Development of Cryopreservation Protocols for Cryobanks of Coconut Zygotic Embryos

The recalcitrant nature of the coconut seed makes it difficult to be stored for the long-term using conventional techniques. Hence, the cryopreservation technique has been considered as a preferred option over field genebanks for the conservation of coconut germplasm. The purpose of this research is to optimize the coconut cryopreservation protocol for large-scale implementation of coconut germplasm conservation; and to upscale the developed protocol for application in the conservation of world-wide coconut genetic resources. The proposed study is part of the international collaboration between Bioversity International and Rural Development Administration of Korea. A preliminary work on coconut cryopreservation was done at Philippine Coconut Authority of the Philippines and 2nd round experiments were tried at Sunchon National University of Korea using Malayan Yellow Dwarf (MYD) as a basal material. Some alternative cryopreservation techniques, i.e., droplet-vitrification of plumular cube (with the plumule and root, 2.5 x 2.5 mm), vitrification of meristematic pole disc (haustorium removed), vacuum-assisted vitrification of intact embryos, and preculture-desiccation of intact embryos were tried using a systematic approach. The preliminary results are: 1) the main bottleneck of coconut cryopreservation is outbreak of bacterial contamination during the preculture; 2) a progressive sucrose preculture with a final concentration of 60% was beneficial to acquire desiccation tolerance; 3) in terms of the material, both intact embryos and plumular cube seems promising in vitrification methods; 4) two protocols were proposed as a candidate for routine implementation of cryobanking for coconut collections: 1) preculture-desiccation of intact embryos; 2) vitrification of plumular cube where samples were progressively loaded with C4-35% (17.5% glycerol + 17.5% sucrose) and C11-60% (30% glycerol + 30% sucrose), followed by dehydration with PVS3 (50% glycerol + 50% sucrose). It is recommended to develop a system to get clean in vitro embryos and to optimize the sample preparation procedure for cryopreservation experiments.