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PLANT TISSUE CULTURE : A PROMISING TOOL OF QUALITY MATERIAL

PRODUCTION WITH SPECIAL REFERENCE TO MICROPROPAGATION OF BANANA

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Biochem. Cell. Arch. Vol. 17, No. 1, pp. 1-26, 2017 www.connectjournals.com/bca ISSN 0972-5075

Review Article
PLANT TISSUE CULTURE : A PROMISING TOOL OF QUALITY MATERIAL
PRODUCTION WITH SPECIAL REFERENCE TO MICROPROPAGATION
OF BANANA
Sugandh Suman
Department of Agricultural Biotechnology & Molecular Biology, Faculty of Basic Sciences & Humanities,
Dr. Rajendra Prasad Central Agricultural University, Pusa, Samastipur - 848 125, India.
e-mail : [email protected]
(Accepted 10 January 2017)

ABSTRACT : In the very fast developing scenario of biological science, the plant tissue culture has taken lead as the most
promising areas of application of biotechnological tools for today and tomorrow agriculture. The areas ranges from
micropropagation of horticultural crops, ornamental and forest trees etc., production of pharmaceutically important compounds,
and plant breeding for improved nutritional value of staple crop plants, including trees for cryopreservation of valuable germplasm.
The rapid production of high quality, disease free and uniform planting stock is only possible through micropropagation. Plant
production can be carried out throughout the year irrespective of season and weather. However micropropagation technology is
expensive as compared to conventional methods of propagation by means of seed, cuttings and grafting etc. Therefore, it is
essential to adopt measures to reduce cost of production. Low cost production of plants requires cost effective practices and
optimal use of equipment to reduce the unit cost of plant production. It can be achieved by improving the process efficiency and
better utilization of resources. Use of ‘Bioreactor’ in plant propagation can increase the speed of multiplication and growth of
cultures and reduce space, energy and labor requirements. The cost of production may also be reduced by selecting several
plants that provide the option for around the year production and allow cost flow and optimal use of equipment and resources.
Quality control is also very essential to assure high quality plant production and to obtain confidence of the consumers. The
selection of explant source, diseases free material, authenticity of variety and elimination of somaclonal variants are some of the
most critical parameters for ensuring the quality of the planting materials. The in vitro culture has a unique role in sustainable
and competitive agriculture, forestry and pharmaceutical industry and has been successfully applied in plant breeding for rapid
introduction of improved plants. Plant tissue culture has become an integral part of plant breeding. At present plant cell culture
has made great advances. Possibly the most significant role that plant cell culture has to play in the future will be in its
association with transgenic plants. The ability to accelerate the conventional multiplication rate can be of great benefit to many
crops/countries where a disease or some climatic disaster wipes out crops. The loss of genetic resources is a common story
when germplasm is held in field gene banks. In vitro storage using plant tissue culture tools and cryopreservation are being
proposed as solutions to the problems inherent in field gene banks. By these means the future generations will be able to have
access to genetic resources for simple conventional breeding programmes, or for the more complex genetic transformation
work. As such, plant tissue culture has a great role to play in agricultural development and productivity. In this review,
important steps of plant tissue culture, its critical precautionary points and commercial applications have been discussed.
As Banana is an important food crop and the second most important fruit crop after mango, a special account has been taken into
consideration to also put on record the steps involved in successful micropropagation of it. Despite the significant commercial
value of the banana crop, the main production constraint is the availability of reliable and safe planting material. The planting
materials obtained through conventional methods (suckers) do not meet the increasing demand for planting and they are of poor
quality. Tissue culture is the approach which can solve these problems. Micropropagation of the planting material is also facing
the different challenges which need to be addressed in order to improve its quality production. Some of the problems which
impair the success of the crop include oxidative browning of the wounded tissues and low number of shoots produce per explant.
This review includes the micropropagation studies of commercially important cultivars of banana in the country, highlights the
challenges encountered in its tissue culture and explores the possibilities of optimization of the in vitro propagation techniques
by using explants from shoot tip.
Key words : Plant tissue culture, micropropagation, banana.
2 Sugandh Suman
1. INTRODUCTION (George, 1993; Suman et al, 2015) which leads to the
The inception of science of plant tissue culture takes development of commercially important improved
its roots from the discovery of cell followed by propounding varieties. Commercial production of plants through
of cell theory. In 1838, Schleiden and Schwann proposed micropropagation techniques has several advantages over
that cell is the basic structural unit of all living organisms. the traditional methods of propagation through seed,
They visualized that cell is capable of autonomy and cutting, grafting and air-layering etc. It is rapid propagation
therefore it should be possible for each cell if given an processes that can lead to the production of virus free
environment to regenerate into whole plant. Based on plants (Garcia-Gonzales et al, 2010). Meristem tip culture
this premise, in 1902, a German physiologist, Gottlieb of banana plants devoid from banana bunchy top virus
Haberlandt for the first time attempted to culture isolated (BBTV) and brome mosaic virus (BMV) were produced
single palisade cells from leaves in knop’s salt solution (El-Dougdoug and El-Shamy, 2011). Higher yields have
enriched with sucrose. The cells remained alive for up to been obtained by culturing pathogen free germplasm in
one month, increased in size, accumulated starch but failed vitro. Increase in yield up to 150% of virus-free potatoes
to divide. Though he was unsuccessful but laid down the was obtained in controlled conditions (Singh, 1992).
foundation of tissue culture technology for which he is Agricultural diversification to meet our future needs
regarded as the “father” of plant tissue culture. Plant call for the adoption of new technologies in agriculture.
tissue culture is the in vitro aseptic culture of cells, tissues, Utilization of the best cultural practices, fertilization, pest
organs or whole plant under controlled nutritional and control measures will not give the necessary results
environmental conditions often to produce the clones of without the use of best planting material. Now a day,
plants (Thorpe, 2007). The resultant clones are true-to tissue culture is adopted as a viable horticultural
type of the selected genotype. The controlled conditions propagation method which has revolutionized the
provide the culture an environment conducive for their horticultural industry. Mass propagation and the
growth and multiplication. These conditions include proper establishment of disease free stock material is
supply of nutrients, pH medium, adequate temperature accomplished by use of this technique (Bachraz, 1998).
and proper gaseous and liquid environment. Plant tissue Tissue culture is a method of vegetative propagation
culture technology is being widely used for large scale based on biotechnology. The plants are derived from stem,
plant multiplication. Apart from their use as a tool of root or leaf tissues and the technology generally aids in
research, plant tissue culture techniques have in recent mass production of desired crop varieties. Tissue culture
years, played an important role in the area of plant is also useful in regeneration of genetically modified cells
propagation, disease elimination, plant improvement and into whole plants as well as in embryo rescue techniques
production of secondary metabolites, using small pieces (Bio Vision, 2008). Ilan and Workafes (2011) have put
of tissue (named explants) to produce hundreds and on record the advantages and disadvantage of this method
thousands of plants in a continuous process. A single as that controlled environment and controlled
explant can be multiplied into several thousand plants in development of the plants enable very rapid multiplication
relatively short time period and space under controlled rate and clean conditions for plant development that
conditions, irrespective of the season and weather on a produce micro-plants free of many pests and diseases.
year round basis (Akin-Idowu et al, 2009). Endangered, The small size of the propagated plants saves nursery
threatened and rare species have successfully been space and plant transport costs. Reproducing the planting
grown and conserved by micropropagation because of materials of vegetatively propagated crops presents
high coefficient of multiplication within less space. In complex problems because of lack of knowledge of
addition, it is also considered to be the most viable phyto-sanitary measures and quarantine issues related
technology for crop improvement by the production of to safe movement of germplasm, plants and planting
somaclonal and gametoclonal variants. The material across national borders, lack of consistent
micropropagation technology has a vast potential to supplies of good quality planting material, variable demand
produce plants of superior quality, isolation of useful for clean planting material, bulkiness and perishability of
variants in well-adapted high yielding genotypes with better planting materials and use of traditional varietal mixtures,
disease resistance and stress tolerance capacities (Brown including local varieties (Wambugu and Kiome, 2001).
and Thorpe, 1995; Suman et al, 2012). Certain type of The prime basic objective of this review is to describe
callus cultures give rise to clones that have inheritable the tissue culture techniques, various developments,
characteristics different from those of parent plants due present and future trends and its application in various
