHIA-18 (ITC1319), SH-3751
(ITC1324), FHIA-21 (ITC1332), FHIA-18 (ITC1412), FHIA-25
(ITC1418)
International Atomic Energy Agency (IAEA) GN 60A (ITC1328), Novaria (ITC1329)
International Institute of Tropical Agriculture PITA-1 (ITC1081), TMPx 2481 (ITC1093), PITA-4 (ITC1094), PITA-9
(IITA) (ITC1110), TMBx 612-74 (ITC1119), PITA-5 (ITC1141), PITA-7
(ITC1195), PITA-11 (ITC1196), PITA-6 (ITC1198), TMPx 4744-1
(ITC1199), PITA-3 (ITC1200), TMPx 5706-1 (ITC1201), PITA-12
(ITC1203), TMPx 7356-1 (ITC1204), PITA-10 (ITC1205), T6
(ITC1247), PITA-8 (ITC1272), TMP2x 1297-3 (ITC1278), TMP2x
1297-3 (ITC1292), TMPx 4479-1 (ITC1293), PITA-14 (ITC1294),
BITA-2 (ITC1296), BITA-3 (ITC1297), TMP2x 1297-3 (ITC1415),
TM2x 2829-62 (ITC1416), PITA-16 (ITC1417), TMB2x 9128-3
(ITC1437)
Instituto Nacional de Investigaciones de IBP 5–61 (ITC1478), IBP 5-B (ITC1479), IBP 12 (ITC1480),
Viandas Tropicales (INIVIT) SH-3436-9 (ITC1283), SH-3436-6 (ITC1284), SH-3436–9 (ITC1318)
Taiwan Banana Research Institute (TBRI) ‘GCTCV-215’ (ITC1271), ‘GCTCV-119’ (ITC1282), ‘GCTCV-215’
(ITC1436), ‘GCTCV-106’ (ITC1442), ‘GCTCV-247’ (ITC1443)
Note: INIBAP is now a program of Bioversity International. Accessions codes are followed by ITC reference numbers
in parentheses.
Source: Data courtesy of Ines Vandenhouwe (Bioversity International, ITC manager).
Table 16.3
Distribution of Improved Varieties of Bananas from the International Transit Center (ITC)
of the International Network for Improvement of Banana and Plantain (INIBAP)
Accession
Breeding Program Number Recipient Countries
Banana Board/ICTA ITC0019 Belgium, Burundi, DR Congo, Germany, Guadeloupe, India, Nigeria,
Tanzania, Uganda, Zimbabwe
ITC0366 Costa Rica, Ethiopia, Guadeloupe, Reunion, United Kingdom
ITC0367 India
ITC0553 Costa Rica, DR Congo, Guadeloupe, India, Marshall Islands, Philippines,
United States
ITC0555 Burundi, Costa Rica, India, Philippines, Reunion, United Kingdom
ITC0581 Kenya, United Kingdom, Zimbabwe
ITC1248 Costa Rica, Guadeloupe, India, Reunion, United Kingdom
ITC1320 Bangladesh, Ecuador, India
CARBAP ITC1342 Cameroon, China, Costa Rica
ITC1344 Belgium, Burundi, Cambodia, Cameroon, China, Colombia, Costa Rica, Côte
d’Ivoire, Cuba, DR Congo, Dominican Republic, Ecuador, France, French
Polynesia, Ghana, Guyana, Haiti, Honduras, India, Indonesia, Jamaica,
Liberia, Malaysia, Marshall Islands, Martinique, Mauritius, Mexico,
Myanmar, Nicaragua, Nigeria, Pakistan, Panama, Papua New Guinea, Peru,
Philippines, Puerto Rico, South Africa, Sri Lanka, Taiwan, Tanzania,
Thailand, Uganda, United States, Venezuela
CIRAD ITC1267 India
ITC1268 France, Guadeloupe, Vietnam
EMBRAPA ITC1194 Guadeloupe, Vietnam
ITC1260 Guadeloupe, India, United States
ITC1261 Belgium, Brazil, Burundi, Cameroon, Colombia, Costa Rica, Cuba, Fiji
Islands, Guadeloupe, Honduras, India, Indonesia, Malaysia, Mexico, Nigeria,
Philippines, Puerto Rico, Saint Lucia, South Africa, Spain, Taiwan, Thailand,
Tonga, Uganda, United States
ITC1262 Belgium, Brazil, Burundi, Cameroon, Colombia, Costa Rica, Cuba,
Dominican Republic, Guadeloupe, Honduras, India, Indonesia, Malaysia,
Mexico, Nigeria, Philippines, Puerto Rico, Saint Lucia, South Africa, Spain,
Taiwan, Thailand, Tonga, Uganda, United States
ITC1263 United States
ITC1302 Australia, Cameroon, Costa Rica, Fiji Islands, Mexico, Puerto Rico, Spain,
United States
ITC1310 Australia, Bangladesh, Cameroon, Costa Rica, Guadeloupe, Malaysia,
Rwanda, Spain, Uganda, United States
ITC1312 Australia, Bangladesh, Cameroon, Costa Rica, Guadeloupe, Puerto Rico,
Spain
ITC1313 Australia, Bangladesh, Cameroon, Costa Rica, DR Congo, Guadeloupe, India,
Puerto Rico, Spain, United States
ITC1314 Australia, Bangladesh, Cameroon, Costa Rica, Dominican Republic,
Guadeloupe, Spain, United States
FHIA ITC0425 Belgium, Guadeloupe, Venezuela
ITC0454 Germany, India, Philippines
ITC0503 Burundi, United Kingdom
(continued)
Accession
ITC0504 Australia, Bangladesh, Barbados, Belgium, Bolivia, Brazil, Burundi,
Cambodia, Cameroon, China, Colombia, Comoros, Congo, Republic of,
Costa Rica, Cuba, Cyprus, DR Congo, Dominican Republic, Ecuador,
Eritrea, Fiji Islands, France, French Polynesia, Gabon, Gambia, Germany
ITC0505 American Samoa, Australia, Bangladesh, Barbados, Belgium, Bolivia, Brazil,
Burundi, Cambodia, Cameroon, China, Colombia, Comoros, Congo,
Republic of, Costa Rica, DR Congo, Dominican Republic, Ecuador, Eritrea,
Fiji Islands, France, French Polynesia, Gabon, Gambia, Germany,
Guadeloupe, Guinea, Haiti, Honduras, India, Indonesia, Ireland, Jamaica,
Kenya, Malawi
ITC0506 American Samoa, Australia, Bangladesh, Barbados, Belgium, Bolivia, Brazil,
Burundi, Cambodia, Cameroon, China, Colombia, Comoros, Congo,
Republic of, Costa Rica, Cuba, DR Congo, Dominican Republic, Ecuador,
Eritrea, Fiji Islands, France, French Polynesia, Gabon, Gambia, Germany,
Ghana, Guadeloupe, Guinea, Guyana, Haiti, Honduras, India, Indonesia
ITC0968 Burundi, Cameroon, Colombia, Costa Rica, Honduras, Nigeria, United
Kingdom, United States
ITC1264 American Samoa, Australia, Bangladesh, Belgium, Bolivia, Brazil, Burundi,
Cambodia, Cameroon, China, Colombia, Comoros, Costa Rica, Côte d’Ivoire,
Cuba, DR Congo, Dominican Republic, Ecuador, Fiji Islands, France, Gambia,
Ghana, Guadeloupe, Honduras, India, Indonesia, Jamaica, Jordan, Malawi,
Malaysia, Mauritius, Mexico, Myanmar, Nicaragua, Nigeria, Oman, Pakistan,
Palau, Papua New Guinea, Peru, Philippines, Reunion, Seychelles, South
Africa
ITC1265 American Samoa, Bangladesh, Bolivia, Brazil, Burundi, Cambodia, Cameroon,
China, Colombia, Comoros, Costa Rica, Côte d’Ivoire, Cuba, DR Congo,
Dominican Republic, Ecuador, Eritrea, France, French Polynesia, Gambia,
Germany, Ghana, Guadeloupe, Honduras, India, Indonesia, Ireland, Jamaica,
Jordan, Madagascar, Malawi, Malaysia, Mauritius
ITC1307 American Samoa, Belgium, Burundi, Cambodia, Cameroon, China, Colombia,
Costa Rica, DR Congo, Dominican Republic, Ecuador, Fiji Islands, France,
Guadeloupe, India, Indonesia, Jamaica, Malawi, Malaysia, Mauritius,
Myanmar, Nicaragua, Oman, Pakistan, Papua New Guinea, Peru, Philippines,
Puerto Rico, Reunion, South Africa, Spain, Sri Lanka, Suriname, Taiwan,
Tanzania, Thailand, Uganda, United Kingdom
ITC1319 American Samoa, Bangladesh, Belgium, Bolivia, Burundi, Cambodia,
Cameroon, China, Colombia, Costa Rica, Côte d’Ivoire, Cuba, DR Congo,
Dominican Republic, Ecuador, Fiji Islands, France, French Polynesia,
Gambia, Germany, Ghana, Guadeloupe, Haiti, India, Indonesia, Malawi,
Malaysia, Martinique, Mauritius, Myanmar, Nicaragua, Nigeria, Oman,
Pakistan, Panama, Papua New Guinea, Peru, Philippines, Puerto Rico,
Reunion, Samoa, South Africa, Spain, Sri Lanka, Taiwan, Tanzania, Thailand,
Trinidad and Tobago, Uganda, Venezuela, Vietnam
ITC1324 Cameroon, France
(continued)
Accession
ITC1332 American Samoa, Australia, Bangladesh, Belgium, Bolivia, Burundi,
Cambodia, Cameroon, China, Colombia, Costa Rica, Côte d’Ivoire, DR
Congo, Dominican Republic, Ecuador, Fiji Islands, France, Gambia, Ghana,
Haiti, Honduras, India, Indonesia, Israel, Jamaica, Malawi, Malaysia,
Mauritius, Mexico, Myanmar, Nicaragua, Nigeria, Oman, Pakistan, Papua
New Guinea, Peru, Philippines, Puerto Rico, Reunion, South Africa, Sri
Lanka, Suriname, Taiwan, Tanzania, Thailand, Trinidad and Tobago, Uganda,
United Kingdom, Venezuela
ITC1350 Australia
ITC1412 China, Comoros, DR Congo, Ecuador, India, Mauritius, Mexico
ITC1418 American Samoa, Bangladesh, Belgium, Burundi, Cambodia, Cameroon,
China, Colombia, Comoros, Costa Rica, Côte d’Ivoire, Cuba, DR Congo,
Ecuador, Fiji Islands, Gambia, Germany, Ghana, Guyana, India, Indonesia,
Jamaica, Jordan, Malawi, Malaysia, Mauritius, Mexico, Myanmar, Nicaragua,
Oman, Pakistan, Papua New Guinea, Peru, Philippines, Puerto Rico, Reunion,
South Africa, Sri Lanka, Suriname, Taiwan, Tanzania, Thailand, Uganda,
Venezuela, Vietnam
IAEA ITC1328 Austria, Colombia, Costa Rica, Israel, United States
IITA ITC1082 United Kingdom, United States
ITC1205 Belgium, Brazil, Cameroon, Colombia, Costa Rica, Cuba, DR Congo,
Dominican Republic, Ecuador, Germany, Ghana, Honduras, India, Israel,
Jamaica, Mauritius, Nicaragua, Nigeria, Puerto Rico, United States
ITC1247 Burundi, Costa Rica, Ecuador, Fiji Islands, Guadeloupe, India, Papua New
Guinea, Reunion, Uganda, United Kingdom
Dominican Republic, Ecuador, Ghana, Honduras, India, Jamaica, Nicaragua,
Nigeria, Uganda, United Kingdom
ITC1278 India, Jamaica, Malaysia, Mauritius, Uganda, Venezuela
Dominican Republic, Ecuador, Germany, Ghana, Honduras, India, Israel,
Jamaica, Mauritius, Nicaragua, Nigeria, Puerto Rico, Uganda, United States
Dominican Republic, Ecuador, Ghana, Honduras, India, Jamaica, Mauritius,
Nicaragua, Nigeria, Puerto Rico, Uganda, United Kingdom, United States
ITC1296 Australia, Belgium, Brazil, Burundi, Cambodia, Cameroon, China, Costa Rica,
Cuba, DR Congo, Dominican Republic, Ecuador, Ghana, Guadeloupe, India,
Indonesia, Jamaica, Madagascar, Malawi, Malaysia, Marshall Islands,
Mauritius, Mexico, Myanmar, Nicaragua, Nigeria, Pakistan, Papua New
Guinea, Philippines, Puerto Rico, Puerto Rico, South Africa, Spain, Sri
Lanka, Taiwan, Tanzania, Thailand, Uganda, United Kingdom, United States
(continued)
Accession
ITC1297 Australia, Belgium, Brazil, Burundi, Cambodia, Cameroon, China, Colombia,
Comoros, Costa Rica, Côte d’Ivoire, Cuba, DR Congo, Dominican Republic,
Ecuador, Ghana, Guadeloupe, India, Indonesia, Jamaica, Madagascar,
Malawi, Malaysia, Marshall Islands, Mauritius, Mexico, Myanmar,
Nicaragua, Nigeria, Pakistan, Panama, Papua New Guinea, Philippines,
Puerto Rico, South Africa, Spain, Sri Lanka, Taiwan, Tanzania, Thailand,
Uganda, United States
ITC1415 India
ITC1416 Dominican Republic, India, Uganda, Venezuela
ITC1417 Australia, Colombia, Costa Rica, DR Congo, Dominican Republic, Ecuador,
Honduras, India, Madagascar, Oman, Pakistan, Peru, Philippines, Puerto
Rico, South Africa, Uganda, Venezuela, Vietnam
ITC1437 China, Dominican Republic, India, Malaysia, South Africa, Venezuela,
Vietnam
INIVIT ITC1283 Austria, Bangladesh, Burundi, Cambodia, Cameroon, China, Colombia, Costa
Rica, Cuba, DR Congo, Dominican Republic, Ecuador, France, Guadeloupe,
Honduras, India, Indonesia, Jamaica, Malaysia, Myanmar, Nigeria, Oman,
Pakistan, Papua New Guinea, Peru, Philippines, Saint Lucia, South Africa,
Spain, Sri Lanka, Taiwan, Tanzania, Thailand, Tonga, Uganda, Zimbabwe
ITC1284 Mexico
ITC1318 Burundi, Venezuela
TBRI ITC1271 Bangladesh, Belgium, Brazil, Burundi, Cameroon, Costa Rica, Cuba,
Dominican Republic, Ecuador, France, Guadeloupe, Honduras, India,
Indonesia, Indonesia, Israel, Madagascar, Malaysia, Mauritius, Mexico,
Pakistan, Philippines, South Africa, Spain, Taiwan, Thailand, Uganda
ITC1282 Bangladesh, Bangladesh, Belgium, Brazil, Burundi, Cambodia, Cameroon,
China, Costa Rica, Cuba, Dominican Republic, Ecuador, France,
Guadeloupe, Honduras, India, Indonesia, Israel, Madagascar, Malaysia,
Mauritius, Mexico, Myanmar, Pakistan, Papua New Guinea, Philippines,
Puerto Rico, South Africa, Spain, Sri Lanka, Taiwan, Thailand, Uganda
ITC1436 Australia
ITC1442 Australia, Bangladesh, Belgium, Cambodia, China, Colombia, India,
Indonesia, Malaysia, Mauritius, Pakistan, Papua New Guinea, Philippines,
Thailand, Vietnam
ITC1443 Australia, Bangladesh, Belgium, Cambodia, China, India, Indonesia, Malaysia,
Mauritius, Myanmar, Oman, Pakistan, Papua New Guinea, Philippines,
Thailand, Vietnam
Source: Data courtesy of Ines Vandenhouwe (Bioversity International, ITC manager).

