Conditions for efficient transformation of tomato (Lycopersicon esculentum M.) cultivars with cry1a(c) gene of Bacillus thuringiensis

Abstract

The efficacy of tomato cultivars Hisar Lalit and Hisar Arun to regenerate on different media combinations and their frequency of transformation with cry 1A(c) gene of Bt was evaluated. Experiments were conducted to develop an efficient and reproducible plant regeneration system in two cultivars which are high yielding. conditions were standardized for various factors which affect transformation efficiency like, preculture of explants, inoculation time and co-cultivation duration, using a binary vector pCAMBIA 1301 harbouring gus as a reporter gene. Agrobacterium-mediated genetic transformation was carried out for the above mentioned tomato cultivars for introduction of Bacillus thuringiensis cry1A(c) gene. For this the binary vector pBin 19 was used which contained cry1A(c) gene and npt II as selectable marker (for kanamycin resistance) controlled by CaMV35S promoter. Both these binary vectors (pCAMBIA 1301 and pBin 19) were mobilized into Agrobacterium tumefaciens strain EHA 105. The explants were precultured for three days, treated with bacterial solution for 20 minutes and then co-cultivated for 48 hours. Then these explants were put under a selection pressure of 100 mg/l kanamycin. The control plants bleached while the transformants containing the cry1A(c) gene of Bt and kanamycin resistance gene were healthy, green and survived. This clearly shows that Bt gene along with selectable marker kanamycin have been integrated into the plant genome. The transformation efficiency was recorded and it was observed that cotyledons of cv. Hisar Arun showed 79 per cent while cv. Hisar Lalit showed 81 per cent transformation frequency. In case of hypocotyl explants there was a reduction in transformation efficiency viz. 66 per cent in case of cv. Hisar Arun and 75 per cent in cv. Hisar Lalit.