to the possibility of occurrence of somaclonal variability fields with particular reference of banana.
 Micropropagation of banana 3
2. TECHNOLOGICAL INNOVATIONS IN a short period, propagation can be carried out throughout
PLANT TISSUE CULTURE the year and the propagating material can be
2.1 Know-How accommodated in a small space, reduction of labour costs
for germplasm maintenance, avoidance of field inspections
In the process of plant cell culture, plant tissues and
& environmental hazards, easy availability of material
organs are grown in vitro on artificial media, under aseptic
for micro propagation and rapid multiplication (Mtui, 2011).
and controlled environment. The technique depends
According to Chadha and Choudhary (2010) various
mainly on the concept of totipotentiality of plant cells
prerequisite steps involved in production of pathogen free
which refers to the ability of a single cell to express the
plantlets by meristem tip culture are: testing of parent
full genome by cell division (Haberlandt, 1902). Along
material for the presence of viruses and similar pathogens
with the totipotent potential of plant cell, the capacity of
(viroids and phytoplasmas), thermotherapy/chemotherapy
cells to alter their metabolism, growth and development
of parent material if disease-free material is not available,
is also equally important and crucial to regenerate the
excision of meristem tip under aseptic conditions, culture
entire plant (Thorpe, 2007). Plant tissue culture medium
of apical dome plus one or two leaf primordia on suitable
contains all the nutrients required for the normal growth
medium to produce plantlets, indexing of plantlets for
and development of plants. It is mainly composed of
presence or absence of viruses, plantlets transferred to
macronutrients, micronutrients, vitamins, other organic
soil, maintenance of pathogen free nuclear plant stocks
components, plant growth regulators, carbon source and
and meristem culture is then followed by in vitro mass
some gelling agents in case of solid medium (Murashige
propagation of the virus-free plants thus obtained.
and Skoog, 1962). Murashige and Skoog medium (MS
medium) is most extensively used for the vegetative 2.2 Technological Protocols
propagation of many plant species in vitro. The pH of In recent past very fast developments have been
the media is also important that affects both the growth taken placed in the area of plant cell culture-cum-
of plants and activity of plant growth regulators. It is microprogation with view of its viable commercial
adjusted to the value between 5.4 - 5.8. Both the solid applications. For the purpose of micropropagation of
and liquid medium can be used for culturing. The planting materials the procedure of it starts with the
composition of the medium, particularly the plant hormones selection of plant tissues (explant) from a disease free
and the nitrogen source has profound effects on the healthy, vigorous mother plant (Murashige, 1974). Any
response of the initial explant. Plant growth regulators part of the plant (leaf, apical meristem, bud and root) can
(PGRs) play an essential role in determining the be used as explant. All the steps can be summarized into
development pathway of plant cells and tissues in culture the following stages as shown in Figure 1 as documented
medium. The auxins, cytokinins and gibberellins are most by Hussain et at (2012).
commonly used plant growth regulators. The type and 2.2.1 Stage 0: Preparation of donor plant- To
the concentration of hormones used depend mainly on enhance the probability of success, the mother plant
the species of the plant, the tissue or organ cultured and should be ex vitro cultivated under optimal conditions to
the objective of the experiment (Ting, 1982). The high minimize contamination in the in vitro culture (Cassells
concentration of auxins generally favors root formation, and Doyle, 2005).
whereas the high concentration of cytokinins promotes
2.2.2 Stage I: Initiation stage- In this stage an
shoot regeneration. A balance of both auxin and cytokinin
explant is surface sterilized and transferred into nutrient
leads to the development of mass of undifferentiated cells
medium. Generally, the combined application of
known as callus.
bactericide and fungicide products is suggested. The
The propagation from meristematic tissue generally selection of products depends on the type of explant to
provides a method of cleaning up material from viruses be introduced. The surface sterilization of explant in
and other systemic pathogen infections. Micropropagation chemical solutions is an important step to remove
is a tissue culture (in vitro) method used for rapid and contaminants with minimal damage to plant cells (Hussain
true to type multiplication of plants on artificial nutrient and Anis, 2009). The most commonly used disinfectants
media under controlled environment and it is the most are sodium hypochlorite (Tilak et al, 2009), calcium
commercially exploited area of plant tissue culture, being hypochlorite (Garcia et al, 1999), ethanol (Singh and
widely used for production of quality planting material in Gurung, 2009) and mercuric chloride (Hussain and Anis,
vegetative propagated species. The most significant 2009). The cultures are incubated in growth chamber
advantages offered by micropropagation are production either under light or dark conditions according to the
of large of disease free propagules from a single plant in
4 Sugandh Suman
method of propagation. proliferation of somatic embryos. Highest efficiency of
2.2.3 Stage II: Multiplication stage- This phase embryonic callus was induced by culturing nodal stem
is to increase the number of propagules under which the segments of rose hybrids on medium supplemented with
number of propagules is multiplied by repeated subcultures various PGRs alone or in combination (Xiangqian et al,
until the desired number of plants is attained (Saini and 2002). The embryonic callus showed high germination
Jaiwal, 2002). rate of somatic embryos when grown on abscisic acid
(ABA) alone. Somatic embryogenesis is not only a
2.2.4 Stage III: Rooting stage- The rooting stage
process of regenerating the plants for mass propagation
may occur simultaneously in the same culture media used
but also regarded as a valuable tool for genetic
for multiplication of the explants. However, in some cases
manipulation. The process can also be used to develop
it is necessary to change media, including nutritional
the plants that are resistant to various kinds of stresses
modification and growth regulator composition to induce
(Bouquet and Terregosa, 2003) and to introduce the genes
rooting and the development of strong root growth.
by genetic transformation (Maynard et al, 2003). A
2.2.5 Stage IV: Acclimatization stage- At this successful protocol has been developed by using this tool
stage, the in vitro plants are weaned and hardened. for regeneration of cotton cultivars with resistance to
Hardening is done gradually from high to low humidity Fusarium and Verticillium wilts (Han et al, 2009).
and from low light intensity to high light intensity. The
3.2 Organogenesis
plants are then transferred to an appropriate substrate
(sand, peat, compost etc.) and gradually hardened under It refers to the production of plant organs i.e. roots,
greenhouse. shoots and leaves that may arise directly from the
meristem or indirectly from the undifferentiated cell
3. RECENT ADVANCES IN PLANT TISSUE
masses (callus). Plant regeneration via organogenesis
CULTURE
involves the callus production and differentiation of
Processes of somatic embryogenesis and adventitious meristems into organs by altering the
organogenesis are the recent development in the area concentration of plant growth hormones in nutrient
application of plant tissue culture by which plant medium. Skoog and Miller (1957) were the first who
regeneration takes place. demonstrated that high ratio of cytokinin to auxin
3.1 Somatic Embryogenesis stimulated the formation of shoots in tobacco callus while
It is an in vitro method of plant regeneration widely high auxin to cytokinin ratio induced root regeneration.
used as an important biotechnological tool for sustained 4. CRITICAL FACTORS INFLUENCING IN
clonal propagation (Park et al, 1998). It is a process by VITRO GROWTH
which somatic cells or tissues develop into differentiated 4.1 Choice of explant
embryos. These somatic embryos can develop into whole
plants without undergoing the process of sexual The tissue which is obtained from the plant to culture
fertilization as done by zygotic embryos. The somatic is called an explant. Based on work with certain model
embryogenesis can be initiated directly from the explants systems, particularly tobacco, it has often been claimed
or indirectly by the establishment of mass of unorganized that a totipotent explant can be grown from any part of
cells named callus (Suman and Kumar, 2016). Plant the plant. In many species, explants of various organs
regeneration via somatic embryogenesis occurs by the vary in their rates of growth and regeneration, while some
induction of embryogenic cultures from zygotic seed, leaf do not grow at all. The choice of explant material also
or stem segment and further multiplication of embryos. determines if the plantlets developed via tissue culture
Mature embryos are then cultured for germination and are haploid or diploid. Also the risk of microbial
plantlet development, and finally transferred to soil. contamination is increased with inappropriate explants.
Somatic embryogenesis has been reported in many plants Thus it is very important that an appropriate choice of
including trees and ornamental plants of different families. explant be made prior to tissue culture. The specific
There are various factors that affect the induction and differences in the regeneration potential of different
development of somatic embryos in cultured cells. A organs and explants have various explanations. The
highly efficient protocol has been reported for somatic significant factors include differences in the stage of the
embryogenesis on grapevine (Jayasankar et al, 1999) cells in the cell cycle, the availability of or ability to
that showed higher plant regeneration sufficiently when transport endogenous growth regulators and the metabolic
the tissues were cultured in liquid medium. Plant growth capabilities of the cells. The most commonly used tissue
regulators play an important role in the regeneration and explants are the meristematic ends of the plants like the
 Micropropagation of banana 5