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17 Molecular Breeding of Other
Vegetatively Propagated Crops
Lessons for Banana
Michael Pillay, Abdou Tenkouano, and Rodomiro Ortiz
Contents
17.1 Introduction........................................................................................................................... 322
17.2 Economic Importance of the Crops....................................................................................... 323
17.2.1 Potato......................................................................................................................... 323
17.2.2 Cassava...................................................................................................................... 323
17.2.3 Sugarcane................................................................................................................... 323
17.3 Breeding Challenges.............................................................................................................. 324
17.3.1 Potato......................................................................................................................... 324
17.3.2 Cassava...................................................................................................................... 324
17.3.3 Sugarcane................................................................................................................... 325
17.4 Production Constraints.......................................................................................................... 325
17.4.1 Potato......................................................................................................................... 325
17.4.2 Cassava...................................................................................................................... 325
17.4.3 Sugarcane................................................................................................................... 326
17.5 Breeding Objectives............................................................................................................... 326
17.5.1 Potato......................................................................................................................... 327
17.5.2 Cassava...................................................................................................................... 327
17.5.3 Sugarcane................................................................................................................... 327
17.6 Conventional versus Molecular Breeding.............................................................................. 328
17.6.1 Molecular Markers.................................................................................................... 329
17.6.1.1 Molecular Markers in Potato...................................................................... 330
17.6.1.2 Molecular Markers in Cassava................................................................... 330
17.6.1.3 Molecular Markers in Sugarcane................................................................ 330
17.6.2 Molecular Maps......................................................................................................... 331
17.6.2.1 Potato.......................................................................................................... 332
17.6.2.2 Cassava........................................................................................................ 332
17.6.2.3 Sugarcane.................................................................................................... 332
17.7 Genetic Transformation......................................................................................................... 333
17.7.1 Achievements and Prospects of Molecular Breeding in Potato, Cassava, and
Sugarcane................................................................................................................... 333
17.7.1.1 Pest Resistance in Potato............................................................................ 333
17.7.1.2 Virus Resistance......................................................................................... 334
17.7.1.3 Abiotic Stress Tolerance in Potato.............................................................. 334
17.7.1.4 Improvement in Nutritional Qualities......................................................... 335
321
17.7.2 Vegetatively Propagated Plants as Biofactories......................................................... 336

17.7.2.1 Sugarcane.................................................................................................... 336
17.7.2.2 Production of Dextran in Transgenic Potato............................................... 337
17.7.2.3 Improved Cassava Starch............................................................................ 337
17.7.3 Risks Associated with Transgenic Crops.................................................................. 337
17.7.3.1 Risk of Transgenic Escape.......................................................................... 338
17.7.3.2 Risks to Other Organisms........................................................................... 338
17.7.3.3 Safety of Biotech Foods.............................................................................. 339
17.7.4 Marker-Free Transgenic Plants..................................................................................340
17.8 Conclusions............................................................................................................................ 341
References....................................................................................................................................... 342
17.1 Introduction
Since the beginning of agriculture about 10,000 years ago, there was a concomitant increase in food
supply with growth in human population. Increases in food production were ascribed to availabil-
ity of new agricultural land for greater food production as well as increased yields of crops due to
breeding. In the past 50 years, increase in food production has matched and outstripped increases
in population growth (Chrispeels and Sadava, 2003). There is uncertainty whether this trend will
continue. The human population is expected to reach 7 billion soon. At the same time agricultural
production is growing at a slower rate of about 1.8% annually (Altman, 1999). The largest increases
in population growth occur in poorer countries where many vegetatively propagated crops play an
important role in maintaining food security and providing income. Countries that fall in this cat-
egory are primarily within the tropical and subtropical regions of Africa, Asia, and Latin America.
Some of the major vegetatively propagated staple foods in these countries include banana, cassava,
potato, sweet potato, and yam. Currently the production of improved cultivars that are nutritionally
acceptable to consumers, with resistance or tolerance to biotic and abiotic stresses, and reduced post-
harvest losses, have been met largely through conventional breeding. The pressure of an increasing
population and consequent increase in demand for food on the one hand and the depletion of arable
land on the other have placed new emphases on conventional plant breeding. However, conventional
breeding—especially of vegetatively propagated crops—poses many challenges.
The rapid development of molecular biology techniques and their application to plant breeding
have resulted in significant genetic gains in agricultural crops, some of which have already entered
the market (James and Krattiger, 1996). Conventional breeding of new improved cultivars heavily
depends on choosing the right plant parents. Breeders typically make a large number of “parent
crosses” of which only a select few eventually result in marketable cultivars. Following through
on each cross is a long process, typically taking 12 to 15 years, and there are no assurances of suc-
cess. Molecular breeding is simply a form of conventional breeding that allows breeders to have a
better understanding of the parents that go into crosses. It is a specialized area of plant science that
relies on collecting genetic information about plants. Molecular breeding may or may not involve
genetic engineering or the development of genetically modified (GM) crops. Two of the basic tools
of molecular breeding are the development of molecular markers that are linked to traits and the
construction of molecular maps.
There have been many attempts at boosting banana breeding efficiency through the use of molec-
ular genetics, but very little progress has been made to date in using molecular tools for selection of
individuals with useful traits of economic importance. Molecular tools have proven very valuable
in genetic and genomic diversity analyses, but breeding applications have been relatively scarce,
with the exception of inferring parent–offspring relationships or ascertaining the dihybrid nature
of progenies from crosses. That molecular breeding has not become routine for banana breeding
is likely due to the complex biology of the species, including polyploidy, heterzygosity, multiple
Molecular Breeding of Other Vegetatively Propagated Crops 323
genomes, and vegetative propagation. In this regard, a study of what has been achieved in other spe-
cies with similar biological characteristics could prove useful for banana breeders of the future.
This chapter examines some aspects of molecular breeding in three major vegetatively propagated
crops: potato, cassava, and sugarcane. It is not the authors’ intention to provide a detailed analysis of
all aspects of molecular breeding of these crops but to examine some key aspects in which molecu-
lar breeding can make useful contributions in the development of new improved cultivars.
17.2 Economic Importance of the Crops

17.2.1 Potato
The potato (Solanum tuberosum L.) is the third major world food crop, exceeded only by wheat
and rice in terms of world production for human consumption (Gebhardt and Valkonen, 2001).
The high yield and low cost of cultivation makes potatoes an important part of the diet in many
cultures. Potato production was recorded as 309 million tons in 2007 (http://www.scri.ac.uk/news/
pgsc, accessed 8 April 2010). It is estimated that by 2020 more than 2 billion people will depend on
potato for food, feed, or income. Potatoes are adapted to grow in a wide range of agro-ecologies and
altitudes and are grown in over 149 countries from latitudes 65° N to 50° S and at altitudes from sea
level to 4,000 m (Hijmans, 2001). Potatoes have been adapted to a wide range of end uses. As well
as being a staple food, the potato is grown as a vegetable for table use, is processed into French fries
and crisps (chips), and is used for dried products and starch production. New uses can be anticipated
for the future such as designer starches (Davies, 1998) and as bioreactors for biopharmaceuticals
(Sonnewald et al., 2003).
17.2.2 Cassava
Cassava (Manihot esculenta Crantz.) is one of the major sources of carbohydrates for more than 600
million in sub-Saharan Africa, Latin America, and Asia. It is the fourth most important tropical
crop worldwide with a production of 162 million metric tons per year. The per capita consumption
of cassava in Africa is approximately 80 kg/capita and is double that number in Central America.
Cassava is drought tolerant, grows well in poor and degraded soils, and the roots can persist in the
soil for 1 or 2 years without decay. This secures rural farmers a carbohydrate source in years with
adverse growth conditions when other crops fail and famine would otherwise prevail (Jorgensen et
al., 2005). Although cassava is cultivated mainly for its roots, the leaves are also consumed by many
African cultures and are regarded as an excellent source of protein and vitamins (Ikoigbo, 1980).
Cassava also has important industrial applications such as the production of animal feed, starch,
and more recently ethanol. Dependence on the crop will not diminish as production is projected to
increase 60% by the year 2020 in response to needs of the growing population that depend on cas-
sava (Scott et al., 2000).
17.2.3 Sugarcane
Sugarcane (Saccharum spp.) is an important vegetatively propagated crop that is cultivated for its
sugar-rich stalks and contributes about 75% of the world’s sucrose supply (McIntyre et al., 2005).
The mature stem can accumulate 12 to 16% of its fresh weight and approximately 50% of its dry
weight as sucrose (Bull and Glasziou, 1963). Sugarcane is cultivated in more than 20 million hect-
ares in tropical and subtropical regions of the world, producing up to 1.3 billion metric tons of
crushable stems (Menossi et al., 2008). Sugarcane has many other uses besides the production of
sugars. Sugarcane production is receiving increased attention for the production of ethanol as an
important source of renewable energy. Sugarcane bagasse is already being used as a fuel source for
sugar mills or for the production of animal feed. Bagasse is also being investigated for use in the
production of paper, as a dietary fiber in bread, and as a wood substitute in the production of com-
posite wood and in the synthesis of carbon fibers (Menossi et al., 2008). Today, sugarcane has many
industrial uses and is one of the most widely used and cheapest domestic products (Jenkins, 1966).
Molasses, a by-product of sugarcane production, is used as a fertilizer for cane soils, stock feed,
alcoholic beverages, vinegar, cosmetics and pharmaceuticals, cleaning preparations and solvents,
and coatings. It is quite possible that further uses of sugarcane will be developed in the future, but
even now it can be seen that sugarcane is a very important and useful plant crop worldwide. The
development of sugarcane as a biological system for large-scale production of value-added products
is also gaining momentum (Lakshmanan et al., 2005) and is discussed later in this chapter.
17.3 Breeding Challenges

