4.

1 Cocos nucifera Coconut

Valerie Hocher, [ean-Luc Verdeil and Bernard Malaurie

IRD/CIRAD Coconut Program, UMR 1098 BEPC, IRD, BP 64501-911 Av.
Agropolis, 34394 Montpellier, Cedex 5, France

1. Introduction length and 30-120 cm deep and continu-

ously generate adventitious roots (Reynolds,
1.1. Botany and history 1988; Persley, 1992). Nutrients and water are
absorbed by the rootlets.
The coconut palm (Cocos nucifera L.) is a rela- The coconut palm 'trunk' is a stem with
tively slow growing woody perennial species. no true bark, no branches and no cambium.
It is the only species in the genus Cocos. All Secondary growth (increased stem diameter)
forms known to date are diploid (2n = 2x = is by secondary enlargement meristem
32). No closely related species with even par- located below the shoot meristem. Growth
tial interfertility has been reported (Bourdeix depends on age, ecotype and edaphic condi-
et al., 2001). The lifespan of a coconut palm tions, but is generally between 30 and 100 cm
can be > 60 years under favourable ecological per annum. The stem is surmounted by a
conditions. Coconuts can grow to a height of crown of approx. 30 compound leaves,
approx. 25 m (Ohler, 1999). which protect the terminal vegetative bud
Optimum growing conditions for coconut and whose destruction causes the death of
are in the lowland humid tropics at altitudes the palm. An adult coconut has virtually as
< 1000 m near coastal areas in sandy, weII- many unopened (20-30) as opened leaves.
drained soils (Persley, 1992); however, Leaves are produced continuously at approx.
coconuts are adaptable to other soil types 1 month intervals.
including coral atolls and soils with moder- The coconut palm is a monoic species.
ate salinity (Batugal, 1999). Coconuts are also Flowering may begin between 3 and 10 years
commonly cultivated several hundred kilo- after planting. Each leaf bears an inflores-
metres inland, e.g. surrounding Lakes cence primordium in its axil. The coconut
Victoria, Tanganyika and Malawi in Africa inflorescence is a spadix, which develops
(Lombard, 2001). Coconuts cannot tolerate within a double sheath referred to as a
temperatures < O°C and ideal growing tem- spathe. When mature, the spadix breaks
peratures range between 24 and 30°C through the spathe and 30-35 spikelets
(Woodroof, 1979; Persley, 1992). emerge, each bearing a large number of male
Coconuts do not form a tap root, but flowers (200-300) with one or two female
develop a fasciculated root system, consist- flowers at the base of each spikelet. Flowers
ing of adventitious roots at the base of the are sessile and follow the trinary organiza-
stem, which typically grow laterally to 2-3 m tion of monocotyledons (Menon and

90
 Cocos nucifera Coconut 91

Pandalai, 1958). Male flowers have three Histological studies have demonstrated digi-
short sepals, three petais, six stamens and tations in the epidermallayer in contact with
one rudimentary pistil. Female flowers are the nu trient reserves, and the existence of
approx. 3 cm in diameter, and are enveloped vascular bundles converging towards the
by small scaly bracteoles endosing three embryonic axis. This villosity displays
sepals and three petaIs, which overlap each numerous structural similarities to stomach
other and surround the spherical pistil. The villi in the digestive system of animaIs
ovary is tricarpous and each carpel has a sin- (Verdeil and Hocher, 2002).
gle ovule. After fertilization, a single ovule Fossil nuts > 15 million years old and
develops and the two others abort or degen- very similar to present-day coconuts have
erate. The inflorescence can be either self- or been discovered in New Zealand and India
cross-pollinated (Bourdeix et al., 2001). (Sauer, 1967, cited by Harries, 1978; De
Pollination is by wind or insects. Taffin, 1998); however, the exact geographic
The appearance of the fruit (size, shape origin of this species is uncertain. In aIl prob-
and colour) varies according to the ecotype ability, the coconut tree was first cultivated
(Bourdeix et al., 2001). The coconut is a either in India or in South-east Asia. The
drupe, whose development requires approx. coconut has attained its highest development
1 year. Only 25 to 40% of the female flowers in terms of variability and number of local
develop into mature nuts and a tree pro- names in South-east Asia.
duces < 100 fruits per annum. After fertiliza-
tion, the husk and shell increase in size and
the cavity of the embryo sac enlarges consid- 1.2. Importance
erably (Menon and Pandalai, 1958). The cav-
ity is filled with a liquid endosperm. After 6 The coconut palm has been referred to as the
months, the solid endosperm develops as a 'tree of life', because of its importance as a
thin and gelatinous layer against the inner subsistence crop in most tropical areas of the
wall of the nut cavity (Ohler, 1999). After 8 world. It is grown on > 11 million ha, 94% of
months and towards the later stages of which are in Asia and the South Pacifie
ripening, the endosperm becomes hard and (Blake, 1990). World production of coconut
white and is surrounded by a hard, brown has been estimated to be 52,940,408 t
testa (Ohler, 1984). The immature endosperm (FAOSTAT, 2004). The leading producers are
is composed of 95% water and < 1% oil, and Indonesia and the Philippines (> 13,000,000 t),
50% water and 30-40% oil at maturity India (9,500,000 t), Brazil (2,833,910 t), Sri
(Ohler, 1984). When ripe, the nut generally Lanka (1,850,000 t), Thailand (1,400,000 t),
falls. The seed, which is one of the largest in Papua New Guinea (570,000 t), Vietnam
the plant kingdom, is characterized by lack (920,000 t) and Mexico (959,000 t). Many
of dormancy and the time necessary for coconut-producing countries are small
development from embryo to plantlets islands in the South Pacifie and Indian
(Blake, 1990; Verdeil, 1993). Oceans and the Caribbean region (Daviron,
Four months are generally required for 1995), where coconut can be grown in harsh
the first leaf to emerge from the husk. A char- environments, such as atolls, and can
acteristic of coconut zygotic embryos is the tolerate swampy and water-deficient areas
substantial development of the haustorium and poor soils. Coconut is an important
(distal part of the cotyledon) within the nut attribute of the rural economy (Punchihewa,
cavity during germination (Menon and 1999), and is cultivated by many farmers on
Pandalai, 1958). This organ invades the nut smaIl landholdings « 4 ha) often in associ-
cavity and establishes intimate contact with ation with other crops (root crops, vegetables,
the endosperm. It enables the hydrolysis of cacao, etc.) (Barrant, 1978; Reynolds, 1988;
the endosperm and the mobilization of nutri- Freud and Daviron, 1994). Only 10% of the
ents required for embryo germination. planted areas constitute commercial planta-
Lipase, protease and saccharase activity have tions. Coconut palm is cultivated mainly for
even been detected (Bertrand, 1994). copra (dried endosperm) production, from
92 V. Hochet et al.

