顽拗型种子对脱水和低温敏感，超低温（液氮）保藏被认为是其长期保藏的唯一方法。维持细胞膜的完整性是种子或组织超低温保藏的关键问题，但其分子基础尚不清楚。膜脂分子组成是影响膜完整性的主要因素。前期发现通过调控膜分子组成能提高顽拗型种子的超低温保藏效果。本项目拟以板栗（顽拗型）、茶（中间型）和豌豆（正常型）为研究对象，比较对照、直接脱水、蔗糖处理、蔗糖加PVS2处理，以及液氮冻存后恢复培养过程，1）通过膜渗漏和激光共聚焦观察膜形态检测膜的完整性；2）用脂类组学检测上述三个代表物种在超低温不同处理过程中11类120种膜脂分子的变化；3）用生物信息学的方法分析膜脂分子变化样式及其与膜完整性的关联；4）克隆上述关联中所对应的膜脂代谢的关键酶基因，并检测其表达与膜完整性的关联；5）用酶抑制剂等调控膜脂分子变化并检测超低温保藏效果以验证上述关联。该研究为将来大量进行种质资源的超低温保藏提供坚实的理论基础。

Cryopreservation is considered to be the only way to long-preserve recalcitrant seeds. Sustain membrane integrity of seeds or tissue are the key issue for the cryopreservation, however, the molecular mechanisms are still unclear. The consistant of lipid molecular of membrane are the main factor for the membrane integrity and our previous work have showed that regulated the membrane molecular consistant could improve the survival rate after crypreservation. In this project, we will use confocal, lipidomics and other molecular method to compare the difference when cryopreserved Castanea mollissima(recalcitrant seed), Camillia sinensis (intermediate seed) and Pisum sativum (orthodox) during various cryopreservation process. The results of this project will provide strong theory for cryopreservation recalcitrant species in the future.