Optimising the PVS2 treatment duration for cryopreserving seeds of the endangered rheophytic orchid <i>Epipactis flava</i> Seidenf

<i>Epipactis flava</i> 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 <i>E</i>. <i>flava</i> 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 <i>E</i>. <i>flava</i> seeds using the vitrification method was 120 min.黄花火烧兰（Epipactis flava）Seidenf.是一种濒危的泰国湿生兰花，亟需采取保护措施以避免其灭绝。本研究旨在开发一套高效的玻璃化超低温保存方案，实现该物种种子的长期储藏。研究人员将成熟种子经装载液预处理后，于25±2℃条件下使用植物玻璃化溶液2（PVS2）分别脱水0、30、60、90及120分钟，随后浸入液氮中保存24小时。超低温保存完成后，将种子接种于BM-1培养基上萌发8周，再将获得的原球茎转接至半固体MS培养基继续培养8周。实验结果显示，经90分钟PVS2脱水并液氮处理的种子，其生活力最高（33.3%）；而脱水120分钟的种子萌发率最高（17.8%），且有5.8%的原球茎发育至成熟阶段（第4、5期）。在脱水120分钟组中，原球茎达到第5期的比例最高，为3.3%。未经过PVS2预处理即开展液氮处理的超低温保存种子，未出现任何萌发现象。综上，本研究结果表明，采用玻璃化法超低温保存黄花火烧兰种子的最优PVS2暴露时长为120分钟。