High frequency transformation and regeneration of transgenic plants in the model legume Lotus japonicus
High frequency transformation and regeneration of transgenic plants in the model legume Lotus japonicus
复制标题
DOI：
10.1093/jxb/48.7.1357
复制
发表时间：
1997-07-01
影响因子：
6.9
通讯作者：
Gresshoff, PM
中科院分区：
文献类型：
--
作者：
Stiller, J;Martirani, L;Gresshoff, PM
由于无法产生大量的转基因豆科植物株系，植物结瘤相关基因的分子分析一直在缓慢进行。本文描述了模式豆科植物日本莲子的高效基因转移和植株再生系统，对一组野生型根瘤菌菌株进行了传染性测试，并选择了毒性最强的菌株9402和AR10作进一步利用。对复合植物毛状根的诱导和结瘤进行了优化，以便在培养皿中进行大规模筛选。接种有效的loti根瘤菌菌株NZP2235后7-12 d，转基因毛状根上有规律地形成3-10个根瘤。毛状根结瘤与野生型根结瘤在形态上无明显差异。为验证毛状根系诱捕共生基因的可行性，采用花菜花叶病毒（CaMV） 35S启动子驱动含有β -葡萄糖苷酸酶（gus, uidA）或荧光素酶（luc）报告基因的二元载体进行了转化实验，结果表明，二元T-DNA与诱导根（Ri） T-DNA的共转移率为70%，gus和luc诱捕载体的阳性表达表明，gus和luc诱捕载体可用于油菜毛状根系的基因标记。为了打开寻找突变表型的可能性，已经开发了一种再生系统，能够在大约5-6个月内从毛状根培养中再生大量转基因植株。同时，对瘤胃芽孢杆菌下胚轴的转化再生进行了改进，在4个月左右就可获得可育的转基因植株。
The molecular analysis of plant genes involved in nodulation has been slowed by the inability to produce high numbers of transgenic legume lines, The high efficiency gene transfer and plant regeneration systems of the model legume Lotus japonicus is described, A collection of wild-type A. rhizogenes strains was tested for infectivity and the most virulent strains, 9402 and AR10, were selected for further use, Growth conditions for plantlets, induction of hairy roots and nodulation of composite plants were optimized for large-scale screening in Petri dishes. A cluster of 3-10 nodules was regularly formed on transgenic hairy roots 7-12 d after inoculation with the effective Rhizobium loti strain NZP2235. There were no apparent morphological differences between nodulation of hairy and wildtype roots. To test the applicability of the hairy root system for the trapping of symbiotic genes, transformation experiments with binary vectors possessing a beta-glucuronidase (gus, uidA) or a luciferase (luc) reporter driven by a cauliflower mosaic virus (CaMV) 35S promoter were performed, The frequency of cotransfer of a binary T-DNA with a root-inducing (Ri) T-DNA was 70%, Positive expression suggests that gus and luc trap vectors can be used for gene tagging in L. japonicus. To open the possibility of searching for mutant phenotypes, a regeneration system has been developed enabling the regeneration of large numbers of transgenic plants from hairy root cultures in about 5-6 months. At the same time, the A. tumefaciens hypocotyl transformation regeneration in L. japonicus has been improved, This new version provides fertile transgenic plants in about 4 months.