which oil is extracted and provides income therefore widely used in food products (mar-
for smallholders in the tropies and subtropies. garine, confectionery, ete.) (Ohler, 1984).
The coconut has been a primary source of With only 4% of the world oil production,
food, drink and shelter for millions of people coconut ranks seventh among oil-bearing
from the earliest days of humankind crops. In the competitive international world
(Batugal, 1999; Punchihewa, 1999). Coconut oil market, the coconut paIm is gradually
farmers are deeply attached to the various being replaced by other oil-seed plants such
products (Punchihewa, 1999), and have con- as soya and oil palm (Freud and Daviron,
tributed to its adaptation to a wide range of 1994). The coconut palm is therefore reverting
environmental conditions. Although signifi- to a multipurpose crop, especially for its fruit.
cant achievements have been made with Several reasons can explain this graduai
respect to the release of high copra-yielding decline: (i) low productivity due to old age of
hybrids (Bourdeix et al., 2001), this progress coconut plantations (two-thirds of the indi-
has yet to reach most coconut producers. viduals are > 60 years old) and insufficient
The coconut is mainly a subsistence crop, replanting; (ii) use of unimproved material
e.g. 70% of the production is consumed and marginal culture practiees; (iii) several
locally in Asia. Every part of the plant can be pests and diseases, e.g. lethal yellowing (LY)
used. Oïl from the fresh nuts is used for food and Cadang-Cadang; (iv) production in areas
preparation in many countries of Asia and often subjected to natural calamities, e.g.
the Pacifie. The kernel can be oven- or sun- typhoons or volcanic eruptions; and (v) low
dried to a moisture content of 6% (copra), prices for coconut oil despite its high quality
and can be conserved for months before oil and lower production (Freud and Daviron,
extraction. Coconut water is a very refresh- 1994). In addition, rapeseed oil, whieh has
ing drink. Endosperm of mature nuts is been genetically modified to produce oil
grated and used in pastries. The woody stem (Laurical®), with a higher content of laurie
is used as a building material and in joinery. acid (37%), has had a significant impact on
The leaves can serve for local handicrafts production. Despite these difficulties and
and as roofing materia1. The processed sap stagnant production for 20 years, coconut oil
provides sugar, syrup and vinegar. The fibres is still important, and there continues to be
from the husk surrounding the nut can be demand for lauric oil for the soap industry
used to manufacture esparto-type goods. (Freud and Daviron, 1994). With the assis-
More ecofriendly than rock wool, these fibres tance of the World Bank, the Philippines has
can also be used as a substrate for growing started a replanting programme using
plants (Bourdeix et al., 2001). improved hybrids, and LY was recently
Plantations were developed throughout declared a national priority for research in
the tropics by the end of the 19th century to Mexico (Aldaba, 1995; INIFAp, 1998). The
satisfy the need for coconut oil for industrial CGIAR has even recognized coconut as
uses (Daviron, 1995), including the extrac- the oil crop most in need of international
tion of glycerine, a component of dynamite. research.
Until the mid-20th century, coconut was the
main oil source in the world market.
Coconut oil is extracted from the dried 1.3. Breeding and genetics
endosperm (copra) and, together with oil
1.3.1. Plant characteristics
palm kernel oil, is the only source of short-
chain fatty acids (from eight to 14 carbon Propagation is entirely by seed. Allogamy
atoms), and a rich source of lauric acid causes a high degree of variability. The
(-48%) (Persley, 1992). It is used in soap breeding cycle is very long (12 to 16 years),
manufacture and in the cosmetic industry with a low number of seeds produced (100 to
(Blake, 1990; Verdeil et al., 1996a). The melt- 200 seeds/tree per annum) and a large recal-
ing point of coconut oil is 24-27°C and citrant seed that makes exchange and conser-
hydrogenation is not required to inhibit ran- vation of germplasm extremely difficult.
cidness because of its stability; coconut oil is These morphological and biological charac-
 Cocos nucifera Coconut 93

