用根癌农杆菌介导法转化大豆萌动子叶节细胞

利用根癌农杆菌转化萌动大豆成熟子叶节获得了高频率的转基因大豆。从萌发12～16 h的大豆种子切取半片种子作为目标组织，对其子叶节组织进行伤处理后，接种于含有pGB载体的LBA4404农杆菌溶液。pGB载体含有除草剂（phosphinothricin, PPT）抗性基因bar和绿色荧光蛋白基因sgfp。将接种的外植体分别在含有3或5 mg/L PPT的芽诱导培养基、3 mg/L PPT的芽伸长培养基及2～4 mg/L PPT的根诱导培养基上进行了筛选。利用萌发5 d的外植体进行PPT浓度筛选的结果表明，芽诱导、芽伸长及根诱导阶段的PPT浓度分别为5、3及3 mg/L时，转化效率达到最大值，为5.3%。PCR和Southern杂交结果证实了外源基因稳定地整合在大豆基因组中。Northern杂交和GFP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。成熟植株对体外喷洒100 mg/L PPT溶液产生抗性。转化效率达8.9%。

Agrobacterium-mediated Transformation of Soybean Germinating Cotyledonary Node Cells

An efficient transformation method for obtaining a high frequency of transformants of soybean Glycine max(L) Merrill] was developed by inoculation of germinating cotyledonary node with Agrobacterium tumefaciens cells.Soybean seeds were germinated for 1216 h,and the cotyledonary node cells of half seeds were inoculated with Agrobacterium tumefaciens cells harboring a binary vector pGB that contained the bar and sgfp genes conferring phosphinothricin(PPT)-resistance and green fluorescent protein(GFP) activity,respectively.The inoculated explants were selected on the shoot initiation,shoot elongation,and root induction media containing PPT,respectively.The optimal selection concentrations of PPT for 5-day-old explants were 5 mg/L for shoot initiation,3 mg/L for shoot elongation,and 3 mg/L for root induction.To determine the effect of the different target tissues on the transformation efficiency,a total of four different cotyledonary nodes germinated for 1216 h,2 day,5 day,and 10 day,respectively, were tested under the optimal selection system.The highest average transformation efficiency(8.9%) of soybean was obtained when the cotyledonary nodes germinated for 1216 h were used as a target tissue,while in case of using the cotyledonary nodes germinated for 5 and 10 days,respectively,as a target tissue,their transformation efficiencies were remarkably reduced.Stable integration and maintenance of the transgenes in the genome of the PPT-resistant plants were confirmed by Polymerase chain reaction and genomic Southern blot analysis.GFP analysis revealed that the transgenes were highly expressed in the leaves and stems of the transformants.Transgenic plants were resistant to 100 mg/L PPT when applied on the leaves,demonstrating their herbicide-resistance.The transformation strategy described in this study will provide a practical protocol to generate diverse transgenic soybean plants.