SpringerPlus 2014-01-01

Transgenic tomato line expressing modified Bacillus thuringiensis cry1Ab gene showing complete resistance to two lepidopteran pests.

Bhupendra Koul, Sugandha Srivastava, Indraneel Sanyal, Bhuminath Tripathi, Vinay Sharma, Devindra Vijay Amla

Index: Springerplus 3 , 84, (2014)

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Abstract

The modified truncated Bt-cry1Ab gene of Bacillus thuringiensis has been used for the development and selection of over expressing transgenic events in a commercially important variety of tomato (Solanum lycopersicum L.) by Agrobacterium-mediated leaf-disc transformation procedure. The integration and inheritance of cry1Ab gene in T0 transgenic plants and their progenies were determined by PCR, RT-PCR and Southern blot hybridization analysis. The toxin expression was monitored by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The transgenic line Ab25 E, expressing 0.47 ± 0.01% Cry1Ab toxin of total soluble protein (TSP) was finally selected in the T4 generation from the segregating population, showing 100% mortality to the second instar larvae of H. armigera and S. litura and minimal damages to leaves and fruits. Southern blot analysis data revealed single copy introgression of cry1Ab gene in highly-expressing Ab25 E transgenic line and expression of Cry1Ab toxin of molecular mass ~65 kDa was evident in Western blot analyses in transgenic plants of T4, T5 and T6 generation. Receptor binding assay performed with partially purified Cry1Ab protein from Ab25 E transgenic tomato line, confirmed efficient protein-protein interaction of Cry1Ab toxin with receptor(s) of both the insects. The higher level of Cry1Ab toxin (≈ 0.47 ± 0.01% TSP) did not affect the normal in vitro regeneration, plant development and fruit yield in this transgenic line. This high expressing Cry1Ab homozygous transgenic line can be a useful candidate in tomato breeding programmes for introgression of important agronomical traits.