teristics impose serious constraints on breed- cultivation system and use. The breeding
ing. There are three groups of coconut palms programme of the Centre de Coopération
- Tall (c. nucifera typica), Dwarf (c. nucifera Internationale en Recherche Agronomique
nana) and hybrids between the two. Tall pour le Développement - Departement
palms represent the more common type and Cultures Pérennes (CIRAD-CP) uses recipro-
account for > 95% of coconut production cal recurring selection as a starting point. The
because of their general superiority in copra method involves exploiting ecotype combin-
production (Woodroof, 1979; Persley, 1992). ing ability and basing phenotypic choices on
Dwarfs are distinguished mainly by slower heritable characters (Gascon and de Nucé de
growth. They generally produce lower qual- Lamothe, 1978) and has been described in
ity copra than Talls and for this reason are detail by de Nucé de Lamothe (1970) and
often not used for large-scale plantings Gascon and de Nucé de Lamothe (1976).
(Woodroof, 1979). Dwarfs exhibit other fea- Genetic improvement involving hybridiza-
tures, e.g. preferential autogamy, reduction tian between ecotypes has resulted in a dou-
in organ size, early maturity and rapid fruit bling of the outputs within 20 years. The best
production. Because of these last two charac- hybrids can increase profits by 20 ta 30%
ters, Dwarfs are very important in breeding within a generation.
programmes (Bourdeix et al., 2001). Genetic gain has been assisted by the
development of reliable hybrid seed produc-
tion techniques using assisted pollination
1.3.2. Breeding objectives
(Wuidart and Rognon, 1981). Hybrids are
The diversity of coconut uses ensures that reproduced on a large scale, e.g. 1 ha of seed-
there is no single ideotype. Breeding objec- bearing trees can produce c. 15,000 seeds per
tives are particularly complex, and include a annum by assisted pollination (de Nucé de
tradeoff between food, cultural habits and Lamothe and Wuidart, 1992). This method is
processing requirements. The highest prior- complex, costly and time consuming (de
ity is increased production of copra per Nucé de Lamothe and Wuidart, 1992),
hectare (Bourdeix et al., 2001). Other impor- requiring emasculation of female parents,
tant objectives include precocity, adaptation conditioning and conservation of pollen
ta certain edaphoclimatic conditions from male parents and manual or assisted
(drought, cold, pH) and resistance ta dis- pollination (Wuidart and Rognon, 1981). The
eases. Several pathogens (see Table 4.1.3), cast of a selected seednut can be as much as
including fungi (Phytophthora spp.), try- US$2-4, which is too expensive for small-
panosomes (heart rot), nematodes (red ring), holders (Verdeil et al., 1998a).
viruses (coconut foliar decay (CFDV)), According ta Baudouin (1999), the effi-
viroids (coconut cadang cadang (CCCVd)) ciency of breeding can be improved as fol-
and phytoplasma (LY) cause heavy lasses. lows: (i) combining genetically distant
The genetic improvement of the coconut genotypes ta increase heterosis; (ii) increas-
relies on exploitation of the variability within ing selectable diversity in breeding popula-
the species. Coconut breeding began in India tions; (iii) using molecular marker and
in 1916 (Harries, 1978), although major quantitative trait loci (QTLs) ta increase
progress was not obtained until the 1960s. selection efficiency using marker-assisted
Currently, 20 centres throughout the tropics selection (MAS); and (iv) using in vitro prop-
are involved in coconut breeding. agation for rapid dissemination of genetic
Hybrids can include: Dwarf X Tall, Tan X gain (Verdeil et al., 1995, 1998a).
Tan or Dwarf X Dwarf (Harries, 1991).
According ta Ohler (1984), breeders and
growers prefer the Dwarf X Tan type because 2. Molecular Genetics
of early maturity, ease of production and
seed whose quality can be readily controlled. The application of MAS in coconut breeding
Nevertheless, other hybrid types can aIsa is urgently needed because desired characters
provide certain advantages depending on the are expressed only after several years of
94 V. Hocher et al.

growth. The use of molecular markers offers 2.2. Linkage mapping and aTL analysis
certain advantages for identifying cultivars
and for determining taxonomie relationships. In coconut, the availability of F] mapping
The studied traits directly reflect variation populations from controlled crosses involv-
that occurs within the genome, they are neu- ing heterozygous parents has allowed link-
tral and their expression is independent of age mapping of identified polymorphisms as
the environment (Lebrun and Baudouin, weIl as the search for QTLs. An initial linkage
2002). Their use should increase the efficiency analysis of the East African Tall (EAT) and
and efficacy of coconut genetic improvement, Laguna Tall (LAGT) coconut types based
especially for germplasm management, geno- entirely on ISTR markers was described by
type identification and MAS of important Rohde et al. (1999). This work was extended
traits. In many species, molecular markers using AFLPs, ISTRs, RAPDs and inter-sample
are being used to create genetic linkage maps sequence repeats (ISSRs), and allowed the
in order to identify markers linked to specifie construction of a linkage map of the Iwo par-
traits that can form the basis for MAS. ents of the cross involving Malayan YeUow
Construction of genetic maps would have Dwarf (MYD) X LAGT, resuIting in 382 iden-
great benefit for coconut. tified markers and 16 linkage groups gener-
ated for each parent and the identification of
QTLs associated with early flowering and
2.1. Markers yield (Herran et al., 2000). In addition, QTLs
for other traits, induding leaf production and
Initial studies on genetic diversity characteri- girth height, were identified for the same
zation involved isozymes or polyphenol mapping population (Ritter et al., 2000).
markers (Carpio, 1982; Canto-Canché et al., AFLP and SSR markers have been used to
1983; Jay et al., 1989; Fernando and construct a linkage map for a coconut type
Gajanayake, 1997; Cardena et al., 1998). The from the Solomon Islands, the Rennell Island
characterization of genetic diversity in TaU (RIT), whieh is used in various breeding
coconut germplasm at the DNA level programmes and as a male parent for com-
(Ashbumer, 1999) has largely replaced these mercial hybrids in the Pacifie (Lebrun et al.,
strategies. Various DNA markers have been 2001). QTL analysis aUowed the identification
used to measure coconut genetic diversity: of loci linked to number of bunches and the
inverse sequence-tagged repeat (ISTR) number of nuts.
(Rohde et al., 1995; Duran et al., 1997); ran- The identification of different QTLs pro-
domly amplified polymorphie DNA (RAPD) vides the first opportunity for MAS in
(Ashburner et al., 1997; Duran et al., 1997; coconut. The most efficient use of MAS
Rodriguez et al., 1997; Wadt et al., 1999); would be to produce parental lines for F]
restriction fragment length polymorphism hybrid production and to search for LY-resis-
(RFLP) (Lebrun et al., 1998, 1999); amplified tant hybrids (Cardena et al., 1999). According
fragment length polymorphism (AFLP) to Ashburner (1999), there is still a basic lack
(Perera et al., 1998); simple sequence repeat of knowledge of the genetics of the species.
(SSR) (Karp, 1999; Perera et al., 1999; Rivera et The large stature, long generation time and
al., 1999; Teulat et al., 2000). Two main low multiplication rate will always hamper
coconut groups have been identified: Indian breeding. Molecular markers can minimize
and Pacifie Ocean. Analysis of DNA poly- but not eliminate these problems.
morphisms has indicated that the Tall and
Dwarf types show different degrees of poly-
morphisms with more polymorphism in TaU 3. Somatie Cel! Geneties
types. Using mierosatellites, a kit for identify-
ing coconut cultivars is under development 3.1. Regeneration
in ORAD and should allow the large-scale
application of molecular fingerprinting of Due to the time required, in order to develop
coconut (Lebrun and Baudouin, 2002). improved selections, micro propagation is
 Cocos nucifera Coeonut 95