Genetic improvement of vegetatively propagated crops through conventional breeding is faced with sev-
eral biological constraints. We examine some of these difficulties below for each of the selected crops.
17.3.1 Potato
Potatoes are polyploid with ploidy ranging from diploid (2n = 2x = 24) to hexaploid (2n = 6x = 72).
Most diploid species are self-incompatible outbreeders, and most of the tetraploids (2n= 4x = 48) are
self compatible and display tetrasomic inheritance, and the hexaploids are mostly self-compatible
allopolyploids that display disomic inheritance (Hawkes, 1990). Crossability groups occur, each
defined by an endosperm balance number, although interspecific pollen–pistil incompatibility and
nuclear-cytoplasmic male sterility can occur (Camadro et al., 2004). The high levels of heterozygos-
ity and severe inbreeding depression in parental clones require very large populations for selection
of individual clones with good agronomic traits. New potato cultivars are exceptionally cumber-
some to develop. Selection for desirable characters is time consuming and it may take up to 20 years
to develop a new cultivar. The genes for resistance to pests, diseases, frost, and some quality traits
such as starch and protein content are introgressed from related wild Solanum species. Wild species
also carry many undesirable traits, such as low yield. The process of getting rid of all the unwanted
traits requires several backcrosses in the breeding process. Cross-pollination for introgression of
traits is only possible with very closely related species that do not contain many of the desired traits.
The multigenic nature and low heritability of some traits also slows down the breeding process.
Potato breeding is also problematic due to its narrow genetic diversity as a result of clonal propaga-
tion (Bradshaw and Mackay, 1994). Genetic improvement through conventional breeding is limited,
since cultivated potatoes are largely tetraploid and are generally vegetatively propagated.
17.3.2 Cassava
Today’s cassava cultivars are regarded as segmental allopolyploids with 2n = 36 chromosomes
(Magoon et al., 1969). Cassava breeding is based mainly on mass phenotypic recurrent selec-
tion. Parental lines are selected mainly on their per se performance for crossing programs. The
high degree of heterozygosity in cassava makes identification of ideal parental material difficult.
Synchronized flowering of selected parents is problematic and when considerable difference in
flowering times exists, delayed planting or pruning of the earliest flowering genotypes must be
practiced. Normally, few seeds are obtained (an average of 1 to 1.5), and acquiring large numbers
of seeds is a labor-intensive and tedious process. It takes about 5–6 years from the time of seed set
to the regional trial stage in cassava breeding (Ceballos et al., 2004). Very little knowledge exists on
the genetics of important agronomic traits in cassava, and precise genetic control is known for rela-
tively few traits. Single-gene control has been demonstrated for leaf lobe width, root surface color,
albinism, stem collenchyma color, stem growth habit, root flesh pigmentation, and male sterility
(Hershey and Ocampo, 1989). There are no confirmed examples of physiological specialization on
a gene-for-gene basis for pests or pathogens. The inheritance patterns of a broad range of agronomi-
cally important traits have been studied. Results to date indicate that nearly all these traits are under
multigenic control, with a high proportion of additive genetic effects (Iglesias and Hershey, 1994).
Genetic variability within cassava for some traits, such as resistance to postharvest deterioration
and virus resistance, is lacking or limited.
17.3.3 Sugarcane
Sugarcane cultivars are both highly aneuploid and polyploid (McIntyre et al., 2005). They typically
contain more than 100 chromosomes. Modern cultivated sugarcane (Saccharum spp.) is a hybrid
complex originating from crosses between S. officinarum (2n = 80, x = 10) and S. spontaneum (2n
= 40–124, x = 8) and in some lineages S. sinensis or S. barberi (D’Hont and Layssac, 1998). Most
cultivated sugarcane cultivars harbor two genomes with about 80% S. officinarum and 20% S.
spontaneum (D’Hont et al., 1996). Sugarcane chromosomes are assigned to one of eight homology
groups (HG) (Aitken et al., 2005). Each HG is estimated to contain between 8 and 14 homo(eo)
logous chromosomes. Little is known about the allelic complexity of homo(eo)logous genes on chro-
mosomes within a HG (McIntyre et al., 2006). The complexity and size of the sugarcane genome is
a major limitation in genetic improvement. Traditional breeding is time consuming and breeding a
new sugarcane cultivar can take more than 15 years from hybridization to commercial cultivation
of the selected cultivar (Arvinth et al., 2009). The breeder has to rely on the initial variation created
during hybridization, and no amount of selection can produce a good cultivar out of a poor cross
(Alwala et al., 2006). The choice of parents to use in crossing is one of the most crucial decisions the
breeder has to make. Enhanced breeding for sucrose accumulation is considered to have reached a
plateau. The employment of new technologies to assist in the association of traits with genetic mark-
ers and genetic maps can aid in achieving further yield increases in breeding programs.
17.4 Production Constraints
17.4.1 Potato
The major diseases infesting the potato crop are late blight, considered to be the most serious potato
disease worldwide, and bacterial wilt (Johnson and Veilleux, 2003). Potato late blight occurs almost
everywhere potatoes are grown. Bacterial wilt or brown rot, caused by Ralstonia [Pseudomonas]
solanacearum, limits potato production worldwide and especially in Asia, Africa, and Latin
America, where it causes severe crop losses in tropical, subtropical, and warm-temperate regions.
The major insect pests include potato tuber moth, Colorado potato beetle, and potato leaf miner
fly. The potato tuber moth is the most damaging pest of potatoes in fields and stores in warm, dry
areas of the world, such as North Africa and the Middle East, Mexico, Central America, and the
inter-Andean valleys of South America. A wide variety of viruses also affect potato production. For
example, a major devastating viral disease is the potato leaf roll virus (PLRV), which causes yield
losses of up to 90% in infected crops. Nematodes especially of the genus Globodera cause major
damage to potatoes.
17.4.2 Cassava
The production constraints of cassava have been detailed in many publications (Jenning and Iglesias,
2002; Kawano, 2003; Ceballos et al., 2004) and will not be dealt with in detail. In summary the
main biotic constraints include viral, bacterial, fungal, and a variety of arthropod pests. There are
a variety of abiotic factors that limit cassava productivity such as drought, low fertility, and acidic
or alkaline soils. Productivity of cassava is very low and there is a huge gap in cassava production
between the ideal (80 tons/ha) and what the average farmer harvests, around 8–12 tons /ha (Taylor
et al., 2004).
17.4.3 Sugarcane
Disease and pests cause considerable economic losses to sugar industries worldwide. Sugarcane
is susceptible to many viral, bacterial, fungal, and phytoplasma diseases, and there are a number
of diseases of unknown etiology (Rott et al., 2000). The major viral pathogens include sugar-
cane mosaic virus (SCMV), Fiji disease virus (FDV), sorghum mosaic virus (SrMV), sugarcane
streak virus (SSV), and sugarcane yellow leaf virus (SCYLV) (Lakshmanan et al., 2005). Viral
diseases cause severe stunting, ratoon stunting, chlorotic streak, Fiji disease, and Sereh disease
(Purseglove, 1979). There are about 160 fungal and 8 bacterial pathogens of sugarcane (Rott et
al., 2000). Major fungal diseases include red rot (Colletotrichum falcatum), root rot (Pythium
graminicolum.), pineapple disease (Thielaviopsis parodoxa), downy mildew (Sclerospora sac-
chari), and smut (Ustilago scitaminea), while the main bacterial diseases include gumming dis-
ease, Xanthomonas vasculorum, in which yellowish stripes occur at the leaf tips and the vascular
bundles exude a yellowish gum when cut, and leaf scald, Xanthomonas albilineans, in which
yellow stripes occur on the leaf blade, many side shoots are produced, and the vascular bundles
of the stalk are red (Purseglove, 1979). Crop loss due to insect pests is estimated to be at 10 to
20% of sugarcane and is a limiting factor in sugarcane production (Avasthy, 1983). The most
destructive insects of sugarcane are stem borers and larvae of several genera of moths. The larvae
burrow into the stem and on emergence cause loss of sucrose and weakened stems. Other pests
include termites and white grubs. Rats are also a problem in many areas since they eat the cane
and introduce pathogens.
17.5 Breeding Objectives

The breeding objectives for most crop plants are similar, with the primary focus being on the con-
tinuous improvement in productivity. Breeding for yield is a major target followed by breeding for
resistance to diseases and pests that have an impact on yield. In addition to yield, disease and pest
resistance, and nutritive and organoleptic qualities, other considerations will have to be added to
breeding parameters. These parameters, according to Sinha and Swaminathan (1984), include
• Increasing the nutritive value of crops by breeding for enhanced proteins, vitamins, and
other essential minerals
• Improving the efficiency of food production for each unit of cultural and solar energy invested
• Producing stable crops over wide environments with resistance to weeds, pests, and pathogens
• Improving and identifying plants as sources of biomass and renewable energy
• Breeding genotypes for optimum response to high, low, and zero inputs
• Breeding efficient plant types for crop-livestock production systems
• Breeding for suitability of crops in drought- and flood-prone areas
Plant breeding aims to improve crop performance or quality and to create new cultivars. However,
new cultivars must preserve the quality characteristics and meet consumer demands. The process of
developing a new cultivar can take up to 14 years in some crops from the first cross to the cultivar
coming to the marketplace. Traditional plant breeding cannot meet all the food requirements of
human beings. Therefore breeding through plant biotechnology is a necessity. The effective merging
of classical breeding with modern plant biotechnology and the novel tools it provides are the gateway
for the continuous increase in agricultural productivity and human survival (Altman, 1999). Some
specific breeding objectives for each of the crops discussed in this chapter are outlined below.
17.5.1 Potato
Breeding efforts for potato are faced with the classical questions “What to breed?” and “How to
breed?” (Bonnel, 2008). As scientific knowledge of the potato increased, the two questions have
become related. The most important characteristics in potato breeding are summarized by Bonnel
(2008) and include:
• Yield, tuber number and tuber size

• Tuber appearance
• Complex quality traits, e.g., low reducing sugar, no oxidation or bruising
• Resistance against nematode and insect pests
• Resistance against pathogens causing late blight, early blight, and scabs, viruses, and
Erwinia spp.
• Resistance against abiotic stresses such as water and heat stress
• Specific traits for niche markets
• Adaptation to specific environments
Bonnel also lists the issues to the question “How to breed potatoes?” (Bonnel, 2008).
17.5.2 Cassava
The breeding objectives for cassava are determined by the ultimate use of the crop. For subsis-
tence use, production stability and cooking quality or starch characteristics are the main traits.
For industrial purposes such as starch production and animal feed, productivity is the main target.
Some morphological traits such as peel color of the roots, the leaf petiole, or the shoot are associ-
ated with good cooking quality. Other root-quality traits include reduced cyanogenic glucosides,
early bulking capacity, higher protein content, and reduced postharvest physiological deterioration
(Ceballos et al., 2004). Breeding for increased yield, multiple pest and disease resistance, desirable
agronomic and consumer preference traits such as early vigor in plant growth (for high foliage yield
for leaf vegetable), appropriate plant architecture, and early bulking of storage roots, combined
with high dry matter content, low cyanide content, and other favorable traits, such as easy peeling,
have been the main breeding objectives. Recently, breeding for improved micronutrient content
has been emphasized. Improvement of this crop has been primarily oriented towards relatively few
traits, most prominently to yield and host-plant resistance to pathogens and pests. The former is a
complex trait with many individual components, each involving numerous biochemical pathways
and progress has been most pronounced in those cases where objectives were relatively simple.
Resistance to pathogens is often determined by fewer genes, allowing for qualitative progress in a
breeding program. Advances in yield potential have been substantial but mainly in more favorable
environments. These gains are often less prominent when translated to farmers’ conditions, but the
products of steady improvement are beginning to have substantial impact (Hershey and Jennings,
1992). Improvements in cassava in terms of adaptation, resistance, productivity, and other traits
have not been exhausted.
17.5.3 Sugarcane
The goal of sugarcane breeding is to produce an economic yield of sugar sustained over several
ratoons. Sugarcane yields the highest number of calories per unit area of any plant (Heiser, 1981),
producing up to 10 tons of sucrose per hectare in Barbados. The highest yields of 22 tons of sucrose
per hectare occur in Hawaii, but the crop takes two or more years to mature there. Recovery of raw
sugar from cane varies from 11 to 13% (Purseglove, 1979). Increasing the yield of sucrose can be
achieved by either increasing biomass at the same concentration of sucrose or increasing the sucrose
concentration. The yield of sucrose has increased over the years mainly due to increase in biomass.
However, increasing the sucrose content is considered to have reached a plateau via conventional
breeding due to the limits in the gene pool (Mariotti, 2002). The employment of new technologies to
assist in the association of traits with genetic markers and genetic maps can aid in achieving further
yield increases by breeding (Dillon et al., 2007). Sugarcanes are highly polyploid, wind-pollinated
outbreeders. They are clonally propagated, highly heterozygous, and intolerant to inbreeding. New
cultivars are sought from the first generation progeny of crosses between clones. The five species
of interest to cane breeders are: (1) S. officinarum (2n = 80) has good sugar quality and low fiber,
although it is susceptible to most of the main diseases, except gumming disease and smut; (2) S.
spontaneum (2n = 40–128) is a source of resistance to many diseases, including “Sereh,” mosaic,
gumming, red rot, and downy mildew; (3) S. barberi (2n = 82–124) is considered the most important
breeding cane and is immune to gumming and mosaic, and resistant to downy mildew, but suscep-
tible to smut and red rot; (4) S. sinense (2n = 82–124) is difficult to breed but has given rise to some
useful breeding lines; and (5) S. robustum (2n = 60–194) has been used to some extent in breeding
lines (Wrigley, 1982). Breeding and selection of cane is not a simple process since viable seeds are
seldom produced. Breeding occurs at the experiment stations, which are able to provide the proper
conditions and techniques required.
17.6 Conventional versus Molecular Breeding