Fig. 1 : Step-wise summary of micropropagation via tissue culture.

Fig. 2 : Steps involved in hybrid plant production via protoplast fusion.

6 Sugandh Suman
stem tip, auxiliary bud tip and root tip because these tissues produced numerous plants (> 50 per explant) when the
have high rates of cell division and either concentrate or thickness of the explant was reduced to 1 to 2 mm. This
produce required growth regulating substances including finding clearly indicates that the explant size plays a key
auxins and cytokinins. Some explants, like the root tip, role in the expression of organogenic potential of the
are hard to isolate and are contaminated with soil cultured tissue. This explants size-based difference in
microflora that become problematic during the tissue organogenic capacity has since been successfully utilized
culture process. Certain soil micro-flora can form tight to develop thin section culture system, a novel approach
associations with the root systems, or even grow within in plant regeneration
the root. Soil particles bound to roots are difficult to remove 4.3 In vitro Environmental Conditions
without injury to the roots, a circumstance that then allows
Some interesting points of basic research that could
microbial attack. These associated microfloras will
improve our understanding and hence our ability to control
generally overgrow the tissue culture medium before
in vitro plant regeneration and development remain under-
there is significant growth of plant tissue. Aerial (above
explored such as by recirculating the liquid culture systems
soil) explants are also rich in undesirable microflora.
it will be feasible to monitor and continuously regulate
However, they are more easily removed from the explant
the medium composition. We need to know more about
by gentle rinsing and the remainder usually can be killed
the dynamics of mineral nutrition in vitro (Williams, 1995).
by surface sterilization. Most of the surface microflora
Light quality is one of the potentially important
does not form tight associations with the plant tissue. Such
environmental factors, as it has been shown to affect the
associations can usually be found by visual inspection as
direction of plant morphogenesis in vitro (Morini et al,
a mosaic, de-colorization or localized necrosis on the
2000) and it also plays role in between gametophytic and
surface of the explant. An alternative for obtaining
sporophytic pathways. Tissue culture processes often
uncontaminated explants is to take explants from seedlings
involves extensive cutting and stress injury of tissues
which are aseptically grown from surface sterilized seeds.
which may cause physiological changes in plants
The hard surface of the seed is less permeable to
affecting the success response (Leon et al, 2001).
penetration of harsh surface sterilizing agents, such as
hypochlorite, so the acceptable conditions of sterilization 5. SCIENTIFIC-CUM-COMMERCIAL
used for seeds can be much more stringent than for APPLICATIONS OF PLANT TISSUE CULTURE
vegetative tissues. TECHNOLOGY
4.2 Explant Size and Thin Section Culture System Today plant tissue culture applications encompass
much more than clonal propagation and micropropagation.
The induction of a desired morphogenic event in
The range of routine technologies has expanded to include
vegetative tissues by appropriate in vitro manipulations
somatic embryogenesis, somatic hybridization, virus
would probably be the most significant advancement in
elimination as well as the application of bioreactors to
plant tissue culture. The success in achieving such
mass propagation and production of secondary
directed morphogenic events are largely determined by
metabolites. The applications of plant tissue culture may
the cultured tissue itself. Several explant-related factors
be categorized into two areas of common interest:
appear to influence the organogenic potential of the
cultured tissue (Benson, 2000). These include growth 5.1 In Agricultural Science
conditions, whole plant physiology and genotype of the Plant tissue culture is used widely in plant science
source plant. In addition, a negative correlation between with its commercial applications as- screening cells rather
the explants size and the number of cells potentially than plants for advantageous characters, e.g. herbicide
available for organogenesis has also been recognized resistance/ tolerance; large scale growth of plant cells in
(Lakshmanan et al, 1995 & 1996, ). In an earlier study, liquid culture inside bioreactors as a source of secondary
Lakshmanan et al (1995) have shown that the production products, like recombinant proteins used as
of orchid protocorms in vitro can be substantially biopharmaceuticals; to cross distantly related species by
improved by manipulating the size of the explant alone. protoplast fusion and regeneration of the novel hybrid;
For example, the number of protocorms produced by thin embryo rescue (the resulting embryo as a result of cross
transverse sections (0.6 mm thick) derived from a single pollination which would otherwise normally die is cultured
shoot tip (6-7 mm long) was 5 times greater than that in a medium to rescue it); for production of doubled
produced by an intact shoot tip (6-7 mm long) cultured monoploid plants from haploid cultures to achieve
under identical conditions. A similar observation was also homozygous lines more rapidly in breeding programs,
made recently in sugarcane. In this crop, leaf explants usually being done by treatment with colchicine which
 Micropropagation of banana 7
causes doubling of the chromosome number; as a tissue (sterile plants) or which have ‘recalcitrant’ seeds that
for transformation, followed by either short term testing cannot be stored for long period of time can successfully
of genetic constructs or regeneration of transgenic plants; be preserved via in vitro techniques for the maintenance
in vitro conservation of germplasm and so on. This of gene banks. Cryopreservation plays a vital role in the
technique is mainly used to conserve plant which do not long-term in vitro conservation of essential biological
produce seeds or which have recalcitrant seeds which material and genetic resources. It involves the storage
cannot be stored under normal storage conditions in seed of in vitro cells or tissues in liquid nitrogen that results in
gene banks. Hence, vegetative propagated crops such cryo-injury on the exposure of tissues to physical and
as root and tubers, ornamentals, medicinal plants and chemical stresses. Successful cryopreservation is often
many other tropical fruits have to be conserved by using ascertained by cell and tissue survival and the ability to
tissue culture methods (Singh and Shetty, 2011). re-grow or regenerate into complete plants or form new
Tissue culture has been introduced into agricultural colonies (Harding, 2004). It is desirable to assess the
practice at a rate without precedent which allows the genetic integrity of recovered germplasm to determine
production and propagation of genetically homogeneous, whether it is ‘true-to-type’ following cryopreservation
disease-free plant material by micropropagation (Day, 2004). The fidelity of recovered plants can be
methodology (Chatenet et al, 2001). Cell and tissue in assessed at phenotypic, histological, cytological,
vitro culture is a useful tool for the induction of biochemical and molecular levels, although, there are
somaclonal variation (Marino and Battistini, 1990). advantages and limitations of the various approaches used
Genetic variability induced by tissue culture could be used to assess genetic stability (Harding et al, 2005; Suman
as a source of variability to obtain new stable genotypes. et al, 2015). Cryobionomics is a new approach to study
Interventions of biotechnological approaches in in vitro genetic stability in the cryopreserved plant materials
regeneration, mass micropropagation techniques and gene Harding, 2010). The embryonic tissues produced by tissue
transfer studies in tree species have been encouraging. culture are cryopreserved for future use or for germplasm
In vitro cultures of mature and/or immature zygotic conservation (Corredoira et al, 2004).
embryos are applied to recover plants obtained from inter- 5.1.2 Embryo culture: It is a type of plant tissue
generic crosses that do not produce fertile seeds (Ahmadi culture that is used to grow embryos from seeds and
et al, 2010). Genetic engineering can make possible a ovules in a nutrient medium. In embryo culture, the plant
number of improved crop varieties with high yield potential develops directly from the embryo or indirectly through
and resistance against pests. Genetic transformation the formation of callus and then subsequent formation of
technology relies on the technical aspects of plant tissue shoots and roots. The technique has been developed to
culture and molecular biology for production of improved break seed dormancy, test the vitality of seeds, production
crop varieties, production of disease (virus)-free plants, of rare species and haploid plants (Holeman, 2009). It is
genetic transformation, production of secondary an effective technique that is employed to shorten the
metabolites, and production of varieties tolerant to salinity, breeding cycle of plants by growing excised embryos and
drought and heat stresses and so on. Details of some results in the reduction of long dormancy period of seeds.
prime applications of tissue culture in agricultural Intra-varietal hybrids of an economically important energy
development are given as- plant “Jatropha” have been produced successfully with
5.1.1 Germplasm conservation : In vitro cell and the specific objective of mass multiplication (Mohan et
organ culture offers an alternative source for the al, 2011). Somatic embryogenesis and plant regeneration
conservation of endangered genotypes (Sengar et al, has been carried out in embryo cultures of Jucara Palm
2010). Germplasm conservation worldwide is increasingly for rapid cloning and improvement of selected individuals
becoming an essential activity due to the high rate of (Guerra and Handro, 1988). In addition, conservation of
disappearance of plant species and the increased need endangered species can also be attained by practicing
for safeguarding the floristic patrimony of the any country embryo culture technique. A successful protocol has been
(Filho et al, 2005). Tissue culture protocols are of prime developed for the in vitro propagation of Khaya
use in the situation for preservation of vegetative tissues grandifoliola, a plant of high economic value for timber
when the targets for conservation are clones instead of wood and for medicinal purposes as well, by excising
seeds, to keep the genetic background of a crop and to embryos culture from mature seeds (Okere and Adegey,
avoid the loss of the conserved patrimony due to natural 2011). Plant tissue culture technology has an important
disasters, whether biotic or abiotic stress (Tyagi et al, application in forestry by offering a mean of propagation
2007). The plant species which do not produce seeds of elite individuals where the selection and improvement
8 Sugandh Suman
Table 1 : Some of secondary metabolites produced in plant cell suspension culture and being
used in pharmaceutical industries.
Secondary metabolite Plant name Reference
Vasine Adhatoda vasica Shalaka and Sandhya (2009)
Artemisinin Artemisia annua Baldi and Dixit (2008)
Azadirachtin Azadirachta indica Sujanya et al (2008)
Cathin Brucea javanica Wagiah et al (2008)
Capsiacin Capsicum annum Umamaheswai and Lalitha (2007)
Sennosides Cassia senna Shrivastava et al (2006)
Ajmalicine Catharanthus roseus Zhao et al (2001)
Secologanin Contin et al (1999),
Indole alkaloids Moreno et al (1993)
Vincristine Lee-Parsons and Rogce (2006)
Stilbenes Cayratia trifoliata Roat and Ramawat (2009)
Berberin Coscinium fenustratum Khan et al (2008)
Sterols Hyssopus officinalis Skrzypek and Wysokinsku (2003)
Shikonin Lithospermum erythrorhizon Tabata and Fujita (1985)
Ginseng saponin Panax notoginseng Zhong et al (1999)
Podophyllotoxin Podophyllum hexandrum Chattopadhyay et al (2002)
Taxane Paclitaxel Taxus chinensis Wang et al (1999)