essential for distribution of selections that Embryogenie cultures are induced from
Emerge from breeding programmes (Verdeil explanted tissues collected from adult
et al., 1998a). Vegetative multiplication of coconut palms on various culture media. At
Elite selections is necessary for producing the Institut de Recherche pour le
homogeneous planting material and thereby Développement (IRD)/CIRAD, the Eeuwens
improving plantation productivity. Moreover, Y3 mineraI solution (Eeuwens, 1976) is used
de nova regeneration of coconut is essential with Morel and Wetrnore's vitamins (1951),
for genetic transformation; however, coconut 40 g/l sucrose, 7.5 g/l agar, 2 to 2.5 g/l
palm is considered to be one of the most activated charcoal and 99.55 to 271.5 fLM
recalcitrant species for in vitro culture 2,4-dichlorophenoxyacetic acid (2,4-D), due
(Georges and Sherrington, 1984; Hocher et al., to the variable sensitivity between palms to
1999). auxin at pH 4.5-5.8 (Verdeil et al., 1999).
Murashige and Skoog medium (1962) (MS)
with the addition of sucrose, activated char-
3.1.1. Somalie embryogenesis
coal and auxin is also employed. The cul-
Somatic embryogenesis involving different tures are usually incubated in the dark at
expIant types has been attempted, including 27°C (Buffard-Morel et al., 1992; Verdeil et al.,
apical meristems (Hagedorn, 1990), young 1994). Activated charcoal is necessary to con-
roots of mature palms Oustin, 1978), stems trol browning, whieh is a major constraint of
and leaves (Pannetier and Buffard-Morel, coconut in vitro culture (Blake and Eeuwens,
1982; Gupta et al., 1984; Raju et al., 1984), 1980, 1981; Pannetier and Buffard-Morel,
zygotic embryos (Bhala-Sarin et al., 1986; 1986; Tisserat, 1990). The effect of activated
Karunaratne and Periyapperuma, 1989; charcoal appears to be due to reversible
Ueda et al., 1993), inflorescences (Eeuwens, adsorption of the auxin and its slow and
1978; Branton and Blake, 1984; Sugimura and graduaI release (Brackpool et al., 1986; Ebert
Salvana, 1989; Verdeil et al., 1989, 1993) and and Taylor, 1990; Ebert et al., 1993; Verdeil et
plumules from mature embryos (Hornung, al., 1999). The auxin 2,4,5-triehlorophenoxy-
1995, 1997; Chan et al., 1998). acetie acid (2,4,5-T) has also been used for
induction of nodular calluses from inflores-
Induction. The primary explants for cence explants (Buffard-Morel et al., 1988;
embryogenic culture must contain meristem- Verdeil and Buffard-Morel, 1995). The histol-
atic tissue, whieh proliferates in the presence ogy of callus has been studied (Buffard-
of an auxin. Immature leaves and inflores- Morel et al., 1992; Verdeil et al., 1992).
cences are the most useful explants, as the Callus grown on media with a gradually
phenotype of the mother tree is already reduced auxin level (Blake, 1990) or with an
known. Inflorescences are generally pre- increase followed by a reduction of auxin
ferred because of a simplified protocol and (Verdeil et al., 1994) will eventually produce
an inflorescence sampling protocol whieh nodular structures (Fig. 4.1.1). that subse-
does not result in death of the tree (Rillo, quentiy develop into proembryos (Fig. 4.1.2).
1989). Plumules (embryo meristem with the Abscisic acid (ABA) appears to affect the for-
first primordium) have been utilized mation of coconut proembryos (Samosir et
(Hornung, 1995, 1997), and this pathway can al., 1999b; Fernando and Gamage, 2000).
be exploited as a model for deveioping pro- Histologïcal studies of Embryogenie cultures
tocols using other explants and to multiply indicate that there are two developmental
the progeny from selected parents (Saenz et pathways. A multieellular pathway occurs
al., 1999). on medium with 2 g/l activated charcoal
Somatic embryogenesis generally occurs and 181-362 fLM 2,4-D (Buffard-Morel et al.,
indirectly by directive induction; however, 1992; Verdeil et al., 1992, 1994), but has also
there is a single report of direct embryogene- been observed on medium containing ABA
sis from leaf explants (Raju et al., 1984), (Fernando et al., 2003). Embryogenie cultures
which is unusual since vascular tissue nor- typically consist of meristematic and pro-
mally produces root primordia (Blake, 1989). embryonic structures. Initially, cells in the
96 V. Hocher et al.

and Maheswaran, 1986; Schwendiman et al.,

1988). There are deep invaginations of the
nuclear envelope, proliferation of dic-
tyosomes and emission of Golgi vesicles,
which is directly related to increased œil
wall thickness (Verdeil et al., 2001). Seven to
14 days after explanting, callose deposition
blocks the plasmodesmata, resulting in phys-
iological isolation. Acquisition of embryo-
genic competence was linked to the
appearance of an outer layer of pectic mater-
ial (mainly non-methyl-esterified) that
Fig. 4.1.1. Coconut embryogenic culture.
entirely coats the embryogenic cells (21 days
after explanting) (Verdeil et al., 2001). Specific
nutrient requirements have been observed
(Oussert et al., 1995a,b; Magnaval et al., 1995,
1997). Tyrosine phosphorylated proteins and
tyrosine kinase activity increase under
induction conditions (Islas-Flores et al.,
2000). A similar observation has been made
during coconut zygotic embryo development
(Islas-Flores et al., 1998, 2000).