Conventional breeding efforts have attempted to address many of the constraints facing vegetatively
propagated crops, but progress has been slow because most of the crops have complex genetic sys-
tems that makes it difficult to breed efficiently. Ploidy manipulations—that is, scaling up and down
chromosome numbers of a species within a polyploid series—facilitate breeding polyploid asexual
crops with their wild genetic resources endowment (Ortiz, 2004). Chromosome sets are manipu-
lated with haploids, 2n gametes, and through interspecific-interploidy crosses. Analytical breeding
schemes rely mainly on ploidy manipulations to “capture” diversity from exotic (wild or nonadapted
germplasm) and use 2n gametes to incorporate this genetic diversity through unilateral (USP; n × 2n
or 2n × n) or bilateral (BSP; 2n × 2n) polyploidization (Ortiz et al., 2009). Haploids are propagules
with the gametophytic chromosome number (n) and 2n gametes possess the sporophytic chromo-
some number of the parental source.
Vegetative propagation is genetically a rather conservative strategy. Clonally propagated
crops have individually limited genetic plasticity, and new variation from natural crosses in
multiclonal fields and introgression from wild species are slow to arise under natural condi-
tions. Consequently, most breeding programs consider recombination as the principal means
of cultivar development. Modern biotechnology can be an important tool in the development of
sustainable agricultural systems (Ferry et al., 2004). GM crops have the potential to contribute
to increased productivity while conserving biodiversity and more sustainable agriculture and the
environment. No set of biological tools has created greater expectations than those generated
by achievements in the area of molecular, cellular, and other technologies broadly described as
biotechnology. Biotechnology provides new tools for overcoming some of the problems for the
genetic improvement of crops.
For example, biotechnology is useful when no resistance for a disease, pest, or virus is found
in the germplasm of a crop. The transgenic approach of introducing foreign genes for these traits
makes it possible to engineer plants with the traits of interest. The rapid and continued development
of tools in biotechnology has introduced fundamental changes in crop improvement research and
broadened the range of potential traits for genetic modification.
Although past achievements in conventional breeding of vegetatively propagated crops are
impressive, it appears that an integrated approach combining both conventional and molecular
breeding strategies is required to make further progress in crops that are characterized by poly-
ploidy, a narrow genetic base, poor fertility, susceptibility to various pests and diseases, and the long
durations its takes to breed elite cultivars (Lakshmanan et al., 2005). Two focus areas of biotechnol-
ogy research in crop improvement include the development of molecular markers and molecular
maps.
17.6.1 Molecular Markers
Molecular markers are discovered by establishing statistical associations between variations in par-
ticular DNA sequences and any traits that may be of interest (Reece and Haribabu, 2007). It is
now generally accepted that molecular markers represent the most significant advance in breeding
technology in the past few decades and are currently the most important application of molecular
biology to plant breeding. There appears to be no resistance to the use of molecular marker tech-
nology in breeding as there is for GM organisms. Research in molecular marker technologies have
blossomed in the last decade because they are essential if marker-assisted selection (MAS) in plant
breeding programs is to be realized. Molecular markers can assist breeders in the process of evalu-
ation and selection and shorten the timescale required for the production of improved cultivars.
MAS eliminates the need for costly and time-consuming field trials to select superior individuals.
Molecular markers enable breeders to detect the presence of multiple alleles that are associated with
a single trait even when the individual alleles do not exert a detectable influence on the expression
of the trait (Reece and Haribabu, 2007). Molecular markers can be used to facilitate decisions made
during crossing, especially when they used to gain knowledge of the genetic diversity in parental
clones. Therefore the search for target traits in many of the vegetatively propagated crops occupies
the minds of many scientists. However, there remains a considerable need for parallel progress in the
understanding of genotype-by-environment interaction and the influence of epigenetic mechanisms.
Nevertheless, even with the current level of understanding, the application of DNA marker technol-
ogy is likely to facilitate the attainment of new objectives in crop research and genetic improve-
ment that have proven difficult to achieve using classical techniques. For example, highly precise
MAS approaches require the development of high-density linkage maps. However, the generation of
highly relevant and precise linkage maps is not routinely achievable in most crops.
DNA-based molecular markers have been used extensively for plant genome analysis, especially
in characterizing genetic diversity, genome fingerprinting, genome mapping, gene localization,
analysis of genome evolution, population genetics, taxonomy, and plant breeding (Sharma et al.,
2005). All marker systems have different advantages and disadvantages in specific applications.
Thus, it is important for molecular breeding programs to develop capacity in several assays in order
that the most suitable system can be chosen and rapidly applied for any particular application. In
addition, different DNA marker assays detect (and are therefore affected by) different types of
genetic variation.
Improved molecular marker systems are required to enhance the adoption of MAS. Several fac-
tors are important when considering MAS, including ease of use, robustness, cost, and linkage to
the trait of interest (de Koeyer et al., 2010). The ideal marker systems for polyploidy crops should
be dosage sensitive and have the ability to distinguish heterozygous genotypes with multiple haplo-
types within the target genomic region by the marker (de Koeyer et al., 2010).
New DNA technologies are constantly being developed especially for human genomic research,
and some of them are being used in plant research. High-throughput technologies based on single
nucleotide polymorphisms (SNPs) or small-scale insertions/deletions (indels) are efficient alterna-
tives for traditional markers (restriction fragment length polymorphism [RFLP], random ampli-
fied polymorphic DNA [RAPD], amplified fragment length polymorphism [AFLP], and so forth)
because of their greater abundance, high polymorphism, ease of measurement, and ability to reveal
hidden polymorphisms where other methods fail (Dillon et al., 2007). SNPs also allow easy and
unambiguous identification of alleles or haplotypes.
A good marker system for polyploid crops should be dosage sensitive and have the ability to dis-
tinguish heterozygous genotypes with multiple haplotypes (de Koeyer et al., 2010). High-resolution
DNA melting (HRM) analysis has been shown to have several advantages over other genotyping
methods (Montgomery et al., 2007; Reed et al., 2007; Erali et al., 2008). The advantages include a
short analysis time and the absence of post–polymerase chain reaction (PCR) sample processing or
separation (de Koeyer et al., 2010). The three ways in which HRM can be used for genotyping and
for variant screening are discussed in de Koeyer et al. (2010). HRM has been used in many crops and
holds great potential for cultivar identification, especially in polyploids, mapping, polymorphism
discovery, mapping candidate genes, and in combination with quantitative trait loci (QTL) or asso-
ciation studies for identifying genomic regions involved in important traits (de Koeyer et al., 2010).
17.6.1.1 Molecular Markers in Potato

More molecular markers are available in potato than for cassava. There are markers for late blight
resistance (Bormann et al., 2004; Bisognin et al., 2005; Colton et al., 2006), resistance to potato virus
Y (Hamalainen et al., 1997, 1998; Kasai et al., 2000; Flis et al., 2005; Gebhardt and Bellin, 2006),
potato virus X (Gebhardt et al., 2006), and to the potato cyst nematode (Niewohner et al., 1995).
Markers for MAS are limited to diploid material and include Ns for PVS resistance (Marczewski et
al., 2002), Ryadg for extreme resistance to PVY, Gro1 for resistance to Globera rostochiensis, Rx1 for
extreme resistance to PVX, and Sen 1 for resistance to potato wart (Gebhardt et al., 2006). Recently
the marker for PVY resistance was validated as a fast and efficient way to select for PVY resistance
(Ottoman et al., 2009). Markers tagging QTLs conferring resistance to P. infestans, Verticillium
dahlia, and V. albatrum have been developed (Simko et al., 2007).
Recently a molecular marker for Verticillium wilt (VW) resistance has been identified in potato
(Bae et al., 2008). Breeding for VW has been difficult because disease symptoms can easily be
confused with natural senescence. Stem plating used to identify the disease is labor intensive and
time consuming. Environmental factors influence disease expression, and great variation exists in
pathogen population from stem to stem. Since the only effective control practice, soil fumigation,
is expensive and has harmful environmental effects, host-plant resistance has been explored as
a method of disease control (Pegg, 1974; Rowe et al., 1987). Most commercial cultivars in the
United States are susceptible, although some exhibit moderate resistance (Rowe and Powelson,
2002). Major resistance genes have been identified for the disease. Inheritance of VW resistance
in cultivated tetraploid potato appears to be polygenic and complex (Hunter et al., 1968; Pavek and
Corsini, 1994) and most cultivars are not highly resistant to VW (Rowe and Powelson, 2002). The
interesting aspect of this study was that tomato Vel and Ve2 gene sequence information was used
to amplify candidate Ve gene orthologs in potato. The marker was tested for association with resis-
tance in segregating populations and was found to be effective for the VW gene in potato.
17.6.1.2 Molecular Markers in Cassava

Molecular markers have made a great contribution to cassava breeding and genetics, in the assess-
ment of genetic diversity, taxonomical studies, understanding the phylogenetic relationships in the
genus, confirmation of ploidy, and the development of genetic maps (Fregene et al., 1997; Lokko et
al., 2004). Very few markers have been identified for traits in cassava and are limited to markers for
host-plant resistance and early yield (Jorge et al., 2000, 2001; Akano et al., 2002).
17.6.1.3 Molecular Markers in Sugarcane