of natural population is not feasible and viable too. 5.1.4 Protoplast fusion : Somatic hybridization
5.1.3 Genetic transformation : Genetic being achieved by protoplasm fusion is an important tool
transformation is the most important aspect of plant cell- of plant breeding and crop improvement by the production
tissue culture that provides the mean of transfer of genes of inter-specific and inter-generic hybrids. It involves the
with desirable trait into host plants with ultimate recovery fusion of protoplasts of two different genomes followed
of transgenic plants (Hinchee et al, 1994). It has a great by the selection of desired somatic hybrid cells and
potential of genetic improvement of various crop plants regeneration of hybrid plants (Evans and Bravo, 1988).
by integrating with plant biotechnology and breeding Practically in the crop improvement programmes
programmes. It has a prioritized promising role for the protoplast fusion is efficiently used as a mean of gene
introduction of agronomically important traits such as transfer with desired trait from one species to another
increased yield, better quality and enhanced resistance (Brown and Thorpe, 1995). Somatic hybrids were
to pests, diseases and abiotic stresses (Sinclair et al, 2004; produced by fusion of protoplasts from rice and ditch
Sharma et al, 2010; Kumar et al, 2016). Genetic reed using electrofusion treatment for salt tolerance
transformation in plants can be achieved by either vector- (Mostageer and Elshihy, 2003).
mediated (indirect gene transfer) or vector less (direct In the recent years, in vitro protoplast fusion tool
gene transfer) method (Sasson, 1993). Among vector has opened a way of developing unique hybrid plants by
dependant gene transfer methods, Agrobacterium- overcoming the barriers of sexual incompatibility. It has
mediated genetic transformation is most widely used for been applicable in horticultural industry to create new
the expression of foreign genes in plant cells. Successful hybrids with increased fruit yield and better resistance to
introduction of agronomic traits in plants was achieved diseases. Successful viable hybrid plants were obtained
by using root explants for the genetic transformation when protoplasts from citrus were fused with other related
(Franklin and Lakshmi, 2003). Regeneration of disease Citrinae species (Motomura et al, 1997). The potential
or viral resistant plants is now achieved by employing of somatic hybridization in important crop plants is best
genetic transformation technique. Successfully transgenic illustrated by the production of intergeneric hybrid plants
plants of potato resistant to potato virus Y (PVY) has among the members of Brassicaceae (Toriyama, 1987).
been developed thus resolving a major threat to potato In wheat crop for the purpose of gene pool recovery and
crop worldwide (Bukovinszki et al, 2007). improvement by resolving the problem of loss of
chromosomes and decreased regeneration capacity,
 Micropropagation of banana 9

Fig. 3 : Shoot tip culture for banana micropropagation: a. sword sucker and explant; b. shooting after apical disabling; c. proliferation; d.
multiple shooting; e. rooting; f. nursery hardening (Source: Singh et al (2011).

successful protocol of protoplasm fusion has been androgenesis refers to the production of haploid plants
established and used for the production of somatic hybrid from young pollen cells without undergoing fertilization.
plants by using two types of wheat protoplast as recipient Sudherson et al. (2008) reported haploid plant production
and protoplast of Haynaldia villosa as a fusion donor of sturt’s desert pea by using pollen grains as primary
(Liu et al, 1988). Steps involved in protoplast fusion are explants via tissue culture. Now a day the haploidy
represented by figure 2. technology has become an integral part of crop
5.1.5 Haploid production: By use of the tissue improvement programmes through plant breeding by
culture techniques it is possible to produce homozygous speeding up the production of inbred lines (Bajaj, 1990)
plants in relatively short time period through the protoplast, and overcoming the constraints of seed dormancy and
anther and microspore cultures instead of conventional embryo non-viability (Yeung et al, 1981). The technique
breeding (Morrison and Evans, 1998). Haploids are sterile has a remarkable use in genetic transformation by the
plants having single set of chromosomes which are production of haploid plants with induced resistance to
converted into homozygous diploids by spontaneous or various biotic and abiotic stresses. Introduction of genes
induced chromosome doubling. The doubling of with desired trait at haploid state followed by chromosome
chromosomes restores the fertility of plants resulting in doubling led to the production of double haploids inbred
production of double haploids with potential to become wheat and drought tolerant plants were attained
pure breeding new cultivars (Basu et al, 2011). The term successfully (Chauhan and Khurana, 2011).
10 Sugandh Suman
Table 2 : Secondary metabolites of commercial importance produced in standard phytochemicals in large volumes but also
plant hairy root culture and being used in pharmaceutical eliminate the presence of interfering compounds that
industries.
occur in the field-grown plants (Lila, 2005). The major
Secondary Plant name Reference advantage of the cell cultures include synthesis of
metabolite
bioactive secondary metabolites, running in controlled
Rosmarinic acid Agastache rugosa Lee et al (2007) environment, independently from climate and soil
Deoursin Angelica gigas Xu et al (2008) conditions (Karuppusamy, 2009). For the industrial
Resveratol Arachys hypogaea Kim et al (2008) production point of view, a number of different types
Tropane Brugmansia candida Marconi et al (2008) of bioreactors have been in use for mass cultivation
Asiaticoside Centella asiatica Kim et al (2007) of plant cells. The first commercial application of
Rutin Fagopyrum esculentum Lee et al (2007) large scale cultivation of plant cells was carried out
Glucoside Gentiana macrophylla Tiwari et al (2007) in stirred tank reactors of 200 liter and 750 liter
Glycyrrhizin Glycyrrhiza glabra Mehrotra et al (2008) capacities to produce shikonin by cell culture of
Shikonin Lithospermum erythrorhizon Fukui et al (1998) Lithospermum erythrorhizo (Payne et al, 1987). A
Glycoside Panax ginseng Jeong and Park (2007) number of medicinally important alkaloids, anticancer
Plumbagin Plumbago zeylanica Verma et al (2002) drugs, recombinant proteins and food additives are
Anthraquinone Rubia akane Park and Lee (2009) produced in various cultures of plant cell and tissues
Silymarin Silybium marianum Rahnama et al (2008) in bioreactors. Advances in the area of cell cultures
Flavonolignan Silybium mariyanm Alikaridis et al (2000)
for the production of medicinal compounds has made
Vincamine Vinca major Tanaka et al (2004)
possible the production of a wide variety of
Withanoloid A Withania somnifera Murthy et al (2008)
pharmaceuticals like alkaloids, terpenoids, steroids,
saponins, phenolics, flavanoids and amino acids
5.2 In Pharmaceutical Science
(Vijayasree et al, 2010; Yesil-Celiktas et al, 2010).
In the era of ever demanding development of Advances in scale up approaches and immobilization
pharmaceutical industry, plant cell tissue culture holds techniques contribute to a considerable increase in the
great promise for controlled production of myriad of useful number of applications of plant cell cultures for the
secondary metabolites (Vijayasree et al, 2010). Plant cell production of compounds with a high added value. Some
cultures have provided an efficient platform for the of the secondary plant products obtained from cell
production of valuable therapeutic secondary metabolites suspension culture of various plants are given in Table 1
by utilizing the merits of whole-plant systems with those (Source: Hussain et al, 2012).
of microbial and animal cell cultures (Hellwig et al, 2004).
5.2.2 Hairy root cultures : This system based on
In the want for alternatives to production of medicinal
inoculation with Agrobacterium rhizogenes has become
compounds from plants, biotechnological approaches,
popular in the last two decades as a method of producing
