!

i

1 propagation of coconut palms Application of in vitro techniques to the conservation and

1

í t

‘ 9 V.jktOCHER, J.L. VERDEIL, A. RIVAL and S/jIAMON

Laboratow GemTrop, ORSTOM, 91 I Avenue Agropolis, BP 5045, 34032 Montpellier Cedex O1 FRANCE. E-niail: hocher@mpl. orstom$

- 1 I

! I

I

. % 1. Introduction

The coconut palm generally considered as “the” symbol of the tropics, and also called the Tree of Life because of its importance as a subsistence crop in most tropical areas of the world. It is grown on more than 10 millions ha in about 90 countries, where it is cultivated mainly for copra (dried endosperm) production, from which oil is extracted. It ensures income for millions of smallholders in the sub-tropical areas and is also a primary source of food, drink and shelter for millions of inhabitants.

I

However, the coconut indusby has several problems. Particularly a decline of productivity and an increasing competition fi-om other oil producing crops. Due to its economic importance in sub-tropical areas, it is now essential to maintain and

biotechnology. This paper will review facilities offered by the application of in vitro methods and how they are applied for the genetic improvement of coconut.

1 improve this culture by using the possibilities offered by breeding and

I . 1. Botanical characteristics of the plant

The coconut palm (Cocos nucifera L.) is a woody perennial monocotyledonous plant with a very slow vegetative development, with growth from single terminal vegetative bud. An adult tree has virtually as many unopened as opened leaves (20 to 30). Each leaf bears a flower primordium in its axil. Each axillary bud produces an inflorescence that consists of 20-30 branches (rachillae), bearing a large number of male flowers with few female flowers at the base of each rachilla. Pollination is by wind or insects and the nut is ripe about 12 months after fertilisation. Natural propagation is entirely by seed. The seed, which is one of the

I

largest in the plant kingdom, is characterised by its lack of dormancy and and the long time required for the development of the embryo into a plantlet (Blake, 1990; Verdeil, 1993).

1.2. Importance of coconut

1.2.1. As a sniallliolder crop

Coconut fits perfectly in a smallholder's economy. More than 90% of the coconut world-wide production is obtained from small plantations with an area less than 4 ha. It is mainly a subsistence crop, for instance, in Asia 70% of the production is 'locally consumed. Every part of the plant can be used. Oil can be extracted from the fresh nuts. Fresh nuts are used for food preparations in many countries of Asia and the Pacific. Coconut water is a very refreshing drink. The endosperm of mature nuts is grated and used in pastry. The woody stem is used as building material and in joinery. The leaves can serve for local handicrafts and as roofing material. The processed sap gives sugar, syrup, alcohol or vinegar. The fibres from the husk surrounding the nut can be used to manufacture esparto-type goods (for a detailed review see The Philippines Recommends for Coconut, 1993).

,

1.2.2. As an indistrial crop

Coconut represents also an economic value of primary importance in lots of countries such as Philippines (11 '%O of exports) or Vanuatu and Samoa islands (around 50% of their exports). The main industrial product obtained íì-om coconut is its oil, extracted from the dried endosperm (copra). Copra oil is an important commercial product world-wide, since, along with oi l palm kernel oil, it is the only source of short-chain fatty acids (from 8 to 14 carbon atoms), and therefore it is greatly sought after by industry for its richness in lauric acid (-48%). It has good lathering properties and is mainly used in soap manufacture, but also in food (margarine, confectionery, etc.) and cosmetic industry (Blake, 1990; Verdeil et al., 1996).

1.3. Drflculties encountered in coconut production

Historically, the importance of coconut culture increased significantly from 1850 with the increase in world-wide exchange of goods. During the First World War, coconut was a highly strategic oleaginous plant, as its oil was the primary source of glycerine, a basic component of dynamite. Then, from 1914 to the end of the 1950s, coconut became the main oil source in the world market. Today, with 4% of the world oil production, coconut ranks only 7* among oil-bearing crops.