The majority of useful traits in sugarcane are multigenic due to the polyploidy nature of the crop.
Quantitative trait loci (QTL) for sucrose content and related traits have been identified (Ming et
al., 2001; Ming, Del Monte, et al., 2002). Comparative analysis of QTL affecting plant height and
flowering between sorghum and sugarcane identified QTL clusters in sugarcane (Ming, Del Monte,
et al., 2002), highlighting the power of genome synteny. The conservation of chromosome organiza-
tion across the grasses has the potential to locate and identify genes of interest in sugarcane. SNPs
have been used to divide a set of 64 expressed sequence tags (ESTs) into two groups related to two
genes family members of 6-phosphogluconate dehydrogenase (Grivet et al., 2001), to reveal the
expression of an Adh2 and two Adh1 genes (Grivet et al., 2003) and the development of codominant
cleaved, amplified polymorphic sequence markers (Quint et al., 2002) and to map several candidate
genes and ESTs (McIntyre et al., 2005). Ming et al. (2001; Ming, Del Monte, et al., 2002) inves-
tigated the genetic basis of traits related to sugar content, plant height, and flowering in interspe-
cific crosses between S. officinarum and S. spontaneum. Numerous QTLs were detected in a few
genomic regions, suggesting that substantially fewer genes may actually be involved in the genetic
control of these traits. Within an interspecific cross, wide phenotype segregation can provide a
favorable setting for QTL detection. By comparison, a study of yield components (plant height,
stalk diameter, stalk number, and Brix) in the selfed progeny of the modern cultivar ‘R570’ revealed
numerous QTL with smaller individual effects (Hoarau et al., 2002). Similarly, Jordan et al. (2004)
detected numerous small QTL for stalk number in sugarcane and found sorghum QTL for tillering
in syntenic positions. The only major gene that has been localized so far in the sugarcane genome is
a rust-resistance gene (Daugrois et al., 1996). This gene (called Brul for brown rust) is currently the
focus of a map-based cloning project (D’Hont et al., 2001; Asnaghi et al., 2004).
17.6.2 Molecular Maps
A molecular map is a construct that places the markers in order indicating the relative distances
between them and assigning them to their linkage groups (Jones et al., 1997). Linkage maps are
based on recombination frequencies, and the distance between points on a genetic map reflects the
recombination frequencies between the points. Linkage maps provide valuable information about
the genomic organization of a species. Maps provide a direct method for showing the positions of
genes and other sequence features and allow selecting genes via detectable markers. Linkage maps
for most crop species were initially developed using morphological markers, the first genetic mark-
ers used in biology. For example, Mendel studied visible traits such as seed and pod color, surface
appearance of seeds and pods, and plant height. In the process of finding more markers, the first
class of markers scored at the molecular level were isoenzymes. The advance of molecular biology
provided a wide spectrum of technologies to assess different markers.
Two diverse approaches are used to facilitate the generation of appropriate linkage maps in
polyploid species: (1) linkage mapping based on diploid relatives and extrapolation to the polyploid
crop, and (2) polyploid mapping based on single-dose markers in populations derived from crosses
between heterozygous tetraploid and diploid genotypes. The more markers there are on a map, the
more likely it is that one will be closely linked to a trait of interest. Several different types of mark-
ers have been used to develop linkage map for crop species, including RFLP, RAPD, minisatellites
or variable number tandem repeats, AFLP, and microsatellites or simple sequence repeats (SSR).
The use of these markers for mapping is discussed adequately in many publications. SSR markers
are advantageous to applied plant breeding because they are codominant, easily assayed, and detect
high levels of polymorphism (Morgante and Olivieri, 1993). For these reasons SSR markers have
become valuable markers to breeders for the purposes of genome and QTL mapping. In addition to
their high polymorphism, SSR are usually single-locus sites, an important feature when dealing with
allopolyploid species. SSR markers have thus become the marker class of choice for the molecular
mapping of many crop species (Roa et al., 2000). While SSRs are standard PCR-based markers and
can be considered as proven anchor-markers, their suitability for high-throughput mapping does not
favorably compare to the new single nucleotide polymorphism (SNP) based genotyping techniques
(Kilian et al., 2005). High-throughput genotyping technologies based on SNP or small-scale inser-
tions/deletions (indels) could become efficient alternative techniques for traditional markers because
of their greater abundance in the genome. However, development of massive SNP resources from
the expressed portion of the genome, amenable to fully automated genotyping, is a difficult task for
polyploidy species (Koebner and Summers, 2003; Somers et al., 2003). Diversity arrays technology
(DArT), for which proof of concept was first reported by Jaccoud et al. (2001), is becoming increas-
ingly adopted in many species. The technology combines a complexity reduction method (Wenzl
et al., 2004) with hybridization-based polymorphism detection using high-throughput, solid-state

platforms and has the potential to generate hundreds of high-quality genomic dominant markers
with a cost- and time-competitive tradeoff (Kilian et al., 2005).
The development of high-resolution, easy-to-use genetic maps, coupled with sequencing and
physical mapping, will revolutionize breeding of vegetatively propagated crops. Highly dense link-
age maps are powerful tools for the localization of both qualitative and quantitative traits, for map-
based cloning of genes, and to develop markers for marker-assisted breeding. They also provide
information for understanding the biological basis of traits (Kriegner et al., 2000).
Constructing genetic maps in polyploids is challenging for several reasons, as outlined in
Cervantes-Flores et al. (2008). Polyploids have multiple chromosomes of the same type and a large
number of possible genotypes are expected in a segregating population due to the larger number of
alleles combining in a particular event. This is especially true in autopolyploid species. The geno-
type of an individual is not always readily inferred through its marker phenotype. The type of ploidy
(allopolyploidy or autopolyploidy) of many crops is unclear, making it difficult to determine patterns
of inheritance (Wu et al., 1992; Ripol et al., 1999). In an autopolyploid species, corresponding chro-
mosomes in the different genome copies are homologs and, therefore, can pair randomly between
each other. In contrast, for allopolyploids chromosomes may originate from two or more different
genomes and during meiosis will pair preferentially to their homologs from the same genome,
and in lower frequency to a homeologous chromosome (Sybenga, 1996; Ramsey and Schemske,
2002). A commonly used approach to construct molecular genetic maps in polyploids is based on
the use of single-dose fragments (SDF) (Wu et al., 1992). Since single-dose markers are markers
present in one parent in a single copy, only half the progeny will carry the marker during gamete
formation. Regardless of the ploidy of the genome, half the progeny will possess this fragment and
half will not. SDFs combined with other mapping strategies such as testcross and pseudo-testcross
approaches, have been used to construct linkage maps in several polyploid species, including potato,
sugarcane, and sweet potato (Cervantes-Flores et al., 2008).
17.6.2.1 Potato
Potato has the densest genetic linkage map and one of the earliest cytogenetic maps among all
plant species (van Os et al., 2006; Iovene et al., 2008). More than 40 major qualitative genes confer-
ring resistance to important diseases and pests have been genetically mapped (Simko et al., 2007;
Ottoman et al., 2009). To integrate these maps, bacterial artificial chromosome (BAC) clones are
being hybridized to the pachytene chromosomes of potato (Iovene et al., 2008).
17.6.2.2 Cassava
The first genetic linkage map for cassava was constructed with predominantly RFLP markers and a
full-sib intraspecific cross (Fregene et al., 1997). The map provided initial tools for genetic analysis
of important traits of cassava (Jorge et al., 2000, 2001; Akano et al., 2002; Okogbenin and Fregene,
2002, 2003). A higher resolution map was developed using SSR markers (Okogbenin et al., 2006).
17.6.2.3 Sugarcane
The identification of single- (present on one chromosome only) and double-dose (present on two
chromosomes) markers for mapping and QTL analysis was a breakthrough in sugarcane genetics.
Despite the structural complexity of the sugarcane genome, genetic maps of sugarcane have been
produced based on single-dose markers (Wu et al., 1992) for the two ancestral species S. sponta-
neum (Al-Janabi et al., 1993; Da Silva et al., 1993, 1995; Ming et al., 1998; Ming, Liu, et al., 2002)
and S. officinarum (Mudge et al., 1996; Guimaraes et al., 1997; Ming et al., 1998; Ming, Liu, et al.,
2002). Genetic maps have also been constructed for two modern sugarcane cultivars, ‘Q165’ (Aitken
et al., 2005) and ‘R570.’ AFLPs and SSRs and resistance gene analogs (Rossi et al., 2003) were later
mapped using a selfed ‘R570’ progeny. The AFLP-based map has more than 1,100 markers with a
total length of 7,800 cM, which represents a coverage of about 46% of the anticipated genome size
(17,000 cM). A saturated map must be developed to be able to efficiently localize major genes or
Mendelian factors involved in quantitative trait loci (QTL). Moreover, QTL mapping in sugarcane is
a real challenge, since many alleles coexist at each locus due to the high polyploidy. At a particular
locus, the effect of an allele should be perceptible only if it exceeds the average effect of all other
segregating alleles in the background but not, as in diploids, if its effect simply exceeds that of a
single alternative allele (D’Hont and Glaszmann, 2001; Hoarau et al., 2002). Two genetic maps of
sugarcane were also constructed using AFLP, SSR, and RFLP markers (Raboin et al., 2006). A new,
brown rust–resistant gene and one controlling stalk color were localized on the map.
17.7 Genetic Transformation
Genetic transformation has become an important tool for crop improvement and especially
for vegetative propagated crops. Genetic transformation is defined as the transfer of any for-
eign gene(s) isolated from plants, viruses, bacteria, or animals into a new genetic background
(Sharma et al., 2005). Successful genetic transformation in plants requires the production of
normal, fertile plants expressing the newly inserted gene(s). The process of genetic transforma-
tion involves several distinct steps: identification of a useful gene, the cloning of the gene into
a suitable plasmid vector, delivery of the vector into the plant cell (insertion and integration),
followed by expression and inheritance of the foreign DNA encoding a polypeptide. Methods of
gene insertion in plants can be achieved by direct gene transfer through particle bombardment
or electroporation, or through biological vectors like a disarmed TI (tumor inducing)-plasmid
of A. tumefaciens. However, this technology has not been realized in all crop plants because
of the lack of suitable transformation methods. The major components for the development of
transgenic plants include (1) the development of reliable tissue culture regeneration systems, (2)
the preparation of gene constructs and transformation with suitable vectors, (3) efficient trans-
formation techniques for the introduction of genes into the crop plants, (4) recovery and mul-
tiplication of transgenic plants, (5) molecular and genetic characterization of transgenic plants
for stable and efficient gene expression, and (6) field evaluation of transgenic plants (Sharma et
al., 2005).
17.7.1 Achievements and Prospects of Molecular Breeding

in Potato, Cassava, and Sugarcane
Protection against diseases caused by fungi, bacteria, and viruses and losses due to insect herbivores
are major challenges for crop improvement and are significant limiting factors in food production
(Akhond and Machray, 2009). Current efforts to improve plant stress tolerance by genetic trans-
formation have resulted in several achievements. Improvement in the nutritional qualities of our
major plant foods is now within reach of crop improvement programs. Plants are also being used as
biofactories for the production of pharmaceuticals, proteins, plastic, and so forth. The following sec-
tion examines some of the achievements and prospects of molecular breeding in plant improvement
programs. There is ample literature on these topics covering all three plants of interest. However,
due to space restrictions, examples covering all plants are not discussed here.
17.7.1.1 Pest Resistance in Potato

A variety of genetic engineering strategies have been used to target insect pests in potato. One of
the most widely used strategies is the use of the cry genes from Bacillus thuringiensis. The cry gene
is part of a large family of homologous genes that encode proteins that are toxic to insects and their
larvae (Davidson et al., 2002). Resistance to a number of insect pests has already been engineered
in potato. They include:
1. Resistance to the Colorado potato beetle and potato tuber moth was derived by expression of
one of the cry endotoxin proteins (Mohammed et al., 2000; Reed et al., 2001; Davidson et
al., 2004).
2. Reduced feeding of the cotton bollworm larvae was observed in plants transformed with
cry1ab gene (Chakrabarti et al., 2000).
3. Transformation of potato with a trypsin inhibitor from cowpea (Bell et al., 2001) and the lectin
genes from snowdrop (Birch et al., 1999) also produced insect resistance in potato.
4. Genes that encode cysteine proteinases inhibitors (cystatins) confer resistance to the potato
cyst nematode (Globodera rostochiensis and G. pallida) (Urwin et al., 2003).
5. The gene chly encoding for chicken lysozyme enzyme enhances resistance to blackleg and soft
rot in potato (Serrano et al., 2000).
17.7.1.2 Virus Resistance

17.7.1.2.1 Cassava
Cassava mosaic disease (CMD) is a major constraint for cassava production in Africa, resulting
in significant economic losses. The disease is caused by a complex of viruses named according to
the regions in which they were discovered and includes the African, East African, and Southern
African cassava mosaic virus. Breeding for resistance to CMD has been a major target in classi-
cal breeding programs. Several attempts have also been made since 2004 to genetically engineer
virus resistance in cassava. Engineering African cassava mosaic disease (ACMV) resistance in
cassava using sense and antisense RNA strategies produced lines with varying levels of resistance
(Chellapan et al., 2004; Zhang et al., 2005). Since then, new approaches for developing transgenic
cassava have been reported using RNA interference technology. RNA interference provides resis-
tance against viruses via post-transcriptional and/or transcriptional gene silencing (PTGS and
TGS) (Waterhouse and Fusaro, 2006). Vanderschuren et al. (2007, 2009) have developed two new
ways of developing transgenic cassava for resistance to CMD. In the first instance they showed that
expression of double-stranded RNA (dsRNA) homologous to virus sequences can interfere with
RNA virus infection in plant cells by triggering RNA silencing (Vanderschuren et al., 2007). More
recently, Vanderschuren et al. (2009) engineered a CMD-susceptible cassava cultivar to express a
hairpin dsRNA homologous to a region of the replication-associated protein coding sequence (Rep/
AC1). This sequence was selected because it is essential for replication of geminiviruses like CMD.
Several of the transgenic cassava lines from these experiments showed resistance to CMD. Similar
technologies could be used for other crops.
17.7.1.2.2 Potato
Research in developing transgenic potatoes for virus resistance began in the 1990s (Lamb and Hay,
1990; Kawchuk et al., 1991). Transformation of potato with the viral coat proteins of potato leaf roll
virus conferred resistance to the virus (Kawchuk et al., 1990). Transformation with virally encoded
proteinase gene sequences produced high levels of resistance to viral infection (Okamato et al.,
1996). Replicase and coat protein genes from potato leaf roll luteovirus and potato Y potyvirus,
respectively, provide resistance to these viruses (Duncan et al., 2002). RNA silencing technology
has also been applied in potato to develop virus-resistant plants. Potato virus Y (PVY) is one of
the most widespread viruses of potato. Missiou et al. (2004) generated transgenic potato that was
resistant to potato virus Y.
17.7.1.3 Abiotic Stress Tolerance in Potato

