Coconut (Cocos nucifera L.) is an economically and socially important palm found throughout the tropical regions of the world. Despite an increased area being planted to coconut, the world average production has not increased in the last 25 years. Several serious lethal diseases have been responsible for production suppression. In the meantime, only conventional breeding approaches, using hand-assisted pollination, have been used to produce new and improved, disease-resistant cultivars. These approaches are often difficult, labor extensive, expensive and very slow to undertake. Therefore, an alternative pathway for coconut improvement needs to be developed. This could involve in vitro culture techniques such as those of embryo culture and somatic embryogenesis. This second technique could be useful for the vegetative propagation of elite, parental lines which have been selected for their superior quality. In addition, somatic embryogenesis could be used for genetic engineering of coconut in future studies. Both approaches could dramatically speeding up breeding programs for the production of coconut planting material with high quality and yield, and resistance to pest and diseases.
Research on somatic embryogenesis of coconut has been underway for close to 40 years. Despite the difficulties in somatic embryogenesis some plantlets have been produced. These plantlets have mainly been generated from zygotic tissues, such as those of intact mature and immature zygotic embryos, sliced mature zygotic embryos as well as plumule tissues isolated from zygotic embryos. However, for coconut, to obtain true-to-type plantlets, somatic tissues have to be used as the starting explant tissue.
Thus, the first series of objectives of the present study were a) to discover the best somatic explant available from coconut seedlings for callus induction and somatic embryogenesis, b) to develop and improve the protocol for somatic embryogenesis from this selected explant, and c) to develop a protocol for producing true-to-type plantlets. The second series of objectives were a) to develop a technique for the collection and importation of coconut immature inflorescences for somatic embryogenic work, and b) to develop and improve the protocol for somatic embryogenesis from immature inflorescence explants. A comparison of the two systems could then be made.
Among all the different somatic explants taken from coconut seedlings, only leaf explants could produce copious amounts of healthy callus that could be used for somatic embryogenic work. Leaf explants produced callus irrespective of the 2,4-D concentration applied. However moderate 2,4-D concentrations (100 to 200 µM) were better able to promote callus production, with 150 µM 2,4-D being optimum. Even using this optimum concentration, callus necrosis was still evident and the rate of callus production was low (only ca. 32 % of the explants producing callus). In addition, the time required to produce callus was very long (ca. 70 days). Various chemical treatments and pre-culture soakings were tested to help overcome these problems. Pre-culture soaking of the leaf explants in either sterile water for up to 60 minutes or filter sterilised ascorbic acid solution for up to 30 minutes were able to improve the percentage of explants undergoing callus production (from 23 to 33 %) but these treatments were unable to reduce the rate of callus necrosis. Therefore, the use of silver thiosulphate (STS ; a chemical which protects tissues from the action of ethylene) was tested to see if it would help prevent necrosis and enhance the rate of callus production. It was found that STS could reduce leaf explant necrosis but could not enhance the rate of callus production. Significant improvements (P<0.05) in callus production (from 30 to 58 %), as well as shorting the time to produce callus (from 70 to 48 days), was obtained when casein hydrolysate and certain polyamines (putrescine, agmatine, β-phenylethilamine, cadaverine or 2,3-diaminopropane) were added into the callus induction medium.
Callus proliferation was also an important step to achieve effective somatic embryogenesis on coconut leaf explants. For optimum callus proliferation to occur, the concentration of 2,4-D had to be maintained at the same level as that used for callus induction (150 µM). The rate of callus proliferation increased (from 85 to 216 %) when casein hydrolysate and certain polyamines (putrescine, agmatine, β-phenilethylamine, cadaverine and 1,3-diaminopropane) were incorporated into the culture medium. Whilst certain polyamines improved the rate of somatic embryo production (from 6 to 16 per plate), casein hydrolysate was unable do the same. The presence of 2,4-D (50 to 100 µM), BAP (20 µM), AbA (45 µM) and PEG (9 mM) was found to be important for the maturation of the somatic embryos to occur. The rate of somatic embryo formation increased when the 2,4-D concentration was decreased (from 150 to 100 or 50 µM) and BAP concentration was increased (from 0 to 20 µM) as well as when both AbA (45 µM) and PEG (9 mM) were incorporated into the maturation medium. Using this system three plantlets have already been obtained. It is expected that this improved protocol will yield more plantlets in the near future as more cultures reach their maturation stage.
When undertaking somatic embryogenesis from immature inflorescences tissue, it is important to ensure the explant tissue is healthy. When such tissues have to be imported from overseas then this is especially important. Coconut immature inflorescence quality was maintained, during a 4-day transportation period, by using one of a number of transportation protocols. Possibly the best technique used involved a cool (5 °C) transportation treatment. However, the benefits of such a system are negated when an Australian Quarantine and Inspection Service requirement of fumigation at 22 °C was undertaken. The cold technique may be most useful when transport between countries is not involved.
Immature inflorescence explants required a higher 2,4-D concentration (200 µM) to initiate callus than that observed for leaf explants (150 µM). In addition, optimum callus proliferation occurred when the 2,4-D concentration was increased to 250 µM (rather than held constant or reduced, as for leaf explants) and casein hydrolysate and certain polyamines (putrescine, agmatine, P-phenilethylamine, cadaverine and 2,3- diaminopropane) were added to the proliferation medium. No plantlets were obtained during the course of study using immature inflorescence explants. However, at the termination of the study immature inflorescence callus cultures were showing a very high meristematic activity that had resulted in the production of many more somatic embryos than had been achieved with leaf callus. Therefore, it could be suggested that, while further improvements in the somatic embryogenic system for leaf explants could facilitate somatic embryogenesis from seedlings, improvements for a somatic embryogenenic system using immature inflorescence explants would be beneficial for clonal propagation of coconut from mature, adult trees.