268

Several reasons can explain this gradual decline:

a. A low productivity due to old age of coconut plantations (two thirds of the palms are over 60 years old), and insufficient replanting rates.

b. The use of unimproved material and the marginal culture practices. c. Several pests and diseases spread (e.g. lethal yellowing in Méxic.0 or cadang-

cadang in the Philippines) and no treatments are yet available. d. Coconuts are generally grown in areas often subje'cted to natural calamities

like typhoons or volcanic eruptions. e. Coconut oil price is low, and despite a higher quality and a lower production

cost for copra oil, there is an increasing competition fi-om oil palm kernel oil and more recently with transgenic rapeseed oil (cultivated in US) which has been genetically modified to produce a higher proportion of lauric acid.

Despite all these diffculties, and an stagnant production in the last 20 years, coconut oil is still econonlically important for different countries, and there is still a high industrial demand for lauric oil, specially for the soap and detergent industry that represents about 50% of the consumption of copra oi l in the OECD countries. As a response, coconut growing countries have started rehabilitation programs for coconut cultivation. For instance, with the help of the World Bank, the Philippines have started a re-plantation prograni using improved hybrids, and the fight against lethal yellowing was recently declared "a national priority" in México.

However, these programs will be successful only if improved plant material (highly productive and/or disease resistantholerant) is produced in high numbers and made easily available to coconut growers.

1.3. How to produce inlprovedplant material?

1.4.1. Conventional breeding

At the beginning, the main objectives of coconut breeding concerned the improvement of productivity of copra per hectare. Now other characters like precocity, resistance to diseases or adaptation to some climatic condition (drought, cold, salinity, etc.) are also sought. Conventional breeding is based on the exploitation of the natural diversity of this species and has led in 20 years to the production of hybrids with a doubled yield of copraha (Gascon and de Nucé de Lamothe, 1978; Baudouin, this volume). Improvement using the best hybrids of each generation can generate a gain in productivity of about 20 to 30% in each new generation.

269

Until now the diffusion of genetic progresses is only possible through the production of hybrid seeds obtained by assisted pollination (Wuidart and Rognon, 1981). Although efficient, this method is nevertheless very time consuming (de Nucé de Lamothe and Wuidart, 1992). In these conditions, the price of a selected seednut can reach 2 to 4 US dollars, which is too expensive for smallholders. Moreover, conventional breeding techniques 'have constraints linked to the morphological and biological characters of this species. There is a frequent allogamy responsible for high variability, a very long breeding cycle (12 to 16 years), a low coefficient of reproduction (100 to 200 seeds/tree/year) with a large and recalcitrant seed that makes exchange and conservation of germplasm difficult, and the lack of natural vegetative propagation.

!

1.4.2. 'In vitro techniques

As a means to overcome these difficulties, in vitro techniques have generated interest as a support to conventional breeding (Santos, this volume), and for more than 20 years, several teams have worked on the application of these techniques to coconut. Nowadays, basically two techniques are used: in vitro culture of zygotic embryos and clonal propagation through somatic embryogenesis.

Embryo cultwe could facilitate the exchange of germplasm by circumventing the constraints linked to the size of the coconut seed and to the lack of dormancy of the embryos. Also its association with cryopreservation should permit the long term conservation of the coconut germplasm through the creation of cryobanks, which are complementary of field collections. In other words, cloning coconut is of the highest importance to help the diffusion of breeding advances by allowing the multiplication of the best genotypes obtained through conventional breeding. Different applications can also be considered.

a. Cloning could be a great help for easier creation of seedgardens. Presently, the selected parents for the generation of selected seeds are reproduced through self-pollination give a population of hybrids with a low multiplication rate (and thus a high price). Cloning the parents would improve the production rate of more homogenous seeds. The diffusion would be done through "mono-clonal" or "bi-clonal" seednuts. The production of improved hybrids would then be easier.

b. Cloning could also allow a rapid multiplication of selected individuals exhibiting resistance or tolerance to important diseases and/or to adverse

270

growing conditions. The fast distribution of resistant planting materials obtained through clonal propagation could contribute to the control of the threatening cadang-cadang or lethal yellowing diseases.

c. Lastly, the cloning of selected elite palms would generate significantly higher and earlier yields than the highly variable traditional planting material derived from seednuts.