specifically plant tissue cultures, are found to have
secondary metabolites synthesized in plant roots (Palazon
potential as a supplement to traditional agriculture in the
et al, 1997). Organized root cultures can make a
industrial production of bioactive plant metabolites (Rao
significant contribution in the production of secondary
and Ravishankar, 2002). During the last decade
metabolites. Most of the research efforts that use
exploration of the biosynthetic capabilities of various cell
differentiated cultures instead of cell suspension cultures
cultures has been carried out by a group of plant scientists
have focused on transformed (hairy) roots.
and microbiologists in several countries (Siahsar et al,
Agrobacterium rhizogenes causes hairy root disease in
2011) particularly adopting either of one of following
plants. The neoplastic (cancerous) roots produced by A.
culture modules.
rhizogenes infection are characterized by high growth
5.2.1 Cell suspension culture : Cell suspension rate, genetic stability and growth in hormone free media
culture systems are used now days for large scale (Hu and Du, 2006). High stability (Giri and Narasu, 2000)
culturing of plant cells from which secondary metabolites and productivity features allow the exploitation of hairy
are extracted. A suspension culture is developed by roots as valuable biotechnological tool for the production
transferring the relatively friable portion of the callus into of plant secondary metabolites (Pistelli et al, 2010). These
liquid medium and is maintained under suitable conditions genetically transformed root cultures can produce high
of aeration, agitation, light, temperature and other physical level of secondary metabolites comparable to that of intact
parameters (Chattopadhyay et al, 2002; Rao and plants (Srivastava and Srivastava, 2007). Nutrient’s
Ravishankar, 2002). Cell cultures cannot only yield defined composition optimizing in hairy root cultures is the critical
 Micropropagation of banana 11
point to gain a high production of secondary metabolites for the recalcitrant species Coffea arabica was recently
(Hu and Du, 2006). Some of the secondary plant products developed using a bioreactor (Etienne-Barry et al, 1999).
obtained from hairy root culture of various plants are The normal and uniform development of coffee embryos
shown in Table 2 (Source: Hussain et al, 2012). achieved with the use of a bioreactor allowed direct
5.3 Innovative Applications sowing of embryos in the field, resulting in rapid crop
establishment. In brief, bioreactors have the potential to
The greatest value of the plant cell/tissue culture lies
improve product quality and substantially reduce the cost
not so much in their application to mass clonal propagation
of micropropagation, but further development of
(micropropagation) only but rather in its role underpinning
technology is required to realize any commercial benefit
development and application in plant improvement,
from this system.
molecular biology and bio-processing, as well as, its
importance in research. The applications of it go well 5.3.2 As in vitro mycorrhization : Traditionally,
beyond the any bound in agriculture and other allied fields aseptic conditions were considered essential for plant
of food productions as follows- tissue culture systems. But now, attention has turned to
the possible beneficial effects of microorganisms in in
5.3.1 As bioreactors : It is a well-established fact
vitro plant cultures. For example, the root endophyte
that most plants in culture grow better in liquid than on
Piriformospora indica promotes explants hardening
solid media. To further enhance the productivity of liquid
(Sahay and Varma, 1999), Psuedomonas spp. can reduce
culture systems, several innovative approaches were
hyperhydricity (Bela et al, 1998) and Bacillus pumilus,
adapted depending on the final product desired and the
Alcaligenes faecalis and Psuedomonas spp. improve
species investigated (Aitken-Christie et al, 1995).
shoot multiplication (Monier et al, 1998). Mycorrhization
Bioreactors for plant culture are the most prominent being
in micropropagation, particularly the use of arbuscular
adapted for a number of species. Since Murashige (1974)
mycorrhizal fungi (AMF), is now gaining momentum due
introduced the basic micropropagation plan, the
to its demonstrated positive impact on post-transplant
application of bioreactors is one of the major developments
performance of in vitro grown plants (Lovato et al, 1996;
that have occurred in the plant tissue culture industry.
Rai, 2001). Improved nutrient uptake, water relations,
Compared to traditional tissue culture techniques,
aeration, soil pH balance (Sylvia, 1998; Bisht et al, 2009)
bioreactor systems offer several advantages; they are
and their potential use as bioregulators (Lovato et al,
time and labour-saving, relatively easy to scale-up, allow
1996) have recently exemplified the research interest in
enhanced growth and multiplication (e.g. by forced
AMF, contributing to the development of effective AMF
aeration) and improved nutrient availability due to the use
production methods, mycorrhization of in vitro plants and
of liquid medium. Several new strategies have been
screening for efficient AMF strains. The potential of
adapted to develop bioreactors suitable for various plant
different AMFs for application in commercial micro-
species and their specific requirements (Aitken-Christie
propagation industries can now be tested using an array
et al., 1995; Paek et al, 2001). As per Lee (2004) the
of tools (Srivastava et al, 2011).
principal systems are-
6. BANANA AS A PRIME-POTENTIAL
I. Aeration-agitation bioreactor
HORTICULTURAL CROP AND ITS
II. Spin filter bioreactor STANDARDIZED MICROPROPAGATION
III. Gaseous phase bioreactor PROTOCOLS
IV. Rotating drum bioreactor Banana evolved in the humid tropical regions of S.E.
V. Air-driven bioreactor Asia with India as one of its centres of origin. Modern
edible varieties have evolved from the two species –
These basic systems have already been used for the
Musa acuminata and Musa balbisiana and their natural
mass production of over 80 crops (Takayama, 1991) and
hybrids, originally found in the rain forests of S.E. Asia.
are now being evaluated for production of several other
During the seventh century AD its cultivation spread to
plant species (Paek et al, 2001). As a plant production
Egypt and Africa. At present banana is being cultivated
technique, bioreactors are far superior to traditional in
throughout the warm tropical regions of the world between
vitro methods for all the species thus far tested. It is
300 N and 300 S of the equator. Banana and Plantains
worth noting that with bioreactors, even the difficult-to-
(Musa spp.) are some of the earliest crop plants having
propagate woody and tree species can be produced
been domesticated by humans. Bananas are consumed
relatively easily at high frequency. For instance, an
as ripe fruit, whereas plantains, which remain starchy
efficient, somatic embryo-based mass propagation system
even when fully ripe, need cooking for palatability and
12 Sugandh Suman
Table 3 : Commercial cultivars of banana popularly propagated in India.
State Varieties grown
Andhra Pradesh Dwarf Cavendish, Robusta, Rasthali, Amritpant, Thellachakrakeli, Karpoora Poovan, Chakrakeli, Monthan and
Yenagu Bontha
Assam Jahaji (Dwarf Cavendish), Chini Champa, Malbhog, Borjahaji (Robusta), Honda, Manjahaji, Chinia (Manohar),
Kanchkol, Bhimkol, Jatikol, Digjowa, Kulpait, Bharat Moni
Bihar Dwarf Cavendish, Alpon, Chinia , Chini Champa, Malbhig, Muthia, Kothia , Gauria
Gujarat Dwarf Cavendish, Lacatan, Harichal (Lokhandi), Gandevi Selection, Basrai, Robusta, G-9, Harichal, Shrimati
Jharkhand Basrai, Singapuri
Karnataka Dwarf Cavendish, Robusta, Rasthali, Poovan, Monthan, Elakkibale
Kerala Nendran (Plantain), Palayankodan (Poovan), Rasthali, Monthan, Red Banana, Robusta
Madhya Pradesh Basrai
Maharashtra Dwarf Cavendish, Basrai, Robusta, Lal Velchi, Safed Velchi, Rajeli Nendran, Grand Naine, Shreemanti, Red Banana
Orissa Dwarf Cavendish, Robusta, Champa, Patkapura (Rasthali)
Tamil Nadu Virupakshi, Robusta, Rad Banana, Poovan, Rasthali, Nendran, Monthan, Karpuravalli, Sakkai, Peyan, Matti
West Bengal Champa, Mortman , Dwarf Cavendish, Giant Governor, Kanthali, Singapuri