Although oxygen is essential for aerobic life, reactive oxygen species (ROS) such as superoxide
anion radicals (–O2–), hydroxyl radicals (–OH), and hydrogen peroxide (H2O2) are produced in all
aerobic cells during metabolism (Asada, 1999; Foyer et al., 1994). Oxidative stress caused by ROS is
one of the major factors that bring about damage in plants exposed to environmental stresses such as
drought, salinity, extreme temperature, or strong light (Lim et al., 2007). The chloroplasts of plants
are a rich source of ROS (Asada, 1999). Two enzymes—superoxide dismutase (SOD) and ascorbate
peroxidise (APX)—are able to detoxify ROS in the chloroplasts. The removal of ROS suggests
that plants will exhibit increased tolerance to environmental stresses. Tang et al. (2006) and Lim et
al. (2007) developed transgenic potato and sweet potato, respectively, in which the genes for SOD
and APX were expressed in the chloroplasts under the control of an oxidative stress-inducible pro-
moter. The plants showed enhanced tolerance to high temperature (42°C for 20 h), oxidative, and
chilling stress. These results suggest that the manipulation of the antioxidative mechanism of the
chloroplasts may be applied in the development of transgenic crop plants with enhanced tolerance
to multiple environmental stresses.
17.7.1.4 Improvement in Nutritional Qualities

Improvement in the nutritional qualities of crops is now within the reach of crop improvement pro-
grams through biotechnology. We provide a few examples of the role of biotechnology in improving
the nutritional qualities of vegetative crops.
17.7.1.4.1 Generation of Cyanogen-Free Transgenic Cassava

Although cassava is used widely as a food crop, it contains toxic glucosides that are harmful to
human health. The roots, leaves, and stems of cassava contain potentially toxic levels of cyanogenic
glucosides (linamarin 95% and lotaustralin 5%). These cyanogens yield cyanide following enzy-
matic hydrolysis (Kakes, 1990; Koch et al., 1992; White et al., 1994). Currently, the cyanogen con-
tent of cassava is reduced to safe levels by maceration, soaking, rinsing, and frying (Siritunga and
Sayre, 2003, 2004). However, many health-related disorders occur when cassava is not pretreated
well before consumption. Chronic low-level cyanide exposure leads to the development of goiter and
tropical ataxic neuropathy, while acute cyanide poisoning is associated with outbreaks of Konzo, a
paralytic disorder and, in some cases, death (Rosling et al., 1992; Tylleskar et al., 1992). The devel-
opment of acyanogenic cassava cultivars will be a safe and effective way of making this food crop
safe and acceptable. Biotechnology has made it possible to identify the two genes that catalyze the
first step in the production of linamarin and lotaustralin (Anderson et al., 2000). These genes were
used in an antisense orientation to transform cultivar ‘MCol 2215’ of cassava. The transformed
plants were shown to have reduced levels of linamarin in the leaves (94%) and roots (99%). Further
studies in reducing cyanogens in cassava showed that transgenic cassava with a 92% reduction of in
cyanogenic glucoside content in tubers and acyagenic leaves could be obtained by RNA interference
that blocked the enzyme in the first committed step in the production of linamarin and lotaustralin
(Jorgensen et al., 2005). These acyanogenic cassava plants represent a safe and more marketable
product, demonstrating the value of biotechnology in making safer foods.
17.7.1.4.2 Nutritional Enhancement of Potato

Because the potato is an important highly nutritious vegetable and staple food, it has become an
excellent target for biotechnological research with regards to nutritional improvement, food com-
positional analysis, and production of dextrans. Improving the nutritional content of potato would
have a significant impact on vitamin and nutrient intake for millions of people who depend on the
crop for food. Potato has been engineered for enhanced nutritional value of specific carotenoids. For
example, overexpression of phytoene synthase resulted in 19-fold higher levels of lutein as well as
higher β-carotene levels (Ducreux et al., 2005), zeaxanthin contents were increased up to 130-fold
through disruption of zeaxanthin catabolism (Römer et al., 2002), and accumulation of astaxanthin
was achieved through expression of a microbial β-carotene ketolase gene (Morris et al., 2006).
Vitamin E (tocopherol) is a powerful antioxidant essential for human health and is synthesized
only by photosynthetic organisms. Humans and other animals cannot manufacture tocopherols and
obtain them from their vegetarian diet. It is estimated that approximately 90% of children and
adults in the United States do not consume the recommended amount of vitamin E (Ahuja et al.,
2004; Drewel et al., 2006). Efforts to enhance total tocopherol levels in plants have met with lim-
ited success but have raised important questions regarding the regulation of the tocopherol biosyn-
thetic pathway. Studying the development and tissue-dependent regulation of vitamin E synthesis
may provide more effective approaches for engineering enhanced vitamin E content in crop plants
(Crowell et al., 2008).
Genetic and molecular approaches were used to determine if increased levels of tocopherols
can be accumulated in potato tubers through metabolic engineering (Crowell et al., 2008). Since
vitamin E synthesis has only been studied in leaves and seeds thus far, potato tuber is a good
model system for studying the accumulation of vitamin E and the regulation of its synthesis in a
below-ground, nonphotosynthetic plant organ. Two transgenes were constitutively overexpressed in
potato: the Arabidopsis thaliana p-hydroxyphenylpyruvate dioxygenase (At-HPPD) and A. thaliana
homogentisate phytyltransferase (At-HPT). Tocopherol levels in the transgenic plants were deter-
mined by high-performance liquid chromatography. In potato tubers, overexpression of At-HPPD
resulted in a maximum 266% increase in x-tocopherol, and overexpression of At-HPT yielded a
106% increase. However, tubers from transgenic plants still accumulated approximately 10- and
100-fold less ct-tocopherol than leaves or seeds, respectively. The results indicate that physiological
and regulatory constraints may be the most limiting factors for tocopherol accumulation in potato
tubers and perhaps in other plants. Studying regulation and induction of tocopherol biosynthesis
should reveal approaches to more effectively engineer crops with enhanced tocopherol content
(Crowell et al., 2008).
17.7.2 Vegetatively Propagated Plants as Biofactories

Bioengineering of plants to produce various useful products has been in progress for over 20 years.
Many crops, including sugarcane and potato, are considered to have potential to be used as biofac-
tories for the production of pharmaceuticals and other useful products. In the following section we
examine how sugarcane and potato are being used for this purpose.
17.7.2.1 Sugarcane
Several efforts are underway to develop sugarcane as a biofactory system. In Australia, research-
ers have developed transgenic sugarcane producing polyhydroxybutyrate, a thermoplastic, and are
also working on other higher-value products (Brumbley et al., 2004). In the United States, efforts
are underway to produce pharmaceutical-grade proteins for human therapeutic use from sugarcane
(Holland-Moritz, 2003). Other research to produce high-value proteins in sugarcane is ongoing in
Brazil, South Africa, and elsewhere. Wang et al. (2005) outlined the advantages of sugarcane for
transgene/product containment that could position the crop as a “secure” platform for production
of pharmaceuticals. Commercial sugarcane is propagated vegetatively. In many places commercial
cultivars do not normally flower, so there is little chance of pollen drift or production of viable seed.
If viable seed were produced, it would not become mixed with seed supplies because commercial
fields are planted with vegetative pieces. Sucrose, the food commodity derived from sugarcane, is
sold as a refined crystal that is essentially free of protein, rather than a whole fruit or vegetable.
In the unlikely event that sugarcane producing a pharmaceutical protein was mixed into the food
supply, the food product (refined sucrose) would remain unaffected. Considering these benefits, sug-
arcane was tested for transgenic production of human granulocyte macrophage colony stimulating
factor (GM-CSF). GM-CSF is a cytokine that is produced in low concentrations by many cell types
throughout the human body (Metcalf, 1991). The purified protein is used for treatment of neutrope-
nia and aplastic anemia and is administered to bone marrow transplant patients to reduce infection
risk by accelerating the response of neutrophils (Dale et al., 1995; Shin et al., 2003). Recombinant
human GM-CSF protein has been produced in mammalian cells (Lee et al., 1985; Wong et al.,
1985a, 1985b; Okamoto et al., 1990), insect cells (Chiou and Wu, 1990), yeast (Ernst et al., 1987;
Balland et al., 1998), bacteria such as Escherichia coli (Libby et al., 1987), plant cell cultures such
as tobacco (James et al., 2000) and rice (Shin et al., 2003), and seeds of tobacco (Ganz et al., 1996;
Sardana et al., 2002) and rice.
Accumulation of GM-CSF protein ranged from undetectable to 0.02% of total soluble protein
in transgenic sugarcane plants. It was further shown that human bone marrow cells (TF-1), which
require GM-CSF for cell division, proliferated when growth media was supplemented with the
extracts from transgenic sugarcane. The sugarcane-produced protein had essentially identical activ-
ity levels as commercially produced GM-CSF. These results suggest that sugarcane may provide a
highly secure system for biofactory of pharmaceutical proteins.
Sugarcane has also been used as a model system for the production of industrials such as poly-
3-hydroxybutyrate (PHB). Brumbley et al. (2003) engineered the genes encoding the enzymes for
PHB production in sugarcane and showed that the leaves accumulated PHB up to 1.2% of their dry
weight without affecting plant growth. Transgenic sugarcane has also been used for the production
of p-hydroxy-benzoic acid that reached up to 7% of dry weight of leaves without any adverse affect
on the growth of the plants (McQualter et al., 2004).
17.7.2.2 Production of Dextran in Transgenic Potato

There is great interest in using plants for the production of novel polymers (Kok-Jacon et al., 2005).
Potato has been used for the production of fructan (Gerrits, 2000) and silk (Scheller et al., 2001) indi-
cating that potato, like sugarcane, can be used as bioreactors for the production of novel polymers.
Dextrans are important extracellular glucans. Commercially, dextran is produced by Leuconostoc
mesenteroides NRRL B-512F via fermentation at a large industrial scale since 1948 (Groenwall and
Ingelman, 1948). Dextrans are used in chromatographic media, as a soil conditioner (Murphy and
Whistler, 1973), and as biodegradable hydrogels (Hennink and van Nostrum, 2002). Dextran-based
gels are used for specific targeting of drugs to the colon, where they are degraded by a secreted
bacterial dextranase. Kok-Jacon et al. (2005) were successful in producing transgenic potato that
produced dextrans concentrations that were almost double that of untransformed plants, providing
further evidence of the power of biotechnology in plants.
17.7.2.3 Improved Cassava Starch

The demand for starch for industrial purposes is on the rise since it is used in paper, textile, medical,
and other applications. Starch is modified in many different ways to meet the criteria for various
applications. Cassava starch consists of two glucan polymers: amylose and amylopectin. Amylose
free-starches have different physicochemical properties compared to those that contain amylase and
they are used for manufacturing different products (Raemakers et al., 2005). For example, amylose-
free potato starch showed a higher granule melting temperature, less retrogradation, and better adhe-
sive properties that that containing amylase (Visser et al., 1997). The presence or absence of amylase
is controlled by the action of one enzyme, granule bound starch synthase I (GBSSI) (Raemakers et
al., 2005). Since conventional breeding of cassava is difficult, the transgenic approach was success-
ful in developing a genotype that produces amylase-free starch (Raemakers et al., 2005).
17.7.3 Risks Associated with Transgenic Crops

The rapid progress of transgenic biotechnology has accelerated the production of transgenic or GM
crops. While GM crops are considered to have great benefits in solving the problems of world food
security, GM technology is also purported to have serious biosafety concerns that have sparked
heated debates. The major issues concerning GM crops include the environmental consequences
of transgene flow from GM to non-GM cultivars and to its wild or weedy relatives. Movement of
genes from one population to another could occur via pollination between individuals of different
populations, seed dispersal between different populations, and through the dispersal of vegetative
organs between different populations.
The release of GM crops for commercial purposes has raised concerns including food, feed,
and human health safety caused by GM products, environmental safety, labeling of GM products,
and detection of possible transgene or derived protein in agricultural products, socioeconomical
and ethics concerns due to application of GM products and technology, regulatory procedures for
GM-related issues, general public perception or acceptance of GM products, and risk assessment
systems in relation to the environmental release and cultivation of GM products (Bao-Rong Lu,
www.icgeb.org/~bsafesrv/pdffiles/Bao-Rong.pdf, accessed 23 April 2010). Consequently, regula-
tory systems have been and are being established in many countries to assess plants produced by
genetic manipulation prior to commercial release. In the following section we examine some of
the risks associated with transgenic crops and the research that is being undertaken to address
these risks.
17.7.3.1 Risk of Transgenic Escape

