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

体细胞胚耐干燥机制及干燥人工种子的应用

基本信息

批准号：
06660003
负责人：
TAKAHATA Yoshihito
金额：
$ 1.28万
依托单位：
Iwate University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06660003/
关键词：
Somatic embryo Desiccation tolerance Artificial seed Abscisic acid Pollen culture Brassica Lea gene LEA遺伝子 Glycine max

项目摘要

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.

本研究通过外源阿坝诱导芸苔属植物小孢子胚的脱水耐性，旨在探讨阿坝诱导脱水耐性的机制，并明确脱水胚作为干燥人工种子的一些特性。1.晚胚发生丰富基因（莱亚）被认为与合子胚的脱水耐性有关。莱亚mRNA在脱水耐受胚中积累，而在脱水敏感胚中不积累。阿坝处理12 h内，胚脱水耐性的诱导和莱亚mRNA的积累同时发生。从油菜和大白菜耐脱水小孢子胚cDNA文库中分离到莱亚基因。对油菜ME-leaN 4克隆和大白菜ME-leaC 4克隆进行了核苷酸序列测定。氨基酸序列预测表明，两个克隆的同源性为82%，两个11残基重复序列在油菜和大白菜中分别出现11次和9次。从油菜基因组文库中分离到莱亚基因，表明莱亚基因的启动子中存在阿坝响应元件（ABRE）。2.为了阐明莱亚基因的功能，将受CaMV 35 S启动子调控的莱亚基因导入烟草和油菜。虽然获得了6株转基因烟草和2株转基因油菜植株，但它们对盐的耐受性没有提高。T1代植株的生长速度略高于非转基因植株。3.干燥胚在-80 ℃保存3年后仍能保持植株再生能力，具有超低温保存能力。用种子分泌物法测定干胚的生活力是一种简便易行的方法。