However, the application of in vitro techniques to coconut largely depends on the regeneration capacity of this species, which is known as one of the more recalcitrant for in vilm culture.

2. Application of in vitro techniques to coconut

2. I . Embryo culture

2.1.1. Embryo rescue

The earliest attempts at coconut tissue culture were actually a form of “embryo rescue” using the embryo Makapuno, a mutant coconut genotype of high price, in the Philippines. In normal nuts the enzyme a-D-galactosidase converts galactomannans to water-insoluble mannans, which are deposit in the endosperm, leaving the centre of the nut filled with coconut water. This enzyme is deficient in the Makapuno nuts so that the endosperm remains as a jelly and fills the whole cavity of the nut. Although the Makapuno coconut contains an apparently normal embryo, the embryo never germinates in situ. The Makapuno coconuts are found as a small proportion of normal h i t on a few palms, generally of the ‘Laguna tall’ ecotype. Such trees are highly prized since the Makapuno coconuts are an expensive delicacy. Such jelly nuts are also found in other countries: Dikiri in Sri Lanka or Kopyor in Indonesia.

Historically, De Guman, who started to rescue the Makapuno embryo conducted the first trials in the Philippines early in the 1960’s. She designed a protocol (De Guzman and Del Rosario, 1964) which has been improved (for a review see Del Rosario, 1998). This protocol is actually routinely applied by several teams and some production units have been recently created in the Philippines (Batugal and Engelmann, 1998; Rillo, this volume).

27 1

2.1.2. Conservation and exchange of germplasm

There is also a considerable interest in embryo culture, since it could lead to an easier and safer method for the exchange of germplasm, and it could be useful for embryo conservation through cryopreservation. Indeed, establishing and maintaining coconut field collections (like for example the one in Côte d'Ivoire) is indispensable but also very costly and open to the risk of disease infection or adverse conditions. The in vitro techniques have several advantages: the use of aseptic conditions, a reduced space for storage is needed, easier and less expensive maintenance and a simplification of the international exchanges.

For germplasm exchange, the FAOAPGRT Technical Guidelines for the Safe Movement of Coconut Germplasm highly recommend the use of zygotic embryos contained in vitro to reduce the risks of introducing diseased material into disease- free areas. Regarding the conservation of coconut germplasm, preliminary experiments have indicated that it is possible to store zygotic embryos in vitro for one year and to successfully germinate them afterwards (Assy Bah and Engelmann, 1993). Long ter& conservation through crypreservation in liquid nitrogen was also demonstrated (Assy Bah and Engelmann, 1992a; 1992b; Engelmann, this volume). This should reduce the loss of important genetic material and is a prerequisite to the creation of coconut germplasm cryobanks.

f

Using in v i ~ o techniques for collecting, exchanging and conserving germplasm requires efficient protocols for in vitro germination, development of the embryos into plantlets, acclimatisation of these plantlets to in vivo conditions and a successful final transfer to the field. Different protocols have been established for embryo culture by various research institutes: ORSTOM/CIRAD (France), PCA (Philippines), CPCRI (India), CICY (México), MART (Tanzania). These protocols have been largely applied (Batugal and Engelmann, 1998; Rillo, this volume), but different problems encountered such as low germination rate, low survival rate of the plantlets specially during ex vitro transfer show that the protocols still need to be improved. Following the last International Workshop on Coconut Embryo Culture (PCA Albay research Centre, Philippines, 27-3 1 October 1997), problems encountered in coconut embryo culture has been clearly identified (low germination rate, heterogeneity of development and a low survival rate of the plantlets) and a joint international program co-ordinated by COGENT has started in order to produce an up-graded in vitro culture and acclimatisation technology (Batugal and Engelmann, 1998).