consumption. Originally crops from humid tropics, they Important cultivars include Dwarf Cavendish, Champa,
have acclimatized to a broad range of climatic conditions. Malbhog, Robusta, Monthan, Poovan, Nendran, Red
While bananas have come to occupy the status of a high banana, Nyali, Safed Velchi, Basrai, Ardhapuri, Rasthali,
value, commercial crop, plantains have remained a staple Karpurvalli, Karthali and Grand Naine etc. Among all
food of many ethnic groups. Irrespective of their these cultivars, Grand Naine, an imported variety from
commercial status, banana and plantains are referred as Israel is gaining popularity and may soon become the
‘Poor man’s apple’. most preferred variety due to its tolerance to abiotic
Banana is globally ranked fourth, next to rice, wheat stresses and good quality bunches. Fruit develops
and maize in terms of gross value of production. It is a attractive uniform yellow colour with better shelf life &
major staple food crop for millions of people as well as quality than other cultivars. Important banana varieties
provides income through local and international trade. cultivated in different states of India are given in table 3.
Among the starchy staple food crops, banana ranks third 6.2 Micropropagation Protocols
with respect to the total production. Though cassava and Traditionally, banana is grown as a perennial crop
sweet potato are positioned as first and second, banana where the plant is allowed to produce continuous shoots
and plantain have almost equal importance in all the from a subterranean stem. But, the yields fall after three
tropical regions of the world. Traditional bananas and other to five years and decline rapidly after ten to fifteen years.
species of family Musaceae have been the major calorie The need to shift to cyclic replacement with a new
source of many ethnic tribes of Africa and Pacific Islands. plantation comprising cycles of one crop and one ratoon
High consumption of bananas has been reported in small has been realized only recently in most Asian countries.
countries of Pacific Islands like Samoa (132 Kcal) and Natural calamities such as typhoons, floods, droughts and
Vanuatu (92 Kcal). Bananas also find importance in the occasional volcanic eruptions cause devastating losses
diet of Caribbean (Haiti and Dominican Republic) and in banana production. The consequent need for fresh
Latin American countries like Ecuador and Brazil (Singh seedlings at regular intervals has led to very large increase
et al, 2011). Banana (Musa sp.) is the second most in the demand for clean planting material. Banana is
important fruit crop in India next to mango. Its year round vulnerable to a number of biotic and abiotic stresses which
availability, affordability, varietal range, taste, nutritive and limit its production, particularly among small and marginal
medicinal value makes it the favorite fruit among all farmers with limited resources. BBTV (Banana bunchy
classes of people. It has also good export potential. top virus), CMV (Cucumber mosaic virus), BSV
6.1 Commercial Cultivars (Banana streak virus) and BBMV (Banana bract
Commercially, bananas are classified as dessert types mosaic virus) are the four most important virus diseases
and culinary types. The culinary types have starchy fruits affecting bananas. In addition, banana is an attractive
and are used in the mature unripe form as vegetables. host for nematodes, particularly Pratylenchus coffeae,
 Micropropagation of banana 13
Meloidogyne incognita, Helicotylenchus multicinctus Ø Fidelity testing and virus indexing at various stages
and Radopholus similis. The pests also spread through of mass multiplication.
transportation of non-quarantined planting material. Insect 6.2.1 Selection of quality explant materials:
pests like, banana weevils (Odoiporus longicollis, and Choice of explant is vital for which purpose well
Cosmpolites sordidus) which till recently had a limited maintained mother plants should be selected. Sword
presence in some states of India have now spread to suckers should be healthy and not less than 60-80 days
Bangladesh and beyond. Disease and pest pressure on of age while the growing meristem should be of 1.0 cm3
Asian bananas is unlikely to lessen in the foreseeable in size.
future (Singh et al, 2011). Tissue culture technology has
6.2.2 Culture medium selection: Success of in
been the foundation of high quality, disease free planting
vitro culture depends largely on the choice of nutrient
material production of banana at a mass scale.
medium, including its chemical composition and physical
Banana is a crop with dual propagation abilities, sexual form (Murashige, 1974). Several media formulations have
through seeds and asexual through suckers. Seed been reported for banana shoot tip culture but nearly half
propagation is common in wild species which are diploid of them are modified MS media (Brown et al, 1995).
and undergo normal meiosis, fertilization and seed set. Other popular media include B5 (Gamborg et al, 1968),
All cultivated commercial bananas are triploid and sterile, SH (Schenk and Hildebrant, 1972), N6 (Chu et al, 1975),
excepting a few parthenocarpic AA and AB diploids. and LS (Linsmaier and Skoog, 1975) media. The culture
Sucker propagation is the only natural means of their media vary in both type and concentration of the
perpetuation; artificial methods of propagation include components, but all have similar basic components of
macropropagation and micropropagation: most commonly growth regulators, nitrogen, carbohydrates, inorganic
by using shoot tips. Micropropagation is the practice of macro and micronutrients, vitamins and organic additives
rapidly multiplying stock plant material to produce a large (table 4). Generally, the cultures are established on a
number of progeny plants under aseptic conditions using separate initiation medium, which has a lower
modern plant tissue culture methods. A common protocol concentration of cytokinin than the multiplication medium
by use of shoot tip as explant has been well documented to which the cultures are subsequently transferred (Jarret
by Singh et al (2011) and given below- et al, 1985; Novak et al, 1989).
The earliest reports of in vitro culture of bananas After autoclave sterilization, the culture medium is
came from Taiwan in the 70’s (Ma and Shii, 1974; Ma et stored in a clean dust free chamber for 1-2 days before
al, 1978). Most commonly shoot tips can be extracted use in order to check for any contamination. Bacterial
from the pseudostem, suckers, peepers, lateral buds or contamination may be observed, particularly during the
even small eyes which contain a shoot meristem (Jarret rainy season. Use of Cefotaxime in the initiation and
et al, 1985; Vuylsteke and De Langhe, 1985). Although subsequent subcultures helps to overcome even latent
all of them behave similarly under in vitro conditions, bacterial contaminations.
peepers and sword suckers are preferred because of their
ease of handling and the minimum damage caused to the 6.2.3 Culture initiation : The sword suckers of 2-
parent stool during their removal. It is always better to 3 months are removed from healthy disease free mother
collect the explants from flowering plants so as to plants for shoot tip culture as given in figure 3a. The
ascertain their trueness to type. suckers are cut to expose the shoot tip of 10 cm3 and cut
further to about 3 cm diameter and 5 cm length. The
The critical steps followed for production of explant should be carefully cut to avoid injury to the
micropropagation based banana planting material are: growing meristem. The shoot tips are washed in tap water
Ø Selection of quality explant material and transferred to a container with 0.1% mercuric chloride
Ø Culture medium selection for 10 min and then to 0.1% cetrimide. Then the shoot
Ø Culture initiation, tips are washed thoroughly under running tap water to
remove all traces of the chemicals. Using sharp sterile
Ø Culture proliferation,
blade, one or two outer juvenile leaves and the corm base
Ø Rooting and primary hardening accompanied by are trimmed out. Afterwards, the shoot tips are washed
rouging, three times in sterile water in aseptic condition (under
Ø Secondary hardening accompanied by rouging, laminar air flow) disinfected with 5% sodium hypochlorite
and later with 0.1% mercuric chloride each for 15
Ø Manuring and plant protection in nursery
minutes. To avoid bacterial contamination, use of
Ø Field planting and initial management
14 Sugandh Suman
Table 4 : The composition of initiation, multiplication and rooting usually maintained at 5.8, which is prone to changes over
media used at the National Research Centre for Banana, culture duration. The optimum incubation temperature
India (NRCB). Source: Singh et al (2011)
should be in the range of 24-26°C. Generally the light
Culture media used at different stages of banana intensity is maintained at 1,500-3,000 lux. Higher levels
micropropagation (for one litre)
of 3,000-10,000 lux during later stages improve the
Ingredients Initiation Shoot Rooting survival rate of plantlets upon transfer to soil. Initially,
medium multiplication medium
medium
the cultures are maintained at 16 h light/8 h dark cycle
and once after rooting they are shifted 14 h light/10 h
Activated charcoal - - 2.50 mg
dark cycle.
Agar - 7.5 g 7.5 g
Decapitation and wounding of shoot tips are carried
Ammonium nitrate 1.65 g 1.65 g 1.65 g out to overcome apical dominance and to encourage
L-ascorbic acid 10.0 mg 10.0 mg - axillary bud proliferation. But injuring the apical bud
6-bezyl amino purine 4.0 mg 4.0 mg - through transverse sections, either four or eight cuts, is a
Boric acid 6.2 mg 6.2 mg 6.2 much preferred method. Injuring the explant encourages
more production of phenols, but it can be kept at minimum
Calcium chloride 440.0 mg 440.0 440.0
using antioxidants like ascorbic acid.
Cobalt chloride 0.025 mg 0.025 0.025
6.2.4 Culture proliferation: First subculture is done
Copper sulphate 0.025 mg 0.025 0.025
after 20-25 days of initiation when the explants turn green
Ferric EDTA 36.7 mg 36.7 36.7 in colour. The cultures are first checked for contamination,
Glycine 2.0 mg 2.0 mg 2.0 mg in general symptoms of fungal contamination appear
Indole-3-acatic acid 1.0 mg 1.0 mg - within one week and bacterial contamination symptoms
Indole-3-butyric acid - - 1.0 mg
like change of medium colour and texture or visible
colonies appear within one week to one month. For
Magnese sulphate 22.3 mg 22.3 22.3
subculturing, the outer dead tissue from the base of explant
Magnesium sulphate 370.0 mg 370.0 370.0 is removed and one or two leaf bases are peeled till the
Myo-inositol 100.0 mg 100.0 mg 100.0 mg fresh meristematic tip gets exposed. The apical meristem
α-Naphaleneacetic acid - - 2.0 mg is cut with two gentle cross incisions and the explant is
Nicotinic acid 0.5 mg 0.5 mg 0.5 mg
transferred to subculture medium. During 20-25 days after
the first subculture, the central meristem produces clusters
Potassium dihydrogen 170.0 mg 170.0 170.0
ortho phosphate
of proliferating buds and one to three axillary buds get
regenerated from the basal parts of explants around the
Phytal gel 2.0 g - -
central apical meristem (Fig. 3b). The number of axiliary
Potassium iodide 0.83 mg 0.83 0.83 buds developed during first and second subculture range
Potassium nitrate 1.9 g 1.9 g 1.9 g from 1 to 5 depending on genomic constitution of the
Pyridoxine hydro 0.5 mg 0.5 mg 0.5 mg variety. In general, diploids like Matti, Anaikomban and
chloride Senna Chenkadali produce more buds than commercial
Sodium molybdate 0.25 mg 0.25 mg 0.25 mg cultivars. Among the latter, the number of buds produced
Sucrose 30.0 g 30.0 g 30.0 g
