Vol. 32, No. 3-4, July-December 2014 577 Transformation of Tomato with cry2AXI gene IJTA © Serials Publications R. Bartakke Ruturaj 1† , A. Naveenkumar 1† , P. Nandeesha 2 , R. Manikandan 3 , N. Balakrishnan 4 , V. Balasubramani 5 , D. Sudhakar 6 and V. Udayasuriyan 7 * ABSTRACT: Tomato (Solanum lycopersicum L.,) is an important vegetable crop extensively damaged by lepidopteran insect pest, Helicoverpa armigera Hubner (tomato fruit borer). In order to mitigate this problem, an attempt was made to generate transgenic tomato plants resistant to fruit borer. In this study, a regeneration protocol was standardized for a local high yielding genotype, cv. PKM1. Out of nine different combinations of media tested, modified MS medium containing B5 vitamins along with zeatin exhibited higher regeneration efficiency. Cotyledonary explants of cv. PKM1 were transformed with an indigenous synthetic cry2AX1 gene encoding insecticidal crystal protein of Bacillus thuringiensis through Agrobacterium mediated transformation. Screening by PCR revealed presence of cry2AX1 gene in five out of nine putative transformants; two of the five PCR positive transformants showed expression of Cry2AX1 protein in qualitative ELISA. The two ELISA positive plants showed low level of mortality in H. armigera and significant reduction in feeding of leaf area, in detached leaf bioassay. Selection of cry2AX1 transgenic tomato plants by quantitative ELISA for high level of gene expression will be useful for getting plants with desirable level of insect protection. Key words: cry2AX1, regeneration, Helicoverpa armigera, tomato transformation 1,3,4,5,6,7 Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore 641003, India. 2 Present address: Indian Institute of Horticultural Research, Bengaluru 560089, India. * corresponding author: udayvar@yahoo.com 1† Authors contributed equally to this work INTRODUCTION Tomato (Solanum lycopersicum L.) is one of the major vegetables which has achieved tremendous popularity over the last century. It is grown worldwide either in the field, greenhouses or net houses. It is very versatile and is grown either for fresh fruits or for processing. At present, India contributes 11 per cent of world tomato production and it is grown in an area of 8,70,000 hectare with productivity of 18.24 MT/hectare in 2012 (FAO Statistical Database, 2014). Tomato is severely damaged by lepidopteran insect Helicoverpa armigera Hubner (fruit borer). Farmers apply numerous insecticidal sprays to manage the pest as it has developed resistance to insecticides (Anonymous, 1990; Kranthi et al., 2000). In the recent past, tomato grower suffers heavy losses because of the very high incidence of H. armigera. Relying generally on chemical insecticides may not be viable as they provide ephemeral benefits, often with adverse side effects, and in some instances, actually worsen farmers’ overall pest problems (Sharma and Ortiz, 2002). Thus, the major challenge is how to increase and sustain crop productivity with less use of insecticides. Evolving insect resistant crop varieties is one of the major breeding objectives of crop plants including tomato. Introduction of genes from wild relatives for resistance to various disease and pest in commercial cultivars of S. lycopersicum by conventional breeding techniques often meets serious difficulties due to high incompatibility barriers to hybridization (Kaul, 1991). Genetic engineering techniques may play a major role in the development of insect/disease resistant cultivars and they are being used in tomato varietal improvement programmes to act against these challenges. Bacillus thuringiensis (Bt) is a spore-forming Gram- positive bacterium. During sporulation, the intracellular insecticidal crystal proteins (Cry proteins) are produced as inclusions. These proteins are toxic to lepidopteran, dipteran, and coleopteran insect larvae (Kuo and Chak, 1996). The Cry protein from Bt has been developed as one of the most