Agrobacterium-Mediated In Planta Transformation of Field Bean (Lablab purpureus L.) and Recovery of Stable Transgenic Plants Expressing the cry1AcF Gene

Abstract

The amenability and reproducibility of a tissue culture-independent Agrobacterium tumefaciens-mediated transformation strategy was analyzed in field bean and the stability of the transgenes was examined. The protocol involves in planta inoculation of embryo axes of germinating seeds and allowing them to grow into seedlings ex vitro. Transformants were raised using a chimeric Bt gene, cry1AcF, and putative transformants were analyzed by PCR for both cry1AcF as well as the nptII genes. Bioassays against Helicoverpa armigera, the major pod borer, showed that several T1 plants performed well with 17% of T1 plants harboring the transgene. Further, enzyme-linked immunosorbent assay (ELISA) and quick dip strip test confirmed the expression of the chimeric Bt toxin. The stability of the transgenes was checked in three generations for integration, expression, and efficacy against the two insects, H. armigera and Spodoptera litura. Southern blot analysis of 10 high expressing plants confirmed the integration of the transgene, whereas single copy integration of the T-DNA in 5 events was also evident. Transcript accumulation of the cry1AcF gene by Northern analysis supported the expression analysis by ELISA. Likewise, Western blot analysis for the NPTII protein further confirmed the transgenic nature of the plants. At the end of the analysis in the T3 generation, five plants from five T1 events were selected as promising. Therefore, the study proved not only the amenability of the field bean to the transformation protocol but also the stability of the introduced genes through three generations.