Maintenance. Embryogenic cultures, irre-

spective of origin, are slow growing and
Fig. 4.1.2. Globular stage coconut somatic nod ular, and proliferation occurs from the
embryos. peripheral region (Buffard-Morel et al., 1992).
Embryogenic cultures are maintained on a
proliferation medium based on MS macro-
cambium-like zones prolifera te, and actively and Nitsch (1969) micro-elements, Morel and
dividing cells give lise to meristematic nod- Wetmore (1951) vitamins, 40 g/l sucrose,
ules that develop a protoderm or epidermis. 2 g/l activated charcoal and 7.5 g/l agar.
Proembryos develop from proembryonic This medium is supplemented with
cells in the periphery; however, if the auxin 271.5-362 IJ.M 2,4-0. Cultures are maintained
concentration is too low, anomalous struc- in darkness and subcultured every 2 months.
tures, e.g. haustorium only, a root pole,
foliar-type somatic embryos, etc., can Maturation. Soma tic embryo development
develop (Branton and Blake, 1983; Brackpool is asynchronous and occurs from < 10% of
et al., 1986). cultures. Regeneration of complete somatic
Another pathway occurs in the presence embryos requires lower 2,4-0 concentrations
of 2-3 g/I charcoal and 362-543 IJ.M 2,4-0, (181-271.5 IJ.M) (Fig. 4.1.3). Thidiazuron
whereby individual embryos deveJop from (TOZ) or 2-isopentenyladenine (2iP) has
single embryogenic cells (Schwendiman et been utilized effectively to stimulate devel-
al., 1988; Verdeil et ni., 1994). In that case, typ- opment (Verdeil et al., 1996b). Somatic
ical proembryos develop according to the embryos are maintained in the dark and sub-
description by Haccius and Phillip (1979). cultured every 2 montJls until shoot emis-
The embryogenic cells have dense cyto- sion. Oifferentiation of tJle shoot meristem of
plasm, a high nucleo-cytoplasmic ratio, a sin- tJle somatic embryo is cytokinin-dependent
gle and voluminous nucJeolus and many (Verdeil et al., 1994) and has been corrobo-
starch and protein reserves. They become rated by the increase in isopentenyl forms of
separated from the culture as a result of cell cytokinin during early soma tic embryo
wall thickening (Lu and Vasil, 1985; Williams deveJopment (Hocher et al., 1998a).
 Cocos nueitera Coconut 97

genesis. Foliar development is very slow and

is sometimes associated with leaf chlorosis.
The physiological status of in vitro shoots has
been studied using in vitro-germinated
zygotic embryos as a mode!. Different photo-
synthetic parameters have been studied
(Triques et al., 1997a,b): (i) chlorophyll fluo-
rescence to determine photosynthetic effi-
ciency; (ii) activities of phosphoenolpyruvate
carboxylase (PEPC) and ribulose 1,5-bispho-
sphate carboxylase/oxygenase (RubisCO)
were determined and the PEPC:RubisCO
Fig. 4.1.3. Somatie embryos during the ratio was used as an indicator of
maturation phase.
autotrophism; (iii) net photosynthesis rate
was estimated through CO 2 exchange mea-
surements; and (iv) chloroplast ulh'astruc-
Germination. Germination of the soma tic ture. A lower rate of net photosynthesis was
embryos occurs on maturation medium con- recorded for in vitro-grown plantlets com-
taining benzyladenine (BA). Gibberellic acid pared with acc!imatized pal ms, possibly due
(GA 3 ) can promote soma tic embryo germina- to lower RubisCO activity together with
tion in the presence of BA (Fig. 4.1.4). lower chlorophyll content compared to accli-
Cultures are transferred to the light after the matized plants (Triques et al., 1998),
development of two to four leaves. Root Santamaria ct al. (1999) demonstrated that
induction can be promoted by naphtha- sucrose lowered RubisCO activity, while
leneacetic acid (NAA). Maturation and slightly increasing the activity of PEPe.
acc!imatization of plantlets are major bottle- Since PEPC/RubisCO is a measure of plant
necks for regeneration by soma tic embryo- photoautotrophy (Desjardins, 1995), these

Fig. 4.1.4. Coeonut somatie plantlets trom the test tube (a) to the greenhouse (b).
98 II. Hocher et al.

results suggest that sucrose inhibits the regeneration from sorne of them.
development of photoautotrophy in vitro. Unfortunately, a low rate of division was
They suggested that sucrose might be impor- observed in coconut protoplast cultures and
tant in early stages of somatie embryo devel- no regeneration was reported.
opment; however, continuous growth in
sucrose-rich medium in later stages could
affect photoautotrophism and also plant per- 3.2. Conservation
formance ex vitro.
In vitro-grown plants (derived from Coconut seeds have no dormancy, causing
zygotic embryos) have reduced capacity to problems in transporting and storing
control water loss compared to field-grown germplasm (Assy-Bah et al., 1987;
plants, due to altered stomatal functioning. Engelmann and Dussert, 2000). Coconut
Ventilation of the culture containers resulted genetic resources are maintained in field col-
in an increased capacity of in vitro-grown lections (Verdeil et al., 1996a) in five coun-
plants to control water loss (Talavera et al., tries: Côte d'Ivoire, Indonesia, India, Papua
2001). These results have implications for in New Guinea and Vanuatu. The Côte d'Ivoire
vitro hardening and acc1imatization. collection is the most important in terms of
genotypic diversity, with 24,962 accessions
including 53 ecotypes (36 Tall types repre-
3. 1.2. Haploids
sented by 20,600 palms and 17 Dwarf types
Haploidy is of great interest considering the represented by 4200 palms) and 12 inter-eco-
allogamy of numerous coconut varieties and type hybrids (Bourdeix et al., 1998; N'Cho et
hybrids (Than-Tuyen and De Guzman, 1983). al., 1998). Ex situ conservation is costly, and
Monfort (1985) and Thanh-Tuyen (1985) collections are subject to diseases and c1i-
reported promising results but no regenera- matic adversity. The Coconut Genetic
tion, and they were unable to recover com- Resources Network (COGENT) was created
plete embryos. More recently Griffis and Litz in 1992 with the support of the International
(1997) obtained proembryos from cultured Plant Genetic Resources Institute (IPGRI) to
anthers, anther filaments and unfertilized bring together 35 producing countries in
ovary cultures on medium containing order to maintain and protect coconut
diethylstilboestrol; however, no further genetic resources (Baudouin et al., 2000; Table
development was reported. 4.1.1). The highest priority is to duplicate
field collections in vitro as pollen and
embryos (Ramanatha Rao and Batugal, 1998)
3.1.3. Protoplast isolation and culture
and to facilitate international exchange of
Haibou and Kovoor (1981) described the germplasm. Short- and medium-term storage
isolation of protoplasts from immature in vitro is essential for conservation of
inflorescence rachillae and microcallus germplasm that is free of known diseases,

