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.
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.
Year 1 (01/07/2002-30/06/2003)
Both the UQ and partner teams made good progress in the early phase of the project.
University of Queensland: Experiments on embryo transplantation, embryo culture, somatic embryogenesis and molecular analysis of tissue-cultured plantlets all took place at UQ in the first year.
The first coconut seedlings, following embryo transplantation, have been successfully produced in the glasshouse. The transplantation technique used still requires improvements and is the subject of overseas collaborative work with the partners.
Large numbers of zygotic embryos have been imported from the Philippines. They have been germinated in vitro and are being used to investigate improved methods of seedling growth, development and establishment. This work at UQ plans to examine the use of CO2 enrichment for improved plantlet formation and greater soil survival.
The UQ researchers have adapted their somatic embryogenesis protocol, previously developed for zygotic tissue explants, and are using it on explants from imported inflorescence tissues. The procedure showed a very low efficiency rate; medium additives such as coconut water and lauric acid improved the rate of success but no significant results have been achieved. From other studies, preliminary results indicate that abscisic acid (AbA) could improve the development of somatic embryos from inflorescence tissues.
A protocol for DNA extraction and purification has been optimised for coconut tissues. Good visualisation of DNA can be obtained using approximately 15 to 20 ng of DNA per reaction. Other DNA samples have been imported from CICY, Mexico and are being analysed for the methylation polymorphisms that may exist between clones.
The team at UQ hosted a meeting in March 2003 to launch a book published by ACIAR. Titled ‘The Coconut Odyssey: The bounteous possibilities of the tree of life’, it was written by Mr Mike Foale, CSIRO, an associate member of the UQ team.
Partners: In Indonesia, PNG, Vietnam and Philippines, the embryo culture research activities are still at an early stage of development. The present work aims are to prepare sufficiently large numbers of germinating embryos for their future work needs on improving seedling establishment rates.
The early studies undertaken include those manipulating the culture medium to aid root system development, for producing a good shoot mass to root mass ratio, and those aiming to develop better acclimatisation steps. The germinating embryos, presently being established, take up to one year before they can be used in such experimentation. Therefore, the results will only become available in the second and third years of the project.
In PNG, work on embryo quality has just been started while one partner in the Philippines has initiated work on somatic embryogenesis.
Meetings at the collaborating partners’ laboratories (PNG, Vietnam and the Philippines) have been undertaken to sharpen the focus of the project program.
A new internet discussion group ‘ACIAR coconut’ has been established to facilitate information exchange among the team members involved in the project.
A method has been developed for the successful transplantation and germination of zygotic embryos from donor to surrogate coconut fruits. This achievement opens the way for the development of a new protocol for raising coconut seedlings following the International exchange of germplasm. Other work has shown that a CO2 fogging step applied to the young germinated coconut embryos can significantly improve their subsequent growth and establishment in soil. This improvement in the embryo culture procedure will enable most, if not all, tissue cultured embryos to be converted into plants in soil. This improved rate of obtaining plants in soil is seen as a very important step forward as the present technique still results in a considerable loss in potentially important germplasm. A protocol to test the genetic fidelity of plants coming from tissue culture has also been developed and used to show that such plants show little or no genetic variation. This is an important observation and indicates the development of a clonal propagation method for coconut is worthy of further study. A final significant development this year has been the improved embryo culture capacity of all partners. Without exception, all partners are now able to undertake embryo culture with a high degree of precision with plantlet production rates as high as 90%. Prior to the start of the project some partners had not been successful in raising any plants using the embryo culture protocol.
