Developing new in vitro micropropagation and cryopreservation techniques in coconut

The coconut palm (Cocos nucifera L.) is the second most important palm crop in the world and its production has an estimated yearly value of US $11.5 billion. Most coconut plantations are owned by smallholders. Currently, there are many old non-productive plantations in need of replanting. However, new quality palms are expensive and will not produce during several years. The price of the planting material is high due to its intensive production process and limited amounts available. Hence, we developed a method to rapidly multiply quality planting material in a clonal way. We also studied the in vitro conservation of coconut cultures because existing field genebanks are threatened by land use disputes, pests and diseases.

The natural growth pattern of the monocot coconut palm prevents the induction of shoots via branching. However, there are rare occurrences of natural branching, suggesting that the artificial induction of branching could be possible, a pathway with less risk with respect to somaclonal variation compared to the current clonal propagation method, somatic embryogenesis. The conservation of coconut genetic resources currently relies on field genebanks, which are under threat, but cryopreservation could mitigate some of these risks. However, existing cryopreservation protocols for coconut are not very efficient and/or not adapted to clonal material.

A first step to take was growing zygotic embryo derived coconut plantlets in vitro, a basic requirement for rapid multiplication and cryopreservation. Amongst many parameters influencing this process, activated charcoal and Y3 medium salts were found to stimulate germination and growth. The reoccurring problem of contamination was solved with a 0.5 % NaOCl sterilization protocol. The horizontal positioning of the embryo on the culture medium during germination, was found to be an important positive factor. Finally, different varieties were shown to grow at different speeds.

The in vitro shoots were used to induce shoot proliferation. We demonstrated that an in vitro plant vertically split into two cultured on Y3 medium containing 1 µM thidiazuron (TDZ) induced the apical meristem to proliferate and form meristematic clumps that could be regenerated into rooted clonal plantlets. This protocol, the first of its kind in coconut tissue culture, is simple and efficient and will be a huge asset to the coconut industry, as the amount of plant material (including meristems), multiplies every month by between 150 and 175 %. During the proliferation of these clumps, the presence of light caused browning probably caused by phototoxicity. However, this browning problem was solved by growing the material in darkness combined with frequent (monthly) subculturing.

TDZ affects multiple pathways, but we determined that its main pathway is a direct cytokinin-like effect probably by binding directly to a cytokinin receptor. We also formulated and tested a new hypothesis regarding TDZ transportation in plant tissues, using coconuts, banana (Musa spp. L.), cactus fig (Opuntia ficus-indica Mill.) and cassava (Manihot esculenta Crantz) plants. We hypothesize that TDZ needs to come into close contact to the meristem to be effective due to its inefficient transport through the plant.

The meristematic clumps can also be used for cryopreservation. After their preculture on 0.3 M sucrose, it was shown that the optimal PVS2 treatment for droplet vitrification ranged between 120 and 240 minutes, resulting in 50 % survival. This protocol is the first successful published and user-friendly cryopreservation protocol for meristematic clumps in the coconut palm.

Finally, clonal plantlets were successfully regenerated and transferred to ex vitro conditions and put in soil, which is an important step for delivering quality material. Whilst improvements are still possible, this PhD presents the whole micropropagation cycle from start (in vitro initiation) to finish (ex vitro material), including methods to conserve this material. This PhD provides thus a lot of novel knowledge regarding coconut tissue culture as well as answers to some of the challenges the coconut industry is currently facing. The provided information may also be of great value for other palms.