Table 4.1.1. Countries with an international coconut genetic resources database (CGRD). Coconut
germplasm collections with passport and characterization data: a French-funded project. Number of
accessions per country. (Adapted from Batugal, 1997, 1999; Baudouin et al., 2000.)
Latin America! South-east
Africa na Caribbean na South Asia na Asia na South Pacifie na
Benin 4 Brazil 16 Bangladesh 4 Indonesia 156 Fiji 11
Côte d'Ivoire 99 Jamaica 60 India 212 Malaysia 92 Papua New Guinea 57
Tanzania 72 Mexico 20 Pakistan 32 Philippines 224 Vanuatu 66
Sri Lanka 78 Thailand 52 Western Samoa 9
Vietnam 31 Solomon Islands 21
Total per region 175 96 326 555 164
na, number of accessions.
 Cocos nueifera Coconut 99

and represents the safest method for interna- mature eoconut embryos could be cryopre-
tional exchange of material (Withers and served after 4 h desiecation in a laminar air
Williams, 1985). It is also a prerequisite for flow followed by immersion for 11-20 h in a
cryogenie storage. cryoprotectant consisting of 600 g/l glucose
Routine techniques for collecting zygotic and 15% glycerol (Assy-Bah and Engelmann,
embryos have been developed, including 1992b). Four coconut varieties (hybrid
field collection, disinfecting and embryo cul- PB121, Indian Ta Il, Cameroon Red Dwarf
ture (Assy-Bah et Ill., 1987; Ril1o, 1995; and Rennell Island Tall) were successfully
Ashburner et al., 1996; Samosir et al., 1999a; cryopreserved with a germination rate of
Karun, 2001; N'Nan et Ill., 2002a). Excised 10-93%, depending on ecotype. These results
embryos can be stored in KCI for up to 14 were validated with West African Tall (WAT)
days before in vitro culture (Assy-Bah ct al., and MW (N'Nan, 1997), and later with ten
1989). Coconut embryo culture was initially more ecotypes (N'Nan ct al., 2003).
developed in the Philippines for embryo res- Plumules have been cryopreserved by
eue of 'Makapuno', a highJy valued encapsulation / dehydration (N'Nan, 1999;
Philippine mutant genotype (De Guzman Malaurie and Borges, 2001; Malaurie et al.,
and Del Rosario, 1964; Del Rosario, 1998). 2002). Plumules were excised and encapsu-
Karunaratne ct al. (1991) used coconut lated in alginate beads, and exposed to dif-
embryo culture to measure drought toler- ferent sucrose concentrations and
ance in Sri Lanka, and were able to sereen a dehydration periods, resulting in 40-80%
large number of genotypes in a short time (2 survival after cryopreservation. Up to 70% of
years). Rillo (1985) used embryo rescue to plumules of sorne ecotypes germinate nor-
screen for disease tolerance. mally following cryopreservation (Malaurie
Different protocols for embryo culture and Borges, 2001; N'Nan et al., 2002b; Fig.
have been described (Del Rosario and De 4.1.5). Hornung et al. (2001) cryopreserved
Guzman, 1976; Karunaratne ct al., 1985; Assy- plumules, and attempted to induce embryo-
Bah, 1986; Sossou et al., 1987; Assy-Bah ct al., genic cultures according to the protocol of
1989; Rillo and Paloma, 1991; Karun ct al., Chan ct al. (998). Other cryopreservation
1993; Ashburner et Ill., 1996; Rillo, 1999). Low techniques, e.g. eneapsulation, osmoprotee-
germination and survival rates of plants ex tion, dehydration and encapsulation, osmo-
vitro indicate that the protocol requires protection and vitrification (Sakai ct al.,
improvement. An international programme 2000), have been applied to plumular tissues,
coordinated by COGENT has begun to focus and shoot deveJopment has been reported
on improving in vitro culture and acclimatiza- (Malaurie et al., 2003).
tion protocols (Ba tugal and Engelmann, 1998). Hybridization and improved nut produc-
Zygotic embryos can be stored in vitro for tion are facilitated by assisted pollination
medium-term periods (6 to 12 months) with- (Wuidart and Rognon, 1981; de Nueé de
out loss of germination (Assy-Bah and
Engelmann, 1993; M'kumbo, 1995).
Development can be suppressed by high
levels of sucrose and activated charcoal
(Assy-Bah, 1992; Verdeil et al., 1998b).
lnereased osmolarity and reduction of nutri-
ent concentration can also impede develop-
ment (Damasco, 2002). None the less,
long-tenn conservation by cryopreserva tion
is essential to reduce the 10ss of important
genetic resources.
Early attempts to cryopreserve coconut
embryos by Bajaj (1984) and Chin et al. (1989)
were not very successful. Assy-Bah and Fig. 4.1.5. Somalie embryo developmenl from
Engelmarul (1992a,b) demonstrated that dehydraled, eneapsulaled and frozen plumule.
100 V. Hocher et al.