272

I I

Í ! 1 1

I

1 I I i l

I i i !

! I I

j

I

i I

I

I

2. I . 3. Other applicaiions

One interesting application of embryo culture was demonstrated by Karunaratne et al. (1991) in Sri Lanka, where coconut embryo culture was used to design a test for tolerance to drought. This technique allowed the test in short time (2 years) of a high' number of genotypes and could also be applied for the search for disease tolerance (Rillo, 1985). It is also important to note that coconut zygotic embryo culture can be use to design physiological studies, which results can be applied to improve the development vitroplants derived from somatic embryos (Rival et al., and Santamaría et al., this volume).

2.2. Clonal propagation

2.2. I . DgfJiczrliies encountered in coconui cloning

The Arecacae family is considered as very poor to respond to in vitro culture. According to Kovoor (1981), it has the difficulties of both the Monocotyledons and the woody crops for in vitro culture. Coconut seems to be one of the more recalcitrant palms (Rillo, 1989a) and is classified according to George and Sherrington (1984), as one of the most recalcitrant species to regenerate in vitro. Among the difficulties encountered, we can list:

a. A slow response in vitro. b. A high heterogeneity of the behaviour of the tissues in vitro. c. An intensive browning of the tissues (due to the oxidation of polyphenols). d. A high rhizogenesis ability and . a low capacity for embryogenesis or

caulogenesis.

2.2.2. Coconut cloning: towards an international coconut network

In vitro culture applied to coconut cloning started as early as in 1981, when Haibou and Kovoor (1981) described the production of protoplasts and callus regeneration from some of them. Unfortunately, no plantlet was obtained. The necessity to maintain the mother tree in the field eliminates the possibility of culturing the vegetative meristem. Floral meristem reversion (Davis, 1969; Blake and Eeuwens, 1980) and neoformation of caulinary meristems after organ fragment culture (Balaga, 1975) were considered, but are now abandoned due to the lack of results. For 20 years, most teams working on coconut cloning have concentrated their efforts on somatic embryogenesis, using different explants. Coconut clonal propagation has been attempted using different types of explants:

273

young roots of mature palms (Justin, 1978), stem and leaf (Pannetier and Buffard- Morel, 1982; Gupta et al., 1984; Raju et al., 1984), embryos (Karunaratne and Periyapperuma, 1989), inflorescences (Branton and Blake, 1984; Ebert and Taylor, 1990; Verdeil et al., 1994). If plantlets were sometimes obtained (mainly from immature leaves and inflorescences), only two or three vegetatively propagated plantlets have been established ex vitro in the field.

The first trials have demonstrated that coconut cloning through somatic embryogenesis is indeed possible but very difficult. Different teams have started to work on coconut somatic embryogenesis all over the world, but after the first very enthusiastic results, a competition between the research groups arose to a certain extent and resulted in a lack of communication that did not benefit research.

!

In 1994, some of the teams (Wye College, United Kingdom; Hanover University, Germany; ORSTOWCIRAD-CP, France; IDEFOWDPO, Côte d'Ivoire; PCA, Philippines; CICY, México) working on coconut embryogenesis joined together under a EC funded project (STD3 ERI3TS3 *CT940298), and this has favoured the achievement important advances. The different phases of the in vitro protocols (callogenesis, initiation of embryogenesis, maturation) have been improved by the different participants of the project and plantlets are now regularly obtained in most laboratories (for a detailed report see Verdeil et al., this volume). Presently, there is an increasing number of institutes working on coconut tissue, and following the recommendations fi-om the International Symposium on Coconut Biotechnology in Mérida, 1997 (see Concluding Remarks, this volume), an international network is currently being established under the coordination of COGENT and IPGRI.

