Agrobacterium-mediated co-transformation method was used to generate marker-free insect resistant transgenic okra plants expressing the cry1Ac gene. The cry1Ac gene was borne on the T-DNA of one plasmid while nptII and uidA (GUS) marker genes were present on the T-DNA of a second plasmid. Putative transgenic plants were screened by histochemical GUS assay for expression of -glucuronidase and 32 transgenic events were positive for GUS in which 21 transgenic events were positive in ELISA for the presence of Cry1Ac protein. Out of 21 Cry1Ac positive T0 events, three events displayed Mendelian inheritance of the transgenes in (9:3:3:1 ratio) T₁ generation for Cry1Ac and GUS. Selected events were chosen for further genetic and molecular analysis. The cry1Ac and marker genes were found to segregate independently, of each other in 10 events in T₁ generation out of 11 Cry1Ac gene inheriting events analysed indicating that the two T-DNAs insertions were genetically unlinked and identification of marker-free plants were possible in these 10 events. The marker-free nature and vector backbone-free Bt events (clean T-DNA insertions carrying cry1Ac gene) were confirmed by Southern analysis using suitable probes. The plants from selected transgenic events were rigorously screened in whole plant insect bioassays using the larvae of shoot and fruit borer, Earias vittella, an important pest of okra. Insect bioassays indicated 100% larval mortality without any infestation in five of the transgenic events and two events showed 5 to 10 percent infestation establishing the insect resistant nature of the transgenic plants. Finally the events inheriting transgenes in Mendelian fashion were characterized further and marker-free and vector backbone-free events were identified showing complete protection from the target pest Earias vittella in whole-plant insect bioassays. Quantification of Cry1Ac protein levels in the plant parts of selected events (lines) was consistent with the results of bioassays. Further, two lines identified in this study met the criteria for inclusion in commercial breeding programs.

农杆菌介导的共转化方法用于产生表达cry1Ac基因的无标记抗虫转基因秋葵植物。cry1Ac基因存在于一个质粒的 T-DNA 上，而npt II 和uidA (GUS) 标记基因存在于第二个质粒的 T-DNA 上。通过组织化学GUS测定筛选假定的转基因植物的β-葡萄糖醛酸酶表达，32个转基因事件对GUS呈阳性，其中21个转基因事件在ELISA中对Cry1Ac蛋白的存在呈阳性。在 21 个 Cry1Ac 阳性 T0 事件中，三个事件显示转基因的孟德尔遗传（比例为 9:3:3:1）T ₁生成 Cry1Ac 和 GUS。选择选定的事件进行进一步的遗传和分子分析。在分析的 11 个 Cry1Ac 基因遗传事件中的 T _{1代中的 10 个事件中，发现}cry1Ac和标记基因彼此独立分离，表明两个 T-DNA 插入在遗传上是不相关的，并且可以鉴定无标记植物这 10 个事件。使用合适的探针通过 Southern 分析证实了无标记的性质和无载体骨架的Bt事件（携带cry1Ac基因的干净 T-DNA 插入）。来自选定转基因事件的植物在全植物昆虫生物测定中使用芽和果蛀虫的幼虫进行严格筛选，Earias vittella，秋葵的重要害虫。昆虫生物测定表明 100% 的幼虫死亡率在五个转基因事件中没有任何侵扰，两个事件显示 5% 到 10% 的侵扰确定了转基因植物的抗虫性。最后，进一步表征了以孟德尔方式遗传转基因的事件，并鉴定了无标记和无载体骨架事件，显示在全植物昆虫生物测定中对目标害虫Earias vittella的完全保护。选定事件（系）的植物部分中 Cry1Ac 蛋白水平的定量与生物测定的结果一致。此外，本研究中确定的两个品系符合纳入商业育种计划的标准。