Coconut tissue culture for clonal propagation and safe germplasm exchange

• Philippine Coconut Authority, Philippines
• Cocoa and Coconut Research Institute, Papua New Guinea
• Research Institute for Coconut Palms, Indonesia
• University of the Philippines at Los Bans, Philippines
• Oil Plants Institute of Vietnam, Vietnam

Coconut is the most important palm of the humid tropics and 96% of the crop is grown by resource-poor smallholders. Its traditional products are copra, coconut oil and copra meat, but it also has the ability to produce a wide variety of food and environmentally friendly non-food products - for domestic use and for export. The coconut palm has also been a stabilising factor in the farming systems of marginal and environmentally fragile environments.
The worldwide production of coconut has not increased in decades, due to the lack of new genetic stocks, devastation of crops from pests and diseases and rapid loss of precious coconut germplasm. Few of the coconut- producing countries have the capacity to characterise and evaluate the field performance of conserved germplasm. Many of them also lack the capacity to exchange disease-free germplasm with other coconut-producing countries because they are unable to undertake embryo culture, they do not have a capability to index diseases and their germplasm export clearances are tedious.

This project aimed to find an improved technique for coconut embryo culture suitable for re-establishing palms from internationally exchanged and cryopreserved embryos. It continued earlier research on the embryogenesis from somatic tissues and developed a protocol for the rapid production of true-to-type clonal palms. It also aimed to develop a technique to genetically analyse plants developed from clonal propagation, embryo culture or cryopreservation. All the partner countries in this project were granted access to the coconut biotechnological techniques developed.

Research teams in the Philippines and Australia evaluated methods to assess the embryo quality and its physiological age, as well as methods for embryo isolation and transport. One team in the Philippines examined the various steps in the embryo culture protocol and investigated how to reduce stress during the tissue culture system, how to improve embryo quality and maturation, and how to assess the appropriate nutritional sources for embryo growth and development. Another team in the Philippines examined plantlet physiology and improved hardening-off technology. There was also collaboration with other international groups involved in embryo culture.
Research teams in the Philippines and Australia studied the quality and physiological status of the explants prior to culture. They sought methods to isolate explants suitable for somatic embryogenesis work. In Australia, the team used cultivars from collaborating countries and investigated how to reduce stress in the tissue culture system, how to improve somatic embryo quality and maturation, and how to assess the appropriate organic carbon source for somatic embryo growth and development.
Also in Australia a research team studied the genetic fidelity of the tissue-cultured plants, examined which steps of the tissue culture procedure led to a genetic change, and attempted to prevent such changes from occurring during the procedure.

The first objective 'to establish an improved embryo culture technique suitable for the re-establishment of palms both from internationally exchanged or cryopreserved embryos' has identified the following improvements in the internationally recognised protocol for embryo culture. The physiological quality of the embryo used for tissue culture work has been identified as an important factor that, in part, determines the rate of seedling production from field-harvested embryos. Big embryos, from 11-month-old fruit of healthy trees produce more and healthier seedlings than those from poor quality embryos. Gibberellic acid (GA3 at 30 to 40 M) placed into the tissue culture medium is able to promote the speed and percentage of embryos germinating. By reducing the medium carbohydrate load (from 45 to 25 grams per litre) at the last seedling subculture step, seedling growth can be improved and the rate of seedling establishment increased.
Plant hormones termed auxins (IBA and NAA) when applied at various times during the early steps of embryo germination will increase root production and this will result in greater ex vitro seedling vigour. A simple acclimatisation method, using a wooden box with a transparent plastic sheet lid, will produce better quality seedlings than those produced using the traditional inverted plastic bag system. Finally, CO2 enrichment applied around young seedlings growing in a nutrient solution will promote seedling growth and development. The use of CO2 is able to cut the time in vitro from 12 to approx. 3-4 months. This represents a massive saving in culture cost and time. Other work has demonstrated that embryo transplantation is another way of producing seedlings from embryos, and to do this a surrogate nut is used as a nurturing host.
Using the new, improved method for embryo culture, it is now possible to consider the possibility of conserving coconut germplasm (in this case as embryos) by cryopreservation. To date the teams have shown that coconut embryos can be kept alive, through a period of cryopreservation (-196oC) if they are appropriately desiccated or dehydrated before being frozen. A dehydration approach adapted for use with coconut embryos gave better survival results after cryopreservation.
The second objective 'to continue work on somatic embryogenesis from somatic tissues with a view to developing a protocol for the rapid production of true-to-type, clonal palms' has identified improvements in the protocol for somatic embryogenesis of coconut. Techniques developed for hardening embryo-derived seedlings are also most useful for establishing somatic seedlings in soil.
The third objective 'to develop a technique for the genetic analysis of plants coming from clonal propagation, embryo culture or cryopreservation' has shown that the clones analysed in this project are genetically similar (stable) at the DNA nucleotide and methylation level following their production by somatic embryogenesis.
The fourth objective 'to transfer all coconut biotechnological techniques, and when they become available, to the COGENT-supported germplasm banks in Indonesia and PNG by way of short courses and training workshops' has presented training activities covering the use of all protocols used in all of the research programs of the project, with an emphasis placed on those used in the embryo culture program. Many other training activities have been undertaken at the partner institutes.