Gene flow and introgression between cultivars and to wild and weedy relatives have always taken
place in most of the important crops in the world (Ellstrand et al., 1999). Consequently, the possibil-
ity of transgene flow from transgenic crop plants to wild relatives is a matter of international con-
cern (Ellstrand et al., 1999; Rieger et al., 2002). Generic studies are being carried out to ascertain
whether transgenes can move from GM crops to plants related to the crop that grow either outside
of cultivation, close to those crops, or as weeds within the crop (Bonnet et al., 2008). The chances
of hybridization with wild relatives of crops’ species vary with the crop. Some plants, such as the
potato, are cultivated in regions that are far away from the center of origin and the likelihood of
transgene escape to wild relatives is remote. However, the situation is different when transgenic
potato are developed and grown within the center of origin of the crop. Therefore each crop has
to be studied to assess its potential for outcrossing with wild relatives and transfer of the trans-
gene (Green et al., 2005). For example, to assess the risk of a transgene escaping from sugarcane
to related species, Bonnet et al. (2008) assessed the literature and reported that there has been a
demonstrated transfer of DNA from Zea mays, Sorghum, Erianthus, and Miscanthus and between
Saccharum species.
Current scientific literature has many similar studies for other crop species that will not be
addressed in this chapter.
17.7.3.2 Risks to Other Organisms

In 2005 insect-resistant crops expressing Bacillus thuringiensis (Bt) toxins represented approxi-
mately 29% of the estimated global area of transgenic crops of about 90 million hectares (James,
2005). Insect-resistant transgenic plants have been suggested to have unpredictable effects on the
biodiversity of the agro-ecosystem, including potential effects on insects’ natural enemies, ben-
eficial in control of crop pests. Although carnivorous as adults, many of these predators may also
consume plant tissues, in particular plant pollen and nectar. The coleoptera are important in terms
of agro-ecological research because of the large number of species in this order and for their role
as biological control agents. Therefore any detrimental impact on this group of insects would be
highly undesirable.
The first evidence that transgenic Bt-corn pollen caused significant mortality of the Monarch but-
terfly appeared in 2000 (Jesse and Obrycki, 2000). Laboratory experiments showed that Monarch
caterpillars fed on milkweed leaves dusted with Bt-corn pollen ate less, grew more slowly, and suf-
fered a higher mortality rate than those fed on leaves with normal pollen or with no pollen at all.
Since then, other studies have shown that Bt toxins do not cause any detrimental effects on some
beneficial coleopterans.
Commercial potato cultivars are highly susceptible to damage by the Colorado potato beetle
(CPB) (Leptinotarsa decemlineata). Conventional breeding of potato has not been successful in pro-
ducing a cultivar with resistance to this major pest. It is estimated that in 2005, 3.2 million pounds of
insecticide were used on 1.2 million acres of potato grown in the United States. The pesticides used
against the CPB are broad spectrum, killing not only the target pest but most of its natural enemies
(Ferry et al., 2007). The effects of transgenic potato expressing the coleopteran-specific Bacillus
thuringiensis–endotoxin Cry3A (Bt Cry3A) on the ladybird beetle Harmonia axyridis and the cara-
bid beetle Nebria brevicollis were investigated via the bitrophic interaction of the adult ladybird
with potato flowers and the tritrophic interaction of the carabid consuming a nontarget potato pest.
Immunoassays confirmed accumulation of the transgene product in potato leaves and floral tissues
at levels of up to 0.01% (pollen) and 0.0285% (anthers) of total soluble protein. Although H. axyridis
and N. brevicollis belonged to the targeted insect order, no significant effects were observed on the
survival or overall body mass change of either beetle. Furthermore, Bt Cry3A had no detrimental
effects on reproductive fitness of either beetle species, either in terms of fecundity or subsequent
egg viability. Behavioral analysis revealed no significant impact of Bt Cry3A on beetle activity or
locomotor behavior. Despite the potential for detrimental effects, in that the ladybird beetles belong
to the targeted order of insects and binding of Cry3A toxin to the gut was demonstrated, Cry3A
expressed in transgenic potato plants had no overall significant acute effects on survival, body mass
change, fecundity, or egg viability of H. axyridis.
Additionally L. oleracea was used as a model nontarget pest to assess the effects of tritrop-
hic prey-mediated exposure to insecticidal proteins expressed in transgenic plants. Despite
the potential for detrimental effects, in that carabids belong to the targeted order of insects,
Cry3A expressed in transgenic potato plants had no overall significant acute effects on survival,
body mass change, fecundity, or egg viability of N. brevicollis. In conclusion, this study shows
that the cultivation of transgenic potato expressing the Bt Cry3A toxin presents a low risk to
coleopteran insects other than the targeted chrysomelid larvae, due to the high specificity of
the toxin.
17.7.3.3 Safety of Biotech Foods

There has been much concern and debate about the safety of GM food for humans. Hence, rigor-
ous risk safety assessment has been recommended prior to commercial cultivation and market
approval of GM crops and products. Shepherd et al. (2006) have outlined the procedures for
assessing the safety of GM crops that are summarized in the following section. The first step in
the risk assessment strategy is to identify appropriate methodologies and approaches to compare
the GM crop or product with its non-GM counterparts. This comparison is the starting point of
the safety assessment, which then focuses on any intended or unintended differences identified.
The concept of substantial equivalence is based on the idea that an existing organism used as
food or feed with a history of safe use can serve as a comparator when assessing the safety of
genetically modified food/feed (OECD, 1993). An important part of this process is to evaluate
any compositional changes that might occur in the GM crop that might influence its nutritional
composition or toxic properties (Kuiper and Kleter, 2003; Howlett et al., 2003). It is important
that such comparisons should only be made with GM and non-GM counterparts grown under
the same regimes and environments. Where substantial equivalence does not occur, this does
not necessarily identify a hazard. Where a trait or traits are introduced with the intention of
modifying composition significantly and where the degree of equivalence cannot be considered
substantial, then the safety assessment of characteristics other than those derived from the intro-
duced trait(s) becomes of greater importance. It should be emphasized that unintended effects,
although known to occur in GM crops, also happen frequently in conventional plant breeding via
point mutations as well as through chromosomal recombination mechanisms (Mohan Jain, 2001).
Furthermore, unintended effects that occur in conventional plant breeding have traditionally been
identified and eliminated by removing lines that show inferior characteristics (such as reduced
yield or altered appearance) to established cultivars (Cellini et al., 2004, and references therein).
GM crops must meet the same criteria.
In substantial equivalence testing, the GM crop is grown side by side with the parental cultivar
or other controls in randomized plots, usually at more than one location. Then selected components
are measured in GM and control plants, and tested statistically for significant differences. Data from
these field trials may also be compared with historical data for conventional cultivars to indicate the
safe range for each component. In practice, the compounds selected are the important nutrients plus
specific antinutrients and toxicants known for that crop.
Targeted analysis of specific compounds that make an important contribution to the nutritional
value or safety of the crop species in question is the currently accepted approach to assess composi-
tional equivalence (OECD, 1993). However, more unbiased profiling technologies such as metabo-
lomics, proteomics, and transcriptomics are considered as emerging technologies that would extend
the breadth of comparative analyses, reduce uncertainty, and identify the need for further risk
assessment (Catchpole et al., 2005). Should new technologies be applied, then the expectation is
that all approaches are properly validated and that statistical analyses have been performed to the
highest standard (Kuiper et al., 2001).
Targeted compositional analysis was carried out on transgenic potato tubers of cultivars ‘Record’
or ‘Desirée’ to assess the potential for unintended effects caused by the genetic modification pro-
cess. The range of transgenic lines analyzed included those modified in primary carbohydrate
metabolism, polyamine biosynthesis, and glycoprotein processing. Controls included wild-type
tubers, tubers produced from plants regenerated through tissue culture (including a callus phase),
and tubers derived from transformation with the empty vector—that is, no specific target gene
included (with the exception of the kanamycin resistance gene as a selectable marker). Metabolite
analysis included soluble carbohydrates, glycoalkaloids, vitamin C, total nitrogen, and fatty acids.
Trypsin inhibitor activity was also assayed. Targeted compositional analysis revealed no consistent
differences between GM clones and respective controls. No construct specifically induced unin-
tended effects. Statistically significant differences between wild-type controls and specific GM
lines did occur but appeared to be random and not associated with any specific construct. Such
significant differences were also found between wild types and both tissue-culture-derived tubers
and tubers derived from transformation with the empty vector. This raises the possibility that soma-
clonal variation (known to occur significantly in potato, depending on genotype) may be responsible
for an unknown proportion of any differences observed between specific GM clones and the wild
type (Shepherd et al., 2006).
17.7.4 Marker-Free Transgenic Plants

