利用改良的草丁膦筛选系统快速而有效筛选转基因大豆(英文)

Rapid And Efficient Selection For Transgenic Soybean Plants With The Improved Glufosinate Selection System

为提高大豆（Glycine max（L．）Merrill）遗传转化效率建立了一种快速而有效的草丁膦筛选系统。将刺伤的子叶节外植体接种于含有pCAMBIA3201载体的LBA4404农杆菌溶液。目前利用草丁膦筛选转基因大豆的常规方法是在含有5mg／L草丁膦的芽诱导培养基和含有3～5mg／L草丁膦的芽伸长培养基上进行筛选。利用此常规筛选方法所获得的大多数植株是非转化体，从而导致转化效率变低，为1．6％；而且这种方法极大地延迟了芽的伸长过程，使多数不定芽的伸长发生在农杆菌处理后的21～41周。为此，对常规草丁膦筛选方法进行了改良。首先将外植体放置在不合有除草剂的芽诱导培养基上培养3周，然后转到含有4mg／L草丁膦的芽伸长培养基上进行筛选。此时，多数不定芽仅在7～12周内便可伸长。当芽长到3～5cm时，从外植体上切下来转到含有1～5mg／L草丁膦的根诱导培养上进行进一步的筛选。结果表明，在添加有3mg／L草丁膦根培养基上，转化效率达到最大值为6．7％。不定芽对根培养基中的除草剂反应迅速，仅在10d天内所有非转化体都变枯死亡。利用这种改良的筛选系统，大多数转基因植株仅在8～16周内便可获得。Southern杂交结果证实了外源基因稳定地整合在大豆基因组中。GUS检测和除草剂抗性分析结果表明，被整合的外源基因在大豆细胞中得到了稳定的表达。

A rapid and efficient glufosinate selection system for obtaining high frequency of transformants insoybean [Glycine max （L.） Merrill] was developed. The cotyledonary node cells were wounded and inoculated with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pCAMBIA3201 that contained a selectable bar gene and a gus reporter gene. The present standard selection based on glufosinate was performed at 5 mg/L glufosinate during shoot induction and at 3-5 mg/L glufosinate during shoot elongation. Many non-transformants were escaped with this standard selection system which resulted in low transformation efficiency of 1.6%, and a delayed shoot elongation with majority of transformed shoots elongation occurred from 21 to 41 weeks after Agrobacterium inoculation, and thus an improved glufosinate selection system was provided in this paper. After 3 weeks on shoot induction medium without glufosinate, the explants were transferred to shoot elongation medium containing 4 mg/L glufosinate for the first selection. The elongation of majority of transformed shoots occurred only from 7 to 12 weeks without glufosinate selection during shoot induction. The transgenic shoots were effectively screened by placing the excised shoots on the root induction medium （RIM containing 3 mg/L glufosinate to facilitate direct uptake of the selective agent that resulted in the high transformation efficiency of 6.7%. Shoots on the RIM rapidly responded to the selective agent applied, all the glufosinate-sensitive shoots were completely necrotic within 10 days after selection. The majority of transgenic plantlets were obtained only 8-16 weeks under the improved selection system. Genomic Southern blot analysis confirmed stable integration of the transgenes in the genome of soybean. Stable expression was confirmed by GUS expression and herbicide application.