2.2.3. The current cloning protocols

Historically, the first starting material used were immature leaves or inflorescences which are considered as the best starting material as the performance of the mother tree is already known. Actually if leaves are still used in some laboratories (ORSTOWCIRAD, PCA, etc.), inflorescences tends to be preferentially used as the in vitro protocol for somatic embryogenesis is then simplified and shorter, but also because PCA designed a process for inflorescence sampling which allow the survival of the tree (Rillo, 1989b). More recently, trials were conducted using the plumule (embryo meristem + first leaves) which have the advantage of being a highly and rapidly reactive material (Hornung, 1995). It could be used as a model fiom which further knowledge could be derived for

274

advancing protocols using other explants, such as inflorescences and leaves, but it is also potentially useful when embryos obtained from selected parents (for example, disease resistant palms) are available in low number (Chan et al., 1998).

Several experimental protocols using inflorescences .or plumules are now available laboratories (see in this volume: Homung and Verdeil; Sáenz et al.). The different phases (callogenesis, initiation and maturation of somatic embryogenesis) are mastered and the regeneration of plantlets is regularly obtained. These advances were also made possible thanks to the use of different techniques such as histology (Buffard-Morel et al., 1992), hormone quantification (Verdeil, 1993; V. Hocher, unpublished results), nutritional studies (Dussert et al., 1995a; 1995b; Magnaval et al., 1995; 1997), photosynthesis characterisation (Triques et al. , 1997a; 1997b; Rival et al., this volume), and search for markers of (Islas-Flores et al., 1998; Köhne and Jacobsen, this volume) that helped to describe the process and to understand and overcome some of the difficulties.

Currently, the main remaining bottleneck is the development of the clonal plantlets and their acclimatisation: the rate of regenerated plantlets is low and they are often weak showing a very slow development. In addition the transfer to ex vitro conditions is often unsuccessful. The improvement of the late phases is necessary if we want, in the short term, to obtain sufficient numbers of somatic plantlets to establish tests in the field. For this purpose, experiments have started on the physiology of vitroplantlets derived from zygotic embryos (Triques et al., 1997a; 1997b; Santamaría et al., this volume). Additionaly, studies for the characterisation of the nut nutrients during germination have started in ORSTOM/CIRAD. Together, these efforts should provide useful information for the improvement of in vitro protocols.

In the long term, another objective is to increase the number of regenerated plantlets. The present efflciency of the protocols allows the production of around 100 plantlets per clone. This should be sufficient to test for the assessment of disease or the plantation of seed gardens for the production of mono- or bi-clonal seeds. For cloning at a higher scale, a longer time would be necessary because there is a need for a multiplication rate of about 1000 plantlets per clone. In that condition, a multiplication phase should be introduced in the protocol (secondary embryogenesis and/or suspension culture).

275

3. Conclusion

Although coconut is one of the most recalcitrant species in in vitro culture, its importance as a subsistence crop in the tropical area justifies the efforts developed for the establishment of reliable in vi~ro culture protocols. The current state of the art is globally positive.

Today embryo culture can be applied to the conservation and high scale exchange of germplasm. In addition, the application of embryo rescue to the Makapuno genotype is entering a commercial phase in the Philippines. Within the next few years, the improvement of the present protocols, and of the cryopreservation process will lead to the creation of cryobanks, which will represent a greater oportunity for the conservation of coconut germplasm by ensuring the duplication of the field collections.

It is hoped that the recent advances in mastering somatic embryogenesis will allow in the short term (probably within 5 years), the application of coconut cloning for the generation of demonstration plots, and concomitantly, within the next 10 years, the evaluation of the performance and conformity of the clonal plantlets. The use of cloned coconut is fmtly thought of as a tool to support conventional breeding. It could be rapidly applied to the production of mono- or bi-clonal seeds and could be useful for the screening of disease toleranthesistant individuals. On the other hand, the propagation of elite individuals will lead to increased yields and homogeneity of plantations in coming years. To reach this longer-tem objective, there is a need for a scaling-up phase of the current regeneration protocols. Mastering coconut cloning would also open the way for genetic engineering that should allow a more precise and directed approach of variety creation, through the introduction of characters such as resistance to disease or adaptabiIity to edaphic conditions.

