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

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

doi:10.1186/2193-1801-3-84

Bhupendra Koul, Devindra Vijay Amla + Show 4 more

Open Access

https://doi.org/10.1186/2193-1801-3-84

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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.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-84) contains supplementary material, which is available to authorized users.

Highlights

Tomato (Solanum lycopersicum L.) is a major vegetable crop plant and extensively consumed either raw or cooked
Leaf disc explants that were kept on MS basal medium for in vitro regeneration served as positive control, while those on kanamycin-supplemented medium served as the negative control
Results of Western blot analysis performed with cell-free protein extracts of T4, T5 and T6 progenies of Ab25 E transgenic line showed single band of molecular weight ~65 kDa hybridizing with the polyclonal Cry1Ab antibodies

Summary

Introduction

Tomato (Solanum lycopersicum L.) is a major vegetable crop plant and extensively consumed either raw or cooked. A wide range of microbial pathogens and insect pests are known to attack tomato, polyphagous lepidopteran insect Helicoverpa armigera, the common fruit borer which primarily damages the fruit, while Spodoptera litura damages the leaves causing severe losses to the crop productivity. There is a possibility of developing stable insect-resistant tomato lines through the expression of insecticidal gene of Bacillus thuringiensis (Bt), as documented successfully in several crop plants like cotton, maize, soybean, rice, canola and potato (Sanahuja et al 2011; Tabashnik et al 2011). Incorporation of cry1Ab insecticidal crystal protein gene in large number of crop plants rice, tomato, maize, sugarcane and cotton have shown considerable protection against different lepidopteran insects and significant enhancement in productivity (Ye et al.2001; Kumar and Kumar 2004; Dutton et al 2005; Arvinth et al 2010; Khan et al 2013)

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