Abstract

ABSTRACT Epipactis flava Seidenf., an endangered Thai rheophytic orchid, urgently requires protection to prevent its extinction. This study aimed to develop an effective vitrification cryopreservation protocol for the long-term storage of E. flava seeds. Mature seeds were pretreated with a loading solution and dehydrated with plant vitrification solution 2 (PVS2) for 0, 30, 60, 90, and 120 min at 25 ± 2°C before immersion in liquid nitrogen for 24 hours. Post-cryopreservation, the seeds were germinated on BM-1 medium for 8 weeks, and then the protocorms were transferred to semi-solid MS medium for another 8 weeks. Seeds treated with PVS2 for 90 min and exposed to liquid nitrogen showed the highest viability (33.3%), while 120 min of dehydration yielded the highest germination rates (17.8%) and 5.8% of the protocorms reached advanced developmental stages (stage 4 and 5). The highest percentage of protocorms at stage 5 (3.3%) was also observed at 120 min of PVS2. No germination occurred in the cryopreserved seeds that had not been exposed to PVS2 prior to the liquid nitrogen treatment. Thus, our results showed that the optimal PVS2 exposure time for cryopreservation of E. flava seeds using the vitrification method was 120 min.

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