利用改良的草丁膦筛选系统快速而有效筛选转基因大豆

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5 mg/L草丁膦的芽诱导培养基和含有3～5 mg/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含有除草剂的芽诱导培养基上培养3周,然后转到含有4 mg/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3～5 cm时,从外植体上切下来转到含有1～5 mg/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3 mg/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.

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

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.