Lamothe and Wuidart, 1992). According to 1999; Nair et al., 1999). A list of treatments
Towill (1985), palms have long-lived pollen; has been proposed for controlling the spread
however, for long-term breeding pro- of these diseases in the technical guidelines
grammes, extended storage of pollen is for the safe movement of coconut
essential (Towill and Walters, 2000). Coconut germplasm (Table 4.1.4). There are no thera-
pollen storage was reported by Whitehead pies for eliminating coconut virus, viroid
(1965) using freeze-drying. Pollen desieca- and phytoplasma diseases of coconut.
tion to 4-5% moisture content over silica gel, Reverse transcription polymerase chain
followed by storage in vacuo in a freezer, reaction (RT-PCR) has demonstrated the
does not cause loss of viability for > 6 presence of LY phytoplasma in embryonic
months (Rognon and de Nucé de Lamothe, tissue, including the plumule (Cordova et
1978). Cryopreservation of pollen is also fea- al., 2003). Exchange of coconut germplasm
sible (Frison et al., 1993; Engelmann, 1999), by means of zygotic embryos corresponds to
and recommendations for collecting, condi- the basie Food and Agriculture Organization
tioning and cryogenie storage of pollen have (FAO)/Inernational Board for Plant Genetic
been reported (Frison et al., 1993). Resources (IBPGR) guidelines for moving
Technical guidelines for the safe move- coconut germplasm (Diekmann, 1997, 1999;
ment of coconut germplasm have been Ramanatha Rao and Batugal, 1998); how-
established (Frison et al., 1993; Diekmann, ever, existing indexing protocols do not pro-
1997; Baudouin, 1998; Table 4.1.2). Indexing vide adequate security. In vitro collections of
techniques for screening germplasm for coconut germplasm are located in six
known diseases is critical, e.g. CFDY, which coconut-producing countries and two
causes foliar decay in Vanuatu, CCCVd in European countries (Table 4.1.5).
the Philippines and LY, a phytoplasma-asso- The establishment of the multi-site
ciated disease, which has caused great dev- International Coconut Genebank (ICG),
astation in the Caribbean region and more hosted by India, Indonesia, Papua New
recently in Ghana (Harrison et al., 1999; Guinea and Côte d'Ivoire for their respective
Rodriguez, 1999). All of these diseases regions, will have the responsibility to con-
(Table 4.1.3) should be prevented from being serve and share a maximum of 200 im-
transferred outside their current area of dis- portant accessions from South and South-
tribution (Frison et al., 1993; Diekmann, east Asia, the Pacific region and Africa and
1997, 1999; Hanold and Randles, 1997; Indian Ocean islands, respectively (Table
Dollet, 1999; Hodgson and Randles, 1999; 4.1.6). The accessions maintained in ICG
Howard and Harrison, 1999; Jones et al., will include: (i) the principal varieties; (ii)

Table 4.1.2. Summary of FAO/IBPGR Technical Guidelines for the Safe Movement of Coconut
Germplasm. General recommendation: to move embryo culture or pollen, not nuts. (Adapted fram
Harrison et al., 1995; Diekmann, 1997; Ramanatha Rao and Batugal, 1998; Dollet et al., 2001 a,b.)
Pathogen Specifie recommendation
CFDV Indexing or exclusion of germplasm from Vanuatu
CCCVd Indexing or exclusion of germplasm fram the Philippines
CtiVd Indexing or exclusion of germplasm from Guam
Viraid-like sequence Indexing or exclusion of germplasm that is moved from countries
where these sequences are known to occur to countries where they
have not yet been reported. Recommendation under revision
LY, phytoplasma Transmission through seed, embryo culture or pollen not reported,
Kerala wilt, phytoplasma but suspicion still exists
Tatipaka disease, phytoplasma A nursery disease which does not occur on adult trees
Blast, phytoplasma
CtiVd, coconut tinangaja viroid.
Table 4.1.3. Causal agent, vector, final disease evolution, geographical distribution of the coconut diseases, and techniques available for indexing (adapted
from Frison et al., 1993; Hanold and Randles, 1997; Diekmann, 1999; Dollet, 1999; Hodgson and Randles, 1999; Howard and Harrison, 1999; Jones et al.,
1999; Naîr et al., 1999; Dollet et al., 2001).
Indexing: Indexing:
Type of Final disease Geographical conventional molecular
disease Disease name Cause Veclor evolution distribution techniques approach

Viral Foliar decay Coconut foliar decay virus Myndus taffini (Cixiidae) ln susceptible coconut palms, Vanuatu, and suspected Dol-blot hybridization and
(CFDV): icosahedral virus planthopper Ihe crown dies within 6 months in other areas complementary iabelled
to 2 years DNAprobe
Viroid Coconut Coconut cadang-cadang viroid Field and seed transmission are 8 to 16 years elapse between first Occurs in certain parts of PAGE MHA. Hybridization analysis
cadang-cadang (CCCVd); circuiar single-stranded observed and pollen suspected. symptoms and death of the paim. the Philippines with radioactive RNA
RNA in a rod-like structure Mechanism of transmission Sorne palms die soon, those that probes (Northern blotting) ;
remains unknown continue to develop never flower Rt-PCR
Coconut Coconut tinangaja viroid (CtiVd); Means of natural transmission Diseased palms decline and die in Guam PAGE Hybridization analysis with
tinangaja single-stranded circular RNA unknown similar manner to cadang-cadang radioactive probe ()
o
Viroid-like - Viroid-Iike sequence similar to Means of natural transmission South Asia to French Northern blotting technique ()
o
sequences but not identical to CCCVd unknown Poiynesia with a complementary RNA (J)

probe specific to CCCVd :J

C
Q.
Mollicute Blast Mycoplasma-like organism (MLO) Reci/ia mica Kramer (Jassidae) Atrica, and South America (p"
and Indonesia for similar Pl
symptoms
Lethal yellowing Phytoplasma
(LY)
Myndus crudus (Cixiidae)
pianthopper; suspicion over
The whole of the crown eventually Africa, Central America
rots and falls off within 3-6 months and Caribbean
different phloem-feeding insects of the appearance of the first
Light or electron
microscopy with
fluorescent staining
Amplification by PCR of the
16-23S rRNA region of
phytoplasma
~
~
for LY in Africa symptoms. Complete destruction (DAPI)
of plantation in Mexico
Rootwillor Mycoplasma-like organism (MLO) Stephanistis typica; Proutista Symptoms appeared only on India (parts of Kerala and Light microscopy with PCR
Kerala will moesla (putative vector) 30-month-old palms. The disease Tamil Nadu states) fluorescent staining
is not lethal, but significantly (DAPI)
reduces production
Tatipaca disease Mycoplasma-like organism (MLO) Unknown The disease is not Iethai, but India (East and West Light microscopy with PCR
significantiy reduces production Godavari, Srikakulam and tluorescent staining
Nellore in Andhra Pradesh) (DAPI)
Heartrot disease Trypanosomatid Pentatomid bugs from the Surinam, Salvador de 40 x 10 phase-contrast None
genus Lincus Bahia Province, north light microscope
Honduras, Trlnidad,
Costa Rica

DAPI, 4'-6-diamidino-2-phenylindole; MHA, Mueller-Hinton agar; PAGE, poiyacrylamide gel electrophoresis; RT·PCR, reverse transcription polymerase chain reaction.
102 V. Hocher et al.