References

1. Assy Bah B and Engelmann F (1992a). Cryopreservation of immature embryos of coconut (Cocos mciJera L.). Cryo-Lett 13:67-74.

2. Assy Bah B and Engelmann F (1992b). Cryopreservation of mature embryos of coconut (Cocos nucifera L.) and subsequent regeneration of plantlets. Cryo-Lett 13:117-126.

3. Assy Bah B and Engelmann F (1993) Medium-term conservation of mature embryos of coconut (Cocos nucifera L.). Plant Cell Tiss Org Cult 33:19-24.

4. Balaga HY (1975). Induction ofbranching in coconut. Kalikasan Philip J Bio1 4:135-140. 5. Batugal P and Engelmann F, Eds. (1998). Coconut embryo in vitro culture. IPGRI-COGENT,

Serdang, Malaysia.

276

6. Blake J (1990). Coconut (Cocos nuc@ra L.): micropropagation. In: YPS Bajaj (Ed.). Biotechnology in Agriculture and Forestry Vol. 10. Legumes and Oilseed Crops I, pp 538-553. Springer-Verlag, Berlin-Heidelberg. Blake J and Eeuwens CJ (1980). Inflorescence tissue as source material for vegetative propagation of coconut palm. In: Proceedings International Conference on Cocoa and Coconuts, Kuala Lumpur

Branton RL and Blake J (1984). Clonal propagation of coconut palm. In: E Pushparajah and CP Soon (Eds.). Cocoa and Coconut: Progress and Outlook, pp 771-780. Incorporated Society of Planters, Kuala Lumpur.

9. Buffard-Morel J, Verdeil JL, and Pannetier C (1992). Embryogenèse somatique du cocotier (Cocos nucifera L.) B partir d'esplants foliaires: étude histologique. Can J Bot 70:735-741.

10. Chan JL, Sáenz L, Talavera C, Hornung R, Robert M and Oropeza C (1998) Regeneration of coconut (Cocos nucifera L.) from plumule explants through somatic embryogenesis, Plant Cell Rep 17:5 15- 521.

I I . Davis TA (1969). Prospects of clonal propagation of the coconut. Ceylon Coconut Planter's Review

12. De Guzman EV and Del Rosario AG (1964). The growth and development of Cocos nucrera L. "Makapuno" embryo in \Ytro. Philip Agricult 48:82-94.

13. de Nucé de Lamothe M and Wuidart W (1992). La production de semences hybrides de cocotier: cas des semences hybrides Nain X Grand. Oléagineux 47:93-96.

14. Del Rosario AG (1998). Status of research and coconut embryo culture and acclimatization techniques in UPLB. In: Batugal P and Engelmann F (Eds.). Coconut embryo in vitro culture, pp 12- 16. IPGRI-COGENT, Serdang, Malaysia.

IS. Dussert S, Verdeil J-L, and Buffard-Morel J (1995a). Specific nutrient uptake during initiation of somatic embryogenesis in coconut calluses. Plant Sci 11 1:229-236.

16. Dussert S, Verdeil IL, Rival A, Noirot M and Buffard-Morel J (1995b). Nutrient uptake and growth of in vitro coconut (Cocosnzicifera L) calluses. Plant Sci 106:185-193.

17. Ebert A and Taylor HF (1990). Assessment of the changes of 2,4-dichlorophenoxyacetic acid concentrations in plant tissue culture media in the presence of activated charcoal. Plant Cell Tiss Org

18. Gascon JF' and de Nucé de Lamothe M (1978) Genetic improvement of the coconut results and prospects. In: Proc Int Confon Cocoa / Coconuts, Kuala Lumpur, 489-499.

