Ignacimuthu S
Entomology Research Institute, Loyola College, Chennai 600 034, India.
Indian J Exp Biol. 2000 May;38(5):493-8.
Agrobacterium mediated transformation of Vigna sesquipedalis was achieved using cotyledonary node explants prepared from 5 days old seedlings germinated on B5 basal medium, and transformed using Agrobacterium tumefaciens strain EHA101, carrying the phosphinothricin-N-acetyltransferase gene and neomycin-3-phosphotransferase-II gene as selectable markers and GUS gene as a screenable marker. Gene transfer was achieved by inoculation of cotyledonary node explants with a bacterial suspension and a further cocultivation with Agrobacterium suspension for 3 days on B5 basal medium. Only 10% of the explants were transformed with EHA101 and exhibited transient expression of GUS genes, while 2% of shoots exhibited stable integration of genes and developed into plants. Transgenic character of tissues was confirmed by GUS assay and Southern analysis. Histological analysis of GUS gene expression directly after cocultivation revealed a high competence of subepidermal cell layers of cotyledonary node and associated cotyledons for transformation with Agrobacterium.
利用在B5基本培养基上萌发5天的幼苗制备的子叶节外植体，通过农杆菌介导实现了长豇豆的转化。使用携带膦丝菌素-N-乙酰转移酶基因和新霉素-3-磷酸转移酶-II基因作为选择标记以及GUS基因作为筛选标记的根癌农杆菌菌株EHA101进行转化。通过用细菌悬浮液接种子叶节外植体并在B5基本培养基上与农杆菌悬浮液进一步共培养3天来实现基因转移。只有10%的外植体被EHA101转化并表现出GUS基因的瞬时表达，而2%的芽表现出基因的稳定整合并发育成植株。通过GUS检测和Southern分析证实了组织的转基因特性。共培养后直接对GUS基因表达进行的组织学分析表明，子叶节和相关子叶的表皮下细胞层对农杆菌转化具有很高的感受态。
