Development of a cryopreservation protocol for Vanilla siamensis: an endangered orchid species in Thailand

BACKGROUND: Vanilla siamensis is listed in Appendix-II of Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) as an endangered species in Thailand. OBJECTIVE: To develop an optimum cryopreservation protocol for V. siamensis. MATERIALS AND METHODS: Protocorms were precultured on solid ½ MS medium with 0.5 M sucrose for 0-7 d. For encapsulation-dehydration, encapsulated protocorms (beads) were dehydrated for 0-6 h. In the case of encapsulation-vitrification, the beads were loaded with a plant vitrification solution 2 (PVS2) at 0°C for 0-90 min. RESULTS: Protocorms precultured for 3 d gave the highest post-cryopreservation survival of 17%. Dehydration of the encapsulated protocorm beads for 4 h gave the highest survival of 33% and a regrowth of 25%. Protocorms subjected to the encapsulation- vitrification method did not survive at all. CONCLUSION: Protocorms precultured with 0.5 M sucrose for 3 d, encapsulated with 3% sodium alginate and dehydrated to a moisture content c. 14% before plunging into LN was a suitable method for the successful cryopreservation of V. siamensis protocorms