Table 4.1.4. Therapy available against the different cocon ut diseases (adapted fram Frison et al., 1993;
Diekmann, 1997).

Disease name Cause Therapy

Foliar decay CFDV None

Coconut cadang-cadang CCCVd None
Coconut tinangaja CtiVd None available
Viroid-like sequence None
Blast Phytoplasma, MLO None
Lethal yellowing (LY) Phytoplasma, MLO Tetracycline, but no elimination of the phytoplasma
from palms
Root will or Kerala wilt Phytoplasma, MLO Tetracycline, but no elimination of the phytoplasma
from palms
Tatipaca disease Phytoplasma, MLO Tetracycline, but no elimination of the phytoplasma
from palms

Table 4.1.5. COGENT member countries concerned in international exchange of cocon ut (Cocos
nucifera L.) germplasm, and expected COGENT member countries (adapted from Batugal, 1997, 1999).

Latin America!
Africa Caribbean South Asia South-east Asia South Pacifie

Côte d'Ivoire Brazil Bangladesh China Cook Islands

Ghana Costa Rica India· Indonesia· Fiji
Kenya Cuba Pakistan Malaysia Kiribati
Mozambique Guyana Sri Lanka· Myanmar Pa pua New Guinea •
Nigeria Haïti Philippines· Solomon Islands
Seychelles Jamaica Thailand Tonga
Tanzania Mexico· Vietnam Vanuatu
Trinidad-Tobago Western Samoa
Possible future members
Comaro Colombia Marshall Islands
Madagascar Dominican Republic Tuvalu
Ecuador
El Salvador
Guatemala
Panama
Venezuela

ln bold, regional cocon ut genebank, also called International Coconut Genebank (ICG).• Number of
countries with in vitro collection (this number reflects more the laboratories involved in tissue culture in
coconut, where United Kigdom (Imperial College, Wye) and France (IRD/CIRAD team, Montpellier) have
an important and active place).

threatened varieties, and varieties with spe- 4. Conclusions

cial traits; (iii) additional diversity dis-
covered during national explorations; and The coconut palm is a major agricultural
(iv) duplicates of accessions from other species and is an important subsistence crop.
regions (Batugal, 1997). In addition, the IeG Since the mid-20th century, a decline in pro-
will undertake field evaluations and share ductivity has occurred worldwide, despite
data and germplasm with member countries the use of improved planting material and
using safe exchange guidelines as prescribed agronomie practices. Biotechnology and its
by FAO and IPGRI (IPGRI, 2000). application to coconut can create new oppor-
 Cocos nucifera Coconut 103

Table 4.1.6. State of the cocon ut germplasm present in the host countries of the regional coconut
genebank. The state of cocon ut germplasm present in Vanuatu is given taking account of its interesting
diversity despite the great risk of genetic erosion caused by coconut foliar disease (CFD). (Adapted from
Baudouin, 199B; N'Cho et al., 199B; Ramanatha Rao and Batugal, 199B.)

Côte d'Ivoire India Indonesia PNG Vanuatu

Ecotype/ Ecotype/ Ecotype/ Ecotype/ Ecotype/
Ecotypes Accession Accession Accession Accession Accession

Tall 36/20,600 6B* + 34**/nc 79/4,337 17/nc 24/2,261

Semi-Tall 2 + O/nc
Dwarf 17/4,200 16 + 12/nc 9/923 6/nc 17/1,OB5
Hybrids 12/nc nc nc nc nc
Indigenous 34 Tall/12 Dwarf
Total accessions 27,962 nc nc nc nc

nc, not communicated; PNG, Papua New Guinea.

*Number of ecotypes collected in different areas outside India; ** number of indigenous Indian ecotypes.

tunities in breeding, cloning, disease control their conversion rate is unacceptable. There is
and germplasm exchange / conservation. a need to better understand the basic botany
COGENT /IPGRI encourages and supports and biochemistry of coconut somatic and
collaboration among various national zygotic embryo development. Studies are
coconut research groups; this is absolutely under way that would characterize genes that
critical as there are insufficient funds to sup- are implicated in the cell cycle regulation of
port the research needs for this crop (Hocher coconut (Sandoval, 2002; Sandoval et al., 2003).
et al., 1998b; Punchihewa, 1999; Rohde et al., Such studies together with genetic transforma-
1999). The development of molecular breed- tion (C. Oropeza, personal communication)
ing tools, e.g. linkage maps and QTLs, should provide opportunities for coconut
should facilitate MAS for the recovery of genetic engineering and improvement.
hybrids with greater productivity and resis-
tance to diseases (Cardefia et al., 1999). Safe
exchange of germplasm can only occur if Acknowledgements
there are accurate methods for detecting and
elimination of diseases. This work is supported by IRD and CIRAD-
Cryobanks for zygotic embryos are a real- CP. The authors would like to thank the fol-
ity (N'Nan et al., 2003), and investigations lowing institutes' for their fruitful
based upon cryopreservation of plumules collaboration: CNRA (Côte d'Ivoire), CICY
will have a great impact on storage and man- and INIFAP (Mexico), PCA (the Philippines),
agement of genetic resources (Hornung et al., Hanover University (Germany), Imperial
2001; Malaurie et al., 2003). College at Wye (United Kingdom), CRI (Sri
Somatic embryogenesis is promising as a Lanka), COGENT and IPGRI. Part of the
means for propagating elite material and for work cited in this chapter was realized with
genetic manipulation. After several decades of European Community (EC) funding (Contract
little success, there are now clonally propa- STD3 ERBTS3*CT940298). We also want to
gated plants in the field (Verdeil et al., 1999). thank the United Nations Educational,
The number of plantlets that have been recov- Scientific and Cultural Organization
ered from somatic embryos remains low and (UNESCO) and BRG for their support.

*BRG, Bureau des Ressources Génétiques, Paris, France; CICY, Centra de Investigaci6n Cientifica de
Yu ca tan, Mexico; CNRA, Centre National de Recherche Agronomique, Côte d'Ivoire; CRI, Coconut
Research Institute; INIFAP, Instituto Nacional de Investigaciones Forestales, Agrfcolas y Pemarias,
Mexico; PCA, Philippines Coconut Authorities.
104 V. Hocher el al.

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