during subculture is high in Cavendish (Robusta, Grand
Naine – AAA genome) group followed by Plantain
Thiamine hydro chloride 0.1 mg 0.1 mg 0.1 mg
(Nendran – AAB genome) and Monthan (ABB genome)
Zinc sulphate 8.6 mg 8.6 8.6 types.
Subsequent subculture is done by trimming the tip of
Cefotaxime (0.1%) in the initiation medium is also emerging axillary buds and removal of dead tissue at the
advised.
base of explant by gentle scratching. Clusters of
Surface sterilized shoot tips are washed three times proliferating buds develop during third and fourth
using sterile water. The outer surface of explant exposed subculture (Fig. 3c). For further subculturing, the explant
to sterilizing agent is removed and the explants trimmed is cut into three to four pieces and each slice with two to
using surgical blade (No. 22) to bring the final size to three proliferating clusters is inoculated to individual
about 3-4 cm length and 1-2 cm diameter (Fig. 3a). The culture bottles. This subculture cycle is repeated at 3-4
explants are inoculated under sterile conditions in 30 ml weeks interval to increase the proliferation rate. During
of initiation medium in a 250 ml glass jar container. pH is fourth and fifth subcultures, a single clump contains about
 Micropropagation of banana 15
15-25 proliferating shoots. After 5-6 subculture cycles, plantlets are transferred from micropots to polybags. Base
the proliferated buds (Fig. 3d) are transferred to rooting substrate is generally soil and sand along with low cost
medium containing IBA and activated charcoal. After a materials like coir pith, sawdust or rice husk. Organic
month, the rooted plantlets are ready for hardening (Fig. manure is either in the form of farm yard manure or poultry
3e). To minimize somatic variation, the subculturing is manure. Even pressed mud, a by-product of sugar
restricted to a maximum of seven cycles when each bottle factories, has been found to provide best substrate for
contains 25-30 plantlets with well developed shoots and secondary hardening along with soil (Vasane et al, 2006).
roots. Experimentally it has been demonstrated that Plantlets from micropots are, dipped in fungicide solution
proliferating shoots can be transferred to polybags (10- (0.1% bavistin) and planted in polybags containing suitable
20 cm size) having rooting media under green house. This substrate. Initially, these are maintained in low light
reduces cost and enhances better establishment. Polybag intensity shade nets and 70% RH. The plants are
provides enough space for plant growth and natural light hardened by gradually increasing the light intensity and
enhances the process of hardening. reducing RH (40%). After 5-6 weeks, the plants become
6.2.5 Rooting and primary hardening ready for field planting having 3-5 well developed leaves
accompanied by rouging : Once the plantlets are ready and a good mass of fibrous roots. During both primary
for shifting outside the laboratory, they are carefully and secondary hardening, the stocks should be rouged
acclimatized to adapt to the green house and later to least for variants at weekly intervals. These could include
protected field conditions (Fig. 3f). During hardening, the vegetative deformities like dwarfism, leaf variegation, and
plantlets undergo physiological adaptation to changing rosette foliage and leaf crinkiness. Other precautions to
external factors like water, temperature, relative humidity be followed are:
and nutrient supply. ü The rooting media should be completely free from
The plantlets from culture vessels/bottles are moved pathogens.
from the laboratory to a room at ambient temperature ü Water used for irrigating the plants should be free
and kept open for 4-6 days. Later they are shifted to from pests and pathogens.
green house for primary hardening where they are first ü Sample plants from each batch should be randomly
gently washed free of agar medium. This is important as virus indexed (at least 10 plants from each batch/
sucrose in agar encourages microorganisms. 8 cm shoots explant)
with 3-4 ramified roots are planted in individual micropots
in a protray. In places where weather is conducive (24- ü While shifting primary hardened plantlets, two
26°C temperature and more than 80% humidity), the longitudinal cuts should be given to the micropots to
plantlets are hardened for 4-6 weeks in mini-sand beds. facilitate further corm growth.
During this period, 90-95% humidity is maintained for 6.2.7 Manuring and plant protection in nursery:
the initial 6-8 days under diffused light. The humidity is Plantlets should be 2-3 weeks old before any fertilizer is
slowly reduced to 70%, light intensity raised to normal applied. 100 ml water containing 0.5 g urea, 2 g
and temperatures brought to 26°C by the end of 6 weeks. superphosphate and 1 g muriate of potash can be applied
Structures used for primary hardening vary with the per plant. The manuring is repeated by doubling the dosage
climatic conditions. These can be highly sophisticated with after three weeks. Spraying of commercially available
UV-stabilized polysheet covering, multiple misting options, micronutrient mixtures during sixth week helps in better
thermal shade net and auto-monitoring of light intensity, establishment both in nursery and field. Strict sanitary
temperature and humidity. On the other hand, the measures are adopted in the nursery to avoid the risk of
structures can be simple with polycarbonate roofing, damage by pests and diseases either through substrate
shade net on all sides with fogger facilities. Temperature, or irrigation water.
RH and light intensities are monitored manually using 6.2.8 Field planting and initial management: 20-
thermometer, hygrometer and lux meter, respectively. 30 cm tall plants with 3-5 broad leaves are ready for
Planting media for primary hardening range from field planting (Figs. 4 & 5). At the time of planting, 10 g
sieved sand augmented with nutrition to mixtures of of Carbofuron is applied per plant. Watering is done soon
cocopeat and Soilrite with fine sand in equal proportions. after field planting as young micropropagated plants are
NPK is provided in liquid form on weekly basis. sensitive to dry weather and heat. Since these are also
highly susceptible to bacterial rot (Erwinia rot), within 3
6.2.6 Secondary hardening accompanied by
days of planting the soil around the plants is drenched
rouging: After primary hardening for 5-6 weeks, the
with 500 ml of 0.1 % Emisson (methyl ethoxy mercuric
16 Sugandh Suman
chloride). Recommended package of practices is strictly species, M. accuminata (‘A’ genome) and M. balbisiana
followed to achieve successful field establishment and (‘B’ genome) resulting into different genomic and ploidy
subsequent vigorous growth (Figs. 4 & 5 & Flow chart levels namely AA, AAA, AAB, ABB, AAAB, AABB
Fig. 6). and ABBB. Tissue culture studies in shoot tips cultures
6.2.9 Testing for genetic fidelity and virus of banana comprising different ploidy levels and having
infection: Virus indexing and genetic fidelity testing are different genome structures, resulted in different forms
important to produce good quality disease free planting of organogenesis. The shoot tips were cultured on MS
material using tissue culture technology by adopting medium supplemented with different concentrations and
standardize protocols. combinations of 2, 4-D, IAA, KIN and BAP. The effect
of ploidy and genome on tissue culture responses have
Shoot tip cultures preserve genetic stability much
been found very significant. Triploids gave the best
better than callus or cell suspension cultures, yet
response followed by tetraploid and diploids for all tissue
somaclonal variation seems to be widespread among
culture responses except somatic embryogenesis for
plants regenerated from banana shoot tip cultures.
which triploids were followed by diploids only. The
Commercial varieties like Robusta, Grand Naine, Dwarf
genotype with more ‘A’ genomes gave better response
Cavendish, Shrimanthi and Madhukar are highly
than those with ‘B’ genome for all tissue culture responses
susceptible to these variations and the off-types number
except somatic embryogenesis as represented in figures
up to 74%. High level of variation is not desirable as it
7(A) & 7(B).
defeats the purpose of clonal reproduction and majority
of the off-types are agronomically inferior to the parental 2. Sugandh S., Kumari R., Sharma V. K. and Kumar
clone. Banana and plantains have a flexible genetic make H. (2015) Isozyme analysis based genetic
and its genetic stability under cultive is strongly influenced fidelity assessment of micropropagated banana
by external factors like growth regulators, duration of plants. J. Applied Natural Sci. 7(2), 579 – 584.
culture etc. Mild stress under in vitro results in reverting Isozyme studies of micropropagated and mother
of the clone to its parental type. For example, Robusta, a plants of banana cvs. Matti, Ney Poovan, Kechulepa,
Cavendish clone frequently reverts to its original type Dwarf Cavendish, Malbhog, Champa, B.B. Battisa and
Dwarf Cavendish which is not acceptable for its low FHIA-1 were done to test their genetic fidelity. The
stature and poor yield. Hence, genetic fidelity testing using banding patterns as revealed by electrophoretic variations
preferably molecular marker techniques is essential to were evaluated with respect to isozymes of acid
ensure the supply of true to type quality planting material. phosphatase, catalase, esterase and peroxidase as
At NRCB, besides phenotype PCR technique with ISSR markers. The genetic fidelity of micropropagated plants
markers is being successfully used for screening off-types and the relationship of the different cultivars were
in banana as in table 5. Flow chart figure 6 summarizes determined by dendrogram using numerical taxonomy and
the various stages in micropropagation of banana. Using multivariate analysis system (NTSYS). A clustered
the protocol, more than 10,000 plants are expected from dendrogram was prepared by unweighted pair group
a single explant at the end of 320 days as per table 6. method using averages (UPGAMA) method. At 87%
7. WORK DONE BY AUTHOR IN THE FIELD similarity, the micropropagated and mother plants were
OF MICROPROPAGATION OF BANANA clustered in four groups reflecting their genomic
constitution. Cvs. Matti (AA) and Dwarf Cavendish
Author has in depth research experience in the field
(AAA) with similar ‘A’ genome were categorized in
of development of protocol of micropropagation of banana
Cluster I. Cluster II comprised of cvs. Ney Poovan (AB),
cultivars of commercial importance and also on the
B.B. Battisa (ABB) and FHIA-1(AAAB) with genomic
influence of ploidy and genome on culture responses
constitution of both ‘A’ and ‘B’ type. Cvs. Champa (AAB)
which is evident by her under given research publications.
and Malbhog (AAB) with similar genome were grouped
1. Sugandh S., Rajak K. K. and Kumar H. (2012) in Cluster III. Cluster IV contained the cv. Kechulepa
Diversity of genome and ploidy in banana and (BB) having only ‘B’ genome. However, there was no
their effect on tissue culture responses. Res. somaclonal variation among the micropropagated plants
Environ. Life Sci. 5(4), 181- 183. and they showed 100% genetic similarity. Thus, the
Banana is one of the most important fruit crop having isozyme studies could be a reliable marker for testing the
a wide range of ploidy and genome constitution. Most of genetic fidelity of micropropagated plants and for
the banana cultivars have originated from inter and intra evaluating the diversity among the banana germplasm.
specific hybridization of two wild diploid (2n=2x=22)
 Micropropagation of banana 17

