冻后黑暗培养修复黄独胚性愈伤组织超低温保存伤害的机理研究

Cryopreservation is considered to be the idealest method of long term conservation of plant germplasm resources. At present, the studies of cryopreservation mostly focus on its operational procedures, submicroscopic structure, genetic stability, proteomics, virus-free and so on. However, many studies indicated that dark culture after cryopreservation was beneficial to improve the survival rate and the regeneration rate of cryopreserved materials, whose causes had no reports. On the base of working foundation obtained in our lab, in this project, we will use embryogenic calli of Dioscorea bulbifera L. (a precious anti-cancer medicinal plant) as experimental materials, and make use of researching techniques, e.g. plant tissue culture, electron microscope observation, high-performance liquid chromatography, Northern blotting hybridization, and suppression subtractive hybridization, etc., to study the cell ultrastructure, the anti-oxidase activity, the contents of metabolite and endogenous hormone, the expression of anti-oxidase genes, the screening and cloning of especially expressed genes, and their function prediction on bio-informatics during dark culture after cryopreservation. The results will provide some elemental materials and firsthand molecular evidences for remission mechanism of dark culture on cryopreservation damage.

超低温保存是植物种质资源长期保存最理想的方法。目前，对超低温保存的研究多集中于操作程序、亚显微结构、遗传稳定性、蛋白组学和脱毒等方面。然而，许多研究表明，冻后黑暗培养有利于提高超低温保存材料的成活率和再生率，而关于其原因尚未见报道。本课题以抗癌药用植物黄独（Dioscorea bulbifera L.）胚性愈伤组织为研究对象，基于已有的工作基础，利用植物组织培养、电镜观察、高效液相色谱、Northern印迹杂交和抑制性消减杂交等技术，研究冻后黑暗培养过程中细胞的亚显微结构、抗氧化酶的活性、代谢产物和内源激素的含量、抗氧化酶基因的表达、特异表达基因的筛选克隆及其生物信息学功能预测，旨在为揭示冻后黑暗培养修复超低温保存伤害的机理提供基础依据和直接的分子生物学证据。

超低温保存是目前植物种质资源离体保存较好的方法。许多研究表明，在超低温保存再培养环节，一定时间的冻后黑暗培养有助于材料的成活。但其机理如何？国内外少见报道。本项目首先建立了黄独无菌培养体系的建立，探明了微型块茎是诱导胚性愈伤组织的较佳材料；其次，本项目在形态解剖、生理生化、转录组、蛋白质组和代谢组方面对微型块茎的诱导形成和低温保存进行了研究；最后，本项目建立和优化了黄独胚性愈伤组织的包埋玻璃化法和小滴玻璃化法超低温保存程序，并在此基础上筛选了其最佳冻后黑暗培养时间，并对冻后黑暗培养的胚性愈伤组织进行了显微和亚显微结构的观察、甲基化水平的分析、抗氧化酶活性和代谢产物含量以及内源激素和多胺含量的检测、抗氧化酶基因的qPCR验证、抑制消减杂交cDNA文库的构建与分析。本项目研究结果可在一定程度上揭示了冻后黑暗培养修复超低温保存伤害的机理，为植物种质资源的超低温保存提供了一些基础资料。