Deole Satish, Padakipatil Sanjeev, Sandhya S R, Nanote Asaram, Jadhav Murlidhar, Bihani Pankaj, Parimi Srinivas, Zehr Usha, Narendran M, Char Bharat R
Mahyco Research Centre, Dawalwadi, Jalna-Aurangabad Road, Jalna, Maharashtra 431203 India.
Physiol Mol Biol Plants. 2021 Oct;27(10):2379-2387. doi: 10.1007/s12298-021-01074-3. Epub 2021 Sep 21.
-mediated co-transformation method was used to generate marker-free insect resistant transgenic okra plants expressing the gene. The gene was borne on the T-DNA of one plasmid while II and (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) T generation for Cry1Ac and GUS. Selected events were chosen for further genetic and molecular analysis. The and marker genes were found to segregate independently, of each other in 10 events in T 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 events (clean T-DNA insertions carrying 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, 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 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.
采用介导共转化法培育出无标记的抗虫转基因秋葵植株，该植株表达了 基因。 基因位于一个质粒的T-DNA上，而II和 （GUS）标记基因则存在于第二个质粒的T-DNA上。通过组织化学GUS检测筛选推定的转基因植株，以检测β-葡萄糖醛酸酶的表达，32个转基因事件GUS呈阳性，其中21个转基因事件ELISA检测Cry1Ac蛋白呈阳性。在21个Cry1Ac阳性的T0事件中，有3个事件在T1代中Cry1Ac和GUS的转基因呈现孟德尔遗传（9:3:3:1比例）。选择的事件用于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，有10个事件的 和标记基因彼此独立分离，这表明两个T-DNA插入在遗传上没有连锁，在这10个事件中可以鉴定出无标记植株。使用合适的探针通过Southern分析确认了无标记性质和无载体骨架的事件（携带 基因的干净T-DNA插入）。使用秋葵的重要害虫——棉铃虫幼虫，对选定转基因事件的植株进行全株昆虫生物测定，进行严格筛选。昆虫生物测定表明，5个转基因事件中幼虫死亡率为100%，没有任何虫害，2个事件显示有5%至10%的虫害，证实了转基因植株的抗虫性。最后，对以孟德尔方式遗传转基因的事件进行了进一步表征，鉴定出无标记和无载体骨架的事件，在全株昆虫生物测定中显示对目标害虫具有完全保护作用。选定事件（品系）植株部分中Cry1Ac蛋白水平的定量与生物测定结果一致。此外，本研究中鉴定的两个品系符合纳入商业育种计划的标准。