Plant transformation is based on the ability to integrate foreign DNA into host-plant genomes and
on the regeneration efficiency of transformed cells into shoots or embryos (Putcha, 2003). The
transformation efficiency for many crops is low and selectable markers genes are used to identify
the transgenic plants. Two types of marker genes are currently being used: a selectable marker
gene (encoding antibiotic resistance, herbicide tolerance, and so on) and a marker gene used for
visual screening of potential transgenics (Akhond and Machray, 2009). Environmentalists and
consumer organizations have raised concerns about the use of the first type of selectable markers.
The concerns include the transfer of herbicide resistance genes to weeds by outcrossing (Dale,
2002). In addition, the presence of resistance genes against antibiotics in food products might the-
oretically lead to the spread of these resistances via the intestinal bacteria in human populations
(Puchta, 2003). These concerns have hindered the testing and widespread use of transgenic crops
in many countries, especially where transgenic crops can potentially increase food productivity.
Therefore studies to avoid markers genes or to eliminate them after use have been conducted, and
a growing number of methods are under development for the elimination of selectable markers
genes. Several methods developed for genetic modification of plants without foreign genes are
described by Akhond and Machray (2009). Native genes and regulatory elements can also be
introduced into plants without using selectable markers by using plant transfer DNAs (P-DNAs)
derived from a crossable donor plant of the same or closely related species (Rommens et al.,
2005, 2007). This approach referred to as the cisgenic or intragenic approach has been used in
the genetic modification of potato (Rommens et al., 2004). Requests have been initiated to alter
the legislation concerning transgenic and cisgenic plants (Havertkort et al., 2008). Transgenic
plants contain genes from plants or other species that could not be introduced through introgres-
sion (crossing). Cisgenic plants contain indigenous genes from crossable species. Cisgenes with
their native promoters introduced into a crop cultivar do not introduce new phenotypic traits into
a species, as they were there already. They do not introduce new fitness traits, so putatively will
not influence the environment or the risks in food or feed in another way than with traditional
breeding. Cisgenesis may even be safer than conventional breeding because it prevents introduc-
tion of genes via linkage drag, which could lead to all kinds of unwanted traits (Haverkort et al.,
2008). It is hopeful that the use of such marker-free techniques will make genetically modified
crops more acceptable to the public.
17.8 Conclusions
Vegetatively propagated crops are staple foods in many of the poorer countries of the world.
The world’s population is headed for 9.2 billion people by 2050. The challenge of doubling food
production in the next 40 years has become a daunting task. We have shown that most vegeta-
tively propagated crops have complex genetic systems, and conventional breeding alone will not
achieve the development of new improved cultivars. Molecular breeding can play an important
role in realizing the full potential of difficult-to-breed vegetatively propagated crops. However,
there appear to be many hurdles. Development of efficient genotype-independent transforma-
tions systems is the ultimate goal for the production of transgenic crops. This remains elusive
due to varying morphogenic responses of some crops. The development of MAS has not reached
the stage where it has become routine in breeding programs. While newer and faster techniques
for developing molecular markers keep emerging, the so-called orphan crops lag far behind in
this MAS.
Although the development of high-density maps for crops such as potato, rice, and so forth is
forging ahead, there is very little research support for development of maps of other crops that feed
the majority of the poor of this world. In this regard researchers of such crops can take advantage
of concepts such as genome snyteny. Comparative mapping of related species has shown that colin-
earity and synteny exist in the arrangement of genes. The degree of genome conservation observed
between species provides the possibility of transferring genetic information and mapping resource
from well-characterized genomes to less studied and more complex genomes (Asnaghi et al., 2000).
For example, knowledge of sugarcane, sorghum, maize, and rice genetic maps enables researchers
to identify chromosome segments corresponding to the region that harbors the rust-resistance gene
in sugarcane (Asnaghi et al., 2000).
This chapter provides evidence of the role of molecular breeding with regards to the development
of crops with disease and pest resistance. Biotechnology is playing an important role in improving
the nutritional qualities of some crops that are also being used as biofactories to produce useful
products for man. One of the major concerns of GM crops is the safety it poses for human health
and for the environment. It is encouraging to see that research to address these problems is ongoing
and strict guidelines are being developed for GM foods.
Genetically modified crops have been commercial on a substantial scale for over 10 years.
There is evidence to show that the technology has reduced pesticide spraying and decreased the
environmental impact associated with herbicide and insecticide use. GM technology has reduced
the release of greenhouse gases and has produced net economic benefits at the farm level. Despite
these benefits, the debate on the adoption of GM foods still continues. Such debates can only act
to stimulate new research such as the development of marker-free transgenic plants and research
on cisgenics.
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18 Future Prospects
Rodomiro Ortiz, Michael Pillay, and Abdou Tenkouano
The preceding chapters of this book deal with several subjects: botany, evolution, genetic resources,
cytogenetics, genetics, plant health management, genetic enhancement, postharvest processing,
micropropagation, and delivery of new cultivars to farming systems. The last chapter provides an
update on molecular breeding of asexual crops from which the next genetic enhancement schemes can
be sought. This brief chapter builds on the overviews given by the specialists in each of the contribut-
ing chapters by summarizing their major findings and providing an outlook on future research.
The most significant advances in Musa botany in recent years are on root systems and bunch
morphology (Chapter 1). Research on the former was driven by the needs of defining an ideotype
for breeding better root systems in Musa at large, but especially for plantains. Experiments during
the second half of the 1990s in the degraded forest of West Africa and in the East African highlands
led to a better understanding of the relationships between root and shoot traits, the variability in
root system sizes, the biophysical effects on root development, and devising alternative methods
for root evaluation. The study of bunch morphology of East African Highland bananas (Lujugira-
Mutika) provided means for clustering them into four morphologically distinct clone sets. The sets
bring together clones that share traits important to farmers and consumers, though they were not
previously used for grouping this diverse Musa germplasm. Further research should focus on find-
ing DNA markers linked to important underground and shoot traits for their further selection. DNA
markers can also be used for gaining insights onto the evolution of bananas and the role that somatic
mutations play on it, which will need to go hand in hand with plant morphology research to establish
sound evolutionary pathways and relationships as a result of both human and natural selection.
In the last two decades, molecular markers have been extensively used for characterization and
classification of Musa germplasm collections (Chapter 2). Nonetheless, these advances brought by
molecular taxonomy will need to consider both complementary and contrasting morphology data,
which will be required to resolve the relationships and phylogeny in Musa. Researchers on this sub-
ject should use numerical taxonomy to bring together morphological and molecular data, which will
be supplemented by cytogenetic methods. Such an integrated approach will allow unraveling rela-
tionships among Musa species and cultivars, which are complicated by interspecific hybridization,
heterozygosity, and polyploidy, that are common in this genus. Priority research with DNA mark-
ers should focus on M. balbisiana to understand its evolution and systematics, and on the parental
contributions of M. acuminata subspecies to the origin of triploid banana and plantain cultivars. A
better understanding and knowledge on Musa origin, domestication, and evolution will facilitate the
genetic enhancement of this crop.
The third chapter deals with the genetic resources of wild-seeded bananas. Recent research on
Musa wild species gave priority to establishing the boundary between sections and among species
therein. Today, Australimusa, Callimusa, Musa (which includes the edible bananas), Rhodochlamys,
and Ingentimusa are the five known sections of Musa. Their species are a great reservoir of impor-
tant resistance traits that can be further used for breeding new edible bananas. Further research with
DNA markers and geo-referenced systems may assist in understanding their demographic history
and dispersal mechanisms, and how they account for Musa genetic diversity among populations and
across geographical regions.
Chapters 4 and 5 provide state-of-the-art knowledge of banana cytogenetics and genetics. Musa
species are among the best examples of organisms whose complex cytogenetic structure ensued
351
through extensive chromosomal rearrangements. Since the 1990s a wealth of knowledge, facilitated
by the reemergence of breeding programs and new tools, particularly ensuing from advances in
molecular biology, became available for genome identification, ploidy analysis, and trait inheritance.
In situ hybridization, DNA markers, and flow cytometry were the tools used for molecular cyto-
genetic research in the last 20 years. The molecular characterization of the Musa genome and the
development of physical maps are therefore suggested topics for further cytogenetic research. The
prospects of elucidating genetic mechanisms controlling important traits will undoubtedly acceler-
ate banana germplasm enhancement in the “omics” era. The sequencing of the banana genome will
bring new knowledge in breeding this crop. With the aid of bioinformatics, inter- and intragenome
comparisons may overcome the lack of high-resolution physical and genetic maps in banana.
Several pathogens (Chapter 6) and pests (Chapter 7) affect the crop and cause yield losses.
Sigatoka leaf spots, Fusarium wilt, banana weevil, and parasitic nematodes are of worldwide impor-
tance, while new emerging diseases such as banana Xanthomonas wilt or banana bract mosaic are
regional constraints for growing bananas in East and Central Africa, and Asia, respectively. Host-
plant resistance remains the cornerstone for banana health management, which along with cultural
practices and biological control provides the means for an integrated approach to overcome many
biotic stresses affecting the crop. Identifying sources of host-plant resistance to the main pathogens
and pests in Musa germplasm for further use in banana breeding will remain an important research
thrust in forthcoming years.
Reproductive biology, which is central to the species breeding system and for success of banana
breeding schemes, is the subject of Chapter 8. Little research has appeared in the literature on Musa
in recent years, though the knowledge of sexual plant reproduction of other species progressed con-
siderably in the same period. Most of the recent research was on West African plantains and East
African Highland bananas. The studies were on male and female fertility, as well as on 2n gametes,
hybridization, seed set, and germination, which are very important traits for the genetic enhancement
of the crop. New research should address why the low seed set occurs in cultivars and how to improve
seed germination rates, which remain among the main bottlenecks for banana crossbreeding.
Not surprisingly, five chapters are devoted to the core of this book: the genetic enhancement of
banana. They provide comprehensive overviews on crossbreeding techniques (Chapter 9), the use
of mutations in cultivar development (Chapter 10), biotechnology approaches (both transgenics and
genomics led) for breeding (Chapter 11), genotype-by-environment interactions (Chapter 12), and
quality improvement and biofortification (Chapter 13). Although significant progress was made in
the past two decades, a few hybrid cultivars (mostly tetraploids) are grown in significant acreages by
banana farmers, and a handful of field experiments are known for transgenic bananas. The focus of
banana breeding seems to be gradually shifting from addressing existing constraints to assessing the
risk potential of emerging threats and preparing to respond to them, particularly under a changing
climate due to global warming that may increase both abiotic and biotic stress incidences and sever-
ity in the crop. Emphasis for banana genetic enhancement should be given to preemptive breeding—
particularly through broadening approaches—to deal with new strains of major pathogens and pests,
as well as other emerging constraints. Nutritional quality should be added as a priority target trait,
along with host-plant resistance and stabilizing yield traits, for banana breeding. Likewise palat-
ability (according to consumers’ preferences) and value-adding traits during postharvest processing
(Chapter 14) cannot be ignored in banana breeding undertakings. High-yielding and stabilizing
traits will allow surplus harvests in small landholders’ fields. They can sell such an excess of fresh
produce to nearby or other markets to improve their incomes. The ongoing analysis and sequencing
of the banana genome, identification of its genes and their expression, recombination, and diversity
will provide more effective and efficient means for the genetic enhancement of this crop. Genomics-
led and evolutionary breeding approaches can indeed pave the way for banana breeding-by-design.
Large-scale propagation methods of true-to-type materials (Chapter 15) are needed for both
banana breeding and delivery systems. More research should focus on low-cost methods for gener-
ating new breeding materials and for delivering outstanding clones to the farmers. The management
Future Prospects 353
of the genotype-by-environment interaction remains crucial in order to have efficient selection

schemes and reach end users, whereas multilocation testing of selected genotypes should be man-
datory prior to release of improved cultivars (Chapter 16). The farmers’ cultural and economic
contexts will also dictate their cultivar choice for adoption. Biophysical and socioeconomic factors
need to elucidate how pest-resistant cultivars can be introduced into the farmers’ cropping system
through association with their own landraces and other crops. Likewise policy research cannot be
ignored because the level of adoption of new banana cultivars appears to be associated with an
induced model of adoption where farmers are able to assess the yield before embarking on the cul-
tivation of the new cultivars and are thereafter supported with educational materials and incentives
for adopting the new technology. Governmental intervention could be often needed through policy
initiatives that should go beyond the ad hoc responses triggered by pest outbreaks.
Molecular breeding in banana has not kept pace with progress that is occurring in some of
the other major food crops. Chapter 17 highlights some of the progress in using biotechnology to
improve some traits in potato, cassava, and sugarcane. Breeding of these vegetatively propagated
crops faces similar constraints as those faced by banana breeders—yet greater progress has been
made in the development of molecular markers for disease and pest resistance and abiotic stresses,
and in constructing molecular maps in the three crops addressed in this chapter. This chapter pro-
vides valuable insights for researchers wishing to improve banana and plantains.

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Banana Breeding

Boca Raton London New York

CRC Press is an imprint of the

No claim to original U.S. Government works

Printed in the United States of America on acid-free paper

International Standard Book Number: 978-1-4398-0017-1 (Hardback)

Chapter 1 General Plant Morphology of Musa..............................................................................1

Chapter 2 Evolution and Genetic Relationships in Banana and Plantains................................... 21

Chapter 3 Genetic Resources for Banana Improvement.............................................................. 41

Chapter 4 Genomes, Cytogenetics, and Flow Cytometry of Musa............................................. 53

Chapter 5 Genetics of Important Traits in Musa......................................................................... 71

Chapter 6 Major Diseases of Banana........................................................................................... 85

Chapter 7 Integrated Pest Management of Banana.................................................................... 121

Chapter 8 Reproductive Biology................................................................................................ 145

Chapter 9 Breeding Techniques................................................................................................. 181

Chapter 10 Mutations and Cultivar Development of Banana...................................................... 203

Chapter 11 Biotechnology in Musa Improvement....................................................................... 219

Chapter 12 Genotype by Environment Interaction and Musa Improvement............................... 237

Chapter 13 Quality Improvement of Cultivated Musa................................................................ 251

Chapter 14 Postharvest Processed Products from Banana.......................................................... 269

Chapter 15 Propagation Methods in Musa.................................................................................. 285

Chapter 16 Hybrid Distribution to Farmers: Adoption and Challenges...................................... 305

Chapter 17 Molecular Breeding of Other Vegetatively Propagated Crops: Lessons for

Chapter 18 Future Prospects........................................................................................................ 351

Abdou Tenkouano (BSc, MSc, PhD) is the director of the

Rodomiro Ortiz, PhD Abdou Tenkouano, PhD

Nicolas Roux, PhD Bradley Till, PhD

David Talengera, MSc

Sword sucker Daughter sucker

Figure 1.1 The morphology of the Musa plant: a mat or stool.

1.2 Botanical Description

1.2.2 The Pseudostem

AAA-EA AAB Plantain ABB Bluggoe

1.2.3 The Suckers

1.2.4 The Leaves

1.2.5 The Inflorescence

1.2.6 Bunch Morphology

AA, AAA ABB

AAB Pome AAB Plantain

1.3 The Underground System

1.3.1 The Corm

1.3.2.1 The Considerable Size of the Musa Root System

1.3.2.2 The Effect of Soil and Climate on Root Systems

1.3.2.3 Root Branching: The Lateral Roots

1.3.2.4 Allocation of Dry Matter to the Shoot and Root System

100% Mbi Egome

1.3.2.5 Genetic Variability in Root Growth and Relationships

1.3.2.6 Alternative Methods for Root System Assessment

1.4 Value of Morphological Variation

1.4.2 Sources of Morphological Variation in Cultivated Musa

2.2 Growth of Global Banana Industry

2.3 Consumption and Utilization of Banana

2.3.1 Importance of Banana as an Edible Fruit

2.3.2 Banana as an Alternative Source of Income

2.3.3 Indigenous Technical Knowledge (ITK)

2.5 Origin and History

2.5.1 Origin of Diploid Bananas

1.3.2.5 Genetic Variability in Root Growth and Relationships