Mechanism of desiccation tolerance of somatic embryos and application of dry artificial seeds

外源ABA诱导了芸苔属小孢子胚的脱水耐性。本研究的目的是探讨ABA诱导干燥耐性的机制，并澄清干燥胚的一些性状，以便作为干燥人工种子利用。胚胎发生晚期，Lea基因被认为与合子胚胎的干燥耐受性有关。在干燥耐受胚中检测到Lea mRNA的积累，而在干燥敏感胚中检测到Lea mRNA的积累。在ABA处理12h内，胚的干燥耐性诱导和Lea mRNA积累同时发生。从油菜和大白菜耐干燥小孢子胚cDNA文库中分离到Lea基因。测定了菜籽ME-leaN4无性系和大白菜ME-leaC4无性系的核苷酸序列。预测的氨基酸序列显示，2个多肽具有82%的同源性，在菜籽和大白菜中分别出现了11次和9次具有一致序列的11个残基重复序列。从油菜基因组文库中分离到Lea基因，发现Lea基因启动子中存在ABA响应元件（ABRE）。为阐明Lea基因的功能，将CaMV35S启动子调控的Lea基因引入烟草和油菜中。虽然获得了6株烟草转基因植株和2株油菜籽转基因植株，但它们的耐盐性并没有增加。但有少数烟草植株在T1代幼苗的生长速率略高于非转基因植株。在80ºC温度下保存的干燥胚在3年内保持植株再生能力，并具有低温保存的能力。种子渗出法可以很容易地测定干燥胚的活力。

Desiccation tolerance of microspore-derived embryos of Brassica spp.are induced by exogenous application of ABA.The objective of this study was to investigate the mechanism by which ABA induces desiccation tolerance and to clear some traits of the desiccated embryos for utilizing as dry artificial seeds.1.Late embryogenesis abundant (Lea) genes are considered to be related to the desiccation tolerance of zygotic embryos. Accumulation of Lea mRNA was detected in desiccation tolerant embryos, but not in desiccation sensitive ones. Both induction of desiccation tolerance of embryos and Lea mRNA accumulation were simultaneously occurred within 12h of ABA treatment. Lea genes were isolated form cDNA libraries derived from desiccation-tolerant microspore embryos of rapeseed and Chinese cabbage. Nucleotide sequence was determined on ME-leaN4 clone of rapeseed and ME-leaC4 of Chinese cabbage. Predicted amino acid sequence of these clones showed that 82% homology was present between two polypeptides, and that two 11 residue repeats with consensus sequences were present 11 and 9 times in rapeseed and Chinese cabbage, respectively. Isolation of Lea gene from genomic library of rapeseed indicated that ABA responsive elements (ABRE) were present in the promoter of Lea gene.2.To elucidate the function of Lea gene, Lea gene which was regulated by CaMV35S promoter was introduced into tobacco and rapeseed. Though 6 tobacco transgenic and 2 transgenic rapeseed plants were obtained, they did not show increased tolerance to salinity. However, a few tobacco plants showed slightly higher growth rates than nontransgenic plants in seedling of T1 generation.3.The desiccated embryos stored at-80ﾟC maintained their ability of plant regeneration for 3 years, and had the ability of cryopreservation. Viability of desiccated embryos was easily able to be determined by seed exudate method.