Source: Singh et al (2011)

Fig. 4: Tissue cultured plantlets at growing stage. Fig. 5 : Tissue cultured plants at fruiting stage.

Fig. 6 : Summary of steps involved in production of quality plantlets of banana by microprogation (tissue culture).

3. Sugandh S. and Kumar H. (2015) adventitious shoots. The maximum differentiation of

Micropropagation of Banana cv. Malbhog. The shoots (92.05%) was observed on MS medium with 0.57
Bioscan 10(2), 647-650. µM IAA and 17.74 ìM BAP. The number of shoots per
Malbhog, one of the most important and delicious culture was 16.75. The subculture of differentiated shoots
local cultivar of banana in Bihar, is on verge of becoming on the same medium resulted in further differentiation
extinct because of panama wilt and non-availability of (91.97%) of more than 15 shoots per culture. The in
disease free quality propagules. The culture of shoot tips vitro developed shoots showed 100% rooting on MS
taken from suckers on Murashige and Skoog (MS) medium supplemented with 4.92 ìM Indole butyric acid
medium supplemented with different concentrations and (IBA). The plantlets were acclimatized and field
combinations of indole acetic acid (IAA) and benzyl transferred. A suitable and efficient protocol for
amino purine (BAP) resulted in differentiation of micropropagation of Malbhog cultivar of banana was
18 Sugandh Suman
Table 5 : List of ISSR primers used for genetic fidelity testing of Table 6 : Success summary table of micropropagation protocol of
banana at National Research Centre for Banana (NRCB), banana (Source: Singh et al, 2011).
Tiruchirapalli 620102, Tamil Nadu, India (Source: Singh
et al, 2011). Particulars stage Duration (days) No. of plants
Initiation 25 1
ISSR Primer Nucleotide sequence Subculture stages 1st 50 3
UBC 807 5’-AGA GAG AGA GAG AGA GT-3’ 2nd 75-80 12
UBC 808 5’-AGA GAG AGA GAG AGA GC-3’ 3rd 100-110 48
UBC 811 5’-GAG AGA GAG AGA GAG AC-3’ 4th 125-130 192
UBC 812 5’-GAG AGA GAG AGA GAG AA-3’ 5th 175-180 760
UBC 818 5’-CAC ACA CAC ACA CAC AG-3’ 6th 200-210 3,040
UBC 830 5’-TGT GTG TGT GTG TGT GG-3’ 7th 225-230 12,160
UBC 834 5’-AGA GAG AGA GAG AGA GYT-3’ Rooting 255-260 11-12,000
UBC 836 5’-AGA GAG AGA GAG AGA GYA-3’ Primary hardening 270-280 11,500-11,000*
UBC 840 5’-GAG AGA GAG AGA GAG AYT-3’ Secondary hardening 310-320 10,000-10,500**
UBC 841 5’-GAG AGA GAG AGA GAG AYC-3’
*Success depends on the sophistication of the hardening structure
UBC 842 5’-GAG AGA GAG AGA GAG AYG-3’
**Somaclones and off-types constitute the major discards.
UBC 850 5’-GTG TGT GTG TGT GTG TYC-3’
UBC 868 5’-GAA GAA GAA GAA GAA GAA-3’ 22.83µM), BAP (4.43, 8.87, 17.74 and 26.61 µM) and
combination of IAA (0.57, 5.71 and 11.42 µM) and BAP
developed: figure 8. (8.87, 13.30 and 17.74 µM). Cultured shoot tips
4. Sugandh S., Rajak K. K. and Kumar H. (2013) differentiated multiple shoots from their bases and the
Micropropagation of Banana cv. B. B. Battisa. rate of shoot multiplication was quite high. The subculture
Biochem. Cell. Arch., 13(2), 349-354. of differentiated shoots on MS medium with 0.57 µM
For banana being the most important fruit of India as IAA and 17.74 µM BAP resulted in further differentiation
whole particularly more popular in Bihar, the requirement of more than 4 shoots per culture. The in vitro developed
of large numbers of quality propagules for plantation rely shoots showed 100% rooting on MS medium with 4.92
on the development of micropropagation protocol for ìM IBA. The regenerated plantlets were transferred to a
important local cultivars. B.B. Battisa (ABB) is an mixture of sand and farm yard manure (1:1) for
important cultivar used as vegetable and in culinary and acclimatization and field transfer. Thus, the present work
wine making. Shoot tips were cultured on MS medium led to the development of a suitable micropropagation
supplemented with different concentrations of IAA (5.71, protocol for cv. Champa: figure 10.
11.42 and 22.83µM), BAP (4.43, 8.87, 17.74 and 26.61 6. Sugandh S., Kumar M. and Kumar H. (2016)
µM) and combination of IAA (0.57, 5.71 and 11.42 µM) Plant regeneration via somatic embryogenesis
and BAP (8.87, 13.30 and 17.74 µM). Cultured shoot from the cultured immature male floral buds of
tips differentiated multiple shoots from their bases and banana genotype ‘Dwarf Cavendish’. Indian J.
the rate of shoot multiplication was quite high. The Life Sci. 13 (2), 000-000.
subculture of differentiated shoots on MS medium with
Immature male floral buds of banana cv. Dwarf
0.57 µM IAA and 17.74 µM BAP resulted in further Cavendish were cultured on Murashige and Skoog (MS)
differentiation of more than 7 shoots per culture. The in medium supplemented with different concentrations and
vitro developed shoots showed 100% rooting on MS combinations of 2,4-D (4.52, 9.05 and 18.10 µM), IAA
medium with 4.92 µM IBA. The regenerated plantlets (5.71, 11.42 and 22.83 µM), Kin (4.65, 9.30 and 18.60
were transferred to a mixture of sand and farm yard µM) and BAP (4.43, 8.87 and 17.74 µM). The culture of
manure (1:1) for acclimatization and field transfer. Thus, the explants resulted in the generation of small spherical,
the present work led to the development of a suitable friable calli. These calli showed embryogenic
micropropagation protocol for cv. B.B. Battisa: figure 9. characteristics and sub-cultured on the same medium for
5. Sugandh S., Rajak K. K., Kishore C. and Kumar further maturation. After 5 months of culture on MS basal
H. (2013) Micropropagation of Banana cv. medium, somatic embryos started to regenerate. The
Champa. Biochem. Cell. Arch. 13(2), 291-295. somatic embryos were transferred to modified MS
Champa (AAB) is another important cultivar known medium for regeneration into plantlets. Among the
for its delicious fruit used as a dessert and making pastries. different combinations tried, MS +9.05µM 2,4-D +
Shoot tips were cultured on MS medium supplemented 18.60µM Kin was found to be the most suitable medium
with different concentrations of IAA (5.71, 11.42 and for somatic embryogenesis. Thus, an efficient protocol
 Micropropagation of banana 19

Fig. 7(A) : Effect of ploidy on different tissue culture responses from shoot tip culture.

Fig. 7(B) : Effect of genome (A & B) on different tissue culture response from shoot tip culture.

Fig. 8 : (a-e) : Explant showing existing

shoot elongation after 15 days of culture
on medium BM8(a), Multiple shoot
formation after 20 days of culture on
medium BM 3 (b), Multiple shoot
formation after 25 days of culture on
medium BM11(b), Root formation after
20 days of culture on medium BM10(d),
Two month-old planelet (e).
20 Sugandh Suman

for plant regeneration via indirect somatic embryogenesis meticulous efforts involved in growing plants in vitro and
have been developed: figure 11. the advances made in plant tissue culture, the application
8. CRITICAL PROBLEMS AND of this technique is still hampered by various physiological
RESEARCHABLE ISSUES and developmental problems. The problems are
It is evident by the literatures that the development summarized by Bairu and Kane (2011) and range from
of tissue culture protocols is a rigorous procedure that abnormal growth to increased genetic variability/instability.
involves optimization of the various chemical, physical Of these, the points of critical attention are:
and environmental factors of cell and tissue growth being i. Shoot-tip necrosis : It is a phenomenon whereby
accomplished by growing plants outside their natural the apical shoot becomes brown and later dies (McCown
environment and growth conditions. Despite the and Sellmer, 1987). The problem of non-pathogenic
 Micropropagation of banana 21

Fig. 11 : Plant regeneration from callus culture in ‘Dwarf Cavendish (a) Explant showing swelling after 20 days of culture on medium
BM14 (b) Explant showing callus formation after 40 days of culture on medium BM21 (c) Explant showing embryogenic callus
formation after 125 days of culture on medium BM17 (d) & (e) Regeneration of somatic embryos into plantlets cultured on modified
MS medium (f) Hardening of regenerated plants in green house.

dieback or shoot-tip necrosis of in vitro cultures has been Some describe fasciation as fusion of organs due to
associated with various culture conditions including auxin- deviation from normal meristematic processes while
cytokinin interactions (Bairu and Kane, 2011). others suggest that it is the result of transformation of a
ii. Fasciation and tissue proliferation: Fasciation single growing point into a line (Clark et al, 1993). Tissue
is abnormal flattening of stems due to failure of the lateral proliferation is a disorder primarily found in
branches to separate from the main stem during growth. micropropagated rhododendrons that is characterized by
22 Sugandh Suman
the formation of abnormal callus-like growths, or tumors worker safety; and higher productivity. Moreover, it is
at or near the crown or base of the plant (Brand and also anticipated that whereas genetic engineering-
Kiyomoto, 1992). molecular technologies offer great opportunity to develop
iii. Epigenetic changes and somaclonal variation: a world that is truly food secure, we must not forget that
The distinction between epigenetic changes and we all - the scientific community, the international
somaclonal variation is that the former involves temporary community, the multinational life science companies and
changes in gene expression as a result of events such as the donor community together with national governments
change in DNA methylation. Such changes are temporary - bear a fundamental responsibility to ensure that the
and often plants reverse to the normal phenotype although developing countries can equitably share in these exciting
some changes may last longer (Brettell and Dennis, 1991) advances that science offers in a way that is safe for
whereas somaclonal variation on the other hand involves their population and environment. This calls for a more
irreversible genetic change originating in cell and tissue open, integrated and collaborative involvement of all the
cultures (Larkin and Scowcroft, 1981). stakeholders of developing country engaged in agriculture
and food production. Bananas are very much a part of
iv. Role of phenolics and plant growth regulators
this opportunity.
in alleviating developmental problems : Limited
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