19. George EF and Sherrington PD (Eds.) (1984). Plant Propagation by Tissue Culture, Handbook and Directory of Conmercial Laboratories, 709 p. Exegetics Eversley Ltd, UK.

20. Gupta PK, Kendurkar SV, Kulkarni W, Shirgurkar MV, and Mascarenhas AF (1984). Somatic embryogenesis and plants from zygotic enxbryos of coconut (Cocos nuciferu L.) in vitro. Plant Cell Rep 3:222-225.

21. Haibou TK and Kovoor A (1 981). Regeneration of callus from coconut protoplasts. In: AN Ra0 (Ed.). Proccedings COSED Symposium on Tissue Culture Of Economically Important Plants, pp149-151. Singapore.

22. Hornung R (1 995). Micropropagation of Cocos i~ziciferu L from plumular tissues excised from mature zygotic embryos. Plantations, Recherche, Développenlent 2 12:38-41.

23, Islas-Flores I, Oropeza C and Hernandez-Sotomayor SMT (1998). Protein phosphorylation during coconut zygotic embryo development, Plant Physiol 118:257-263.

24. Justin SHFW (1978). Vegetative propagation of coconut. In: Report East Malling Research Station

25. Karunaratne S and Periyappema K (1989). Culture of immature embryos of coconut, COCOS

26. Karunaratne S, Santha S and Kovoor A (1991). An in vltro assay for drought-tolerant coconut

7.

1978, pp 549-556. 8.

711-5.

Cult 20:165-172.

1977, pp 175-176.

nzrcifera L.: callus proliferation and somatic enibryogenesis. Plant Sci 62:247-253.

germplasm. Euphytica 53 :25-3 O.

277

27. Kovoor A (1981). Palm tissue culture: state of the art and its application to the coconut. FAO Plant Production and Protection, Paper 30. FAO, Rome

28. Magnaval C, Noirot M, Verdeil JL, Blattes A, Huet C, Grosdemange F, and Buffard-Morel J (1995). Free amino acid composition of coconut (Cocos nucrera L.) calli under somatic embryogenesis induction conditions. J Plant Physiol 146:155-161.

29. Magnaval C, Noirot M, Verdeil J-L, Blattes A, Huet C, Grosdemange F, Beulé T, and Buffard-hlorel J (1997). Specific nutritional requirements of coconut calli (Cocos nucrera L.) during somatic embryogenesis induction. J Plant Physiol 150:719-728.

30. Pannetier C and Buffard-Morel J (1982). Premiers résultats concernant la production d'embqons somatiques ?i partir de tissus foliaires de cocotier, Cocos nucifera. Oléagineux 37:349-354.

31. Raja CR, Prakash Kumar P, Chandramohan M and Iyer RD (1984). Coconut plantlets from leaf tissde cultures. Plantation Crops 12:75-81.

32. Ri110 EP (1985). Cadang-Cadang research in vitro mechanical inoculation of different coconut cultivars and hybrids with ccRNA. Annual Report Philippine Coconut Authority, p 93.

33. Ri110 EP (1989a). A frame-work for co-ordination of application of tissue cultures techniques for a breakthrough in vegetative propagation of coconut. COCOTECH, XXVI Meeting, pp 15-19. Bangkok, Thailand.

34. Ri110 EP (1989b). A non-destructive technique for collecting immature inflorescences for tissue culture. Philip J Coconut Stud 14:16-17.

35. The Philippines Recommends for Coconut (1993). The Coconut Commitee 1992 (Series No 2- B/1993) PCARRD, PARRFI and PCRDF, Los Baños, Philippines, 234 p.

36. Triques K, Rival A, Beulé T, Puard M, Roy J, Nato A, Lavergne D, Havaux M, Verdeil JL, Sangare A and Hanion S (1997a). Photosynthetic ability of in vitro grown coconut (Cocos nucijéFa L.) plantlets derived from zygotic embryos. Plant Sci 127:39-5 1.