The physiological quality of the embryo used for tissue culture work is an important factor that, in part, determines the rate of seedling production from those embryos. Recent studies have shown that bigger embryos produce more healthy seedlings than the smaller ones. In addition gibberellic acid (GA3 at 30 to 40 M) placed into the tissue culture medium is able to promote the rate and final percentage of embryo germination. The efficiency of the embryo culture system has been further improved with a step that screens out all of the non-viable embryos from the tissue culture system after 6 to10 weeks of culture. This saves up to 20 % of the total resources needed to undertake the embryo culture procedure. Reducing the medium carbohydrate load (from 45 to 25 g L-1), at the last seedling subculture step has been shown to have no negative effects on its subsequent growth but this action does reduce the chance of the seedlings becoming infected and performing poorly at a later stage of growth. Auxins (IBA and NAA) applied at various times during the early steps of the embryo culture procedure were found to increase root production and this resulted in greater ex vitro seedling vigour. Removal of the haustorial tissue during the early steps of embryo culture was also able to improve seedling growth and ex vitro establishment. A simple acclimatization method, using a wooden box with a transparent plastic sheet lid, produced better quality seedlings than those produced using the traditional inverted plastic bag system presently used for seedling production.
Other results have shown that as early as 3 months into the in vitro culture procedure seedlings are capable of autotrophic growth if their roots are able to access a Hoaglands nutrient solution. However, the subsequent growth of these nutrient-nurtured seedlings was often slower than that seen for seedlings growing in the traditional way using the Y3 nutrient medium. Finally, CO2 enrichment around young seedlings (one unfolded leaf stage) growing in nutrient solution, could promote seedling growth back to that seen in the control. This action was able to cut the time in vitro from 12 to about 3 or 4 months. This represents a massive saving in culture cost and time. Earlier work demonstrated that embryo transplantation is a possible method for producing seedlings from isolated embryos using surrogate nuts as nurturing hosts. This work, undertaken in Australia on poor quality supermarket nuts is now being repeated using quality fruit and embryos in Indonesia (ICOPRI). At this stage, there has been some difficulty in germinating the transplanted embryos in the surrogate host nuts with contamination hard to contain. A seed priming method is now being used to promote the germination of the transplanted embryo. At this stage >200 primed embryos of Palu Tall have been transplanted into either Mapanget Tall or Palu Tall. Germination will be followed over the coming months. Using the new, improved methods of embryo culture, it is now possible to start looking at the possibility of conserving embryos by cryopreservation and reviving them by embryo culture.
Results so far show that coconut embryos can be kept alive, through a period of cryopreservation (placement in liquid nitrogen, -196oC), if they are appropriately desiccated and pre-treated prior to freezing. The initial rates of success are now being improved using modifications to the standard method. Research on somatic embryogenesis is focussing on steps to select appropriate explant tissues and cell types from within those explants for the production of callus. Among inflorescence calli types identified, white, round and shiny, smooth surfaced ones are more likely to be highly embryogenic than rough surfaced, friable calli masses. Multiplication of these highly embryogenic calli masses is possible by cutting the cultures into smaller pieces (c. 0.5 g) and culturing them onto a medium containing 199 M 2,4-D and doubling the casein hydrolysate concentration (0.6 g L-1). ABA was found to be useful in promoting the formation of somatic embryos when applied I month later, to either on pointed structures (0.5 to 1.0 cm) or pointed embryos (2 to 3 cm. These ABA-treated embryos showed further development when they were subcultured onto an ABA-free medium where they germinated at a rate of 4.5%.
A protocol to test the genetic fidelity of plants coming from tissue culture has now been developed and used to study 20 clonal plants. So far 10 AMP primers have been used to screen c. 750 DNA markers for nucleotide or DNA methylation polymorphisms, in four clonal plants coming from each of five donor ortets. No DNA methylation or DNA nucleotide polymorphisms could be identified. These results indicate that the clones analysed in this project are genetically similar (stable) at the DNA nucleotide and methylation level following their production by somatic embryogenesis. A training was undertaken at UQ from 4th to 15th October 2004 attended by three participants: Mr. Alfred Kembu from CCI, PNG, Dr. Pablito Magdalita from UPLB, the Philippines and Ms. Vu Thi My Lien from OPI, Vietnam. The training covered all of protocols used in all of the research programs of the project, with an emphasis placed on those used in the embryo culture program particularly the use a CO2 fogging step to improve the physiological health and growth of in vitro growing seedlings prior to their establishment in soil. The participants were provided with hands-on experience with a range of advanced tissue culture techniques as well as being very active in technique development and project discussions. Other training activities were also undertaken at OPI, ICOPRI Indonesia and PCA the Philippines.
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.