Development of marker-free insect resistant transgenic okra (Abelmoschus esculentus L. Moench) expressing the cry1Ac gene and identification of vector backbone-free events.

Abstract

Agrobacterium-mediated co-transformation method was used to generate marker-free insect resistant transgenic okraplants 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) wasconsistent with the results of bioassays. Further, two lines identified in this study met the criteria for inclusion in commercial breeding programs.