37. Triques K, Rival A, Beulé T, Dussert S, Hocher V, Verdeil JL and Hamon S (1997b). Developmental changes in carboxylase activities in in vifro cultured coconut zygotic embryos: comparison with corresponding activities in seedlings. Plant Cell Tiss Org Cult 49:227-23 1.

38. Verdeil JL (1993). Etude de la régénération du Cocotier (Cocos nucifera L.) par embryogenkse somatique à partir d'eqAants inflorescentiels. PhD thesis, l'Université Paris VI, 156 p.

39. Verdeil JL, Huet C, Grosdemange F and BufFard-Morel J (1994). Plant regeneration from cultured immature inflorescences of coconut (Cocos nucifera L.): evidence for somatic embryogenesis. Plant CellRep 13:218-221.

40. Verdeil JL, Assy Bah B, Bourdeix R, N'Cho YP, Hocher V, BufFard-Morel J and Sangaré A (1996). Le cocotier In: C Teisson (Ed.). Biotechnologies Végétales: Intégration chez les Plantes Tropicales 1, pp 125-141. CNED-AUPELF, Paris.

41. Wuidart W and Rognon F (1981). La production de semences de cocotier. Oléagineux 36:131-138.

278

Current Advances in Coconut Biotechnology

Edited by

C. OROPEZA Centro de Iiiwstigocidn CieritFcn de firrrrtn'iz (Mérida)

J.L. VERDEIL ORSTOM- CIRA D (Montpellier)

G.R. ASHBURNER liistitirte of Sirstaiiinble Irrigated Agi.icirltirre (Tatiira)

R. CARDEÑA Centro de Iii\*estigncin'ii CieiitFca de fircathi (Mérida)

and

J.M. SANTAMARÍA Ceniio de Iiiwstigcicióiz Cieritfica de Hicatn'ri (Mérida)

4 . .-

KLUWER ACADEMIC PUBLISHERS DORDRECHT / BOSTON / LONDON

i,

i

1 -

. . . , . . .,)_.. - - . . . , . .

ISBN 0-7923-5823-6

Published by Kluwer Academic Publishers, P.O. Box 17,3300 AA Dordrecht, The Netherlands.

Sold and distributed in North, Central and South America by Kluwer Academic Publishers, 101 Philip Drive, Norwell, MA 02061, U.S.A.

In all other countries, sold and distributed by Kluwer Academic Publishers, P.O. Box 322,3300 AH Dordrecht, The Netherlands.

Pritited oli ncid-frce ptrper

All Rights Reserved O 1999 KIuwer Acadeniic Publishers No part of the material rcotected by this copyright notice may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying recording or by any information storage and retrieval system, withoui written permission from the copyright owner.

Printed i n the Netherlantas.

Current Plant Science and Biotechnology in Agriculture

Scientific Editor R.J. Summerfield, The University of Reading, Department of Agriculture, l? O. Box 236, Reading RG6 2AT Berkshire, UK

Scientific Advisor)? Board D.F. Bezdicek, Washington State Universiv, Pirlltnan, USA J. Denecke, University of York, York, UK J. Hamblin, The University of Western Aiistralia, Nedlands, Aiistrdia H.-J. Jacobsen, Universitnt Hannover; Hannove6 Gertnatty

Aims and Scope The book series is intended for readers ranging from advanced students to senior research scientists and corporate directors interested in acquiring in-depth, state-of-the-art knowledge about research fin- dings and techniques related to all aspects of agricultural biotechnology. Although the previous volu- mes in the series dealt with plant science and biotechnology, the aim is now to also include volumes dealing with animal science, food science and microbiology. While the subject matter will relate more particularly to agricultural applications, timely topics in basic science and biotechnology will also be explored. Some volumes will report progress in rapidly advancing disciplines through proceedings of symposia and workshops while others will detail fundamental information of an enduring nature that will be referenced repeatedly.

I

The titles published in this series qre lisred at the end of this vollime.

i

.- ,