Genetic Manipulation Of Tomato (Lycopersicon Esculentum) Using Wga Gene Through Agrobacterium Mediated Transformation

Abstract

The major problems faced in the field of Agriculture are loss in crop yield caused by insects, Herbs (weeds), viruses and the pathogens and the pests associated loss is about 14% of total agricultural production. The use of pesticides has resulted in adverse effect on the beneficial organisms and other plant parts such as leaves, fruits and has reached pollution levels, which has become a major concern for environmentalists. Therefore, products of crops resistant to insects have been the first priority in crop biotechnology. Genetic transformation has led the possibility of transforming crops for enhanced resistance to insects through the use of insect control protein gene-WGA (Wheat Germ Agglutinin), a glycoprotein with molecular weight of 34000 Da. Toxic effect appears to be mediated through binding of the lectins (WGA) to glycoproteins in the insect leading to the disruption of gut epithelial cells and are believed to be “natures own insecticides”. The present study involves preparation of recombinant pGPTV vector having WGA gene, which was transferred to E. coli DH5α basic strain. The recombinant vector was transferred to Agrobacterium tumefaciens strain LBA4404 using helper strain through triparental mating. The recombinant vector having Agrobacterium was infected with tomato leaf discs through co-cultivation and the leaf discs were transferred to selection media containing Kanamycin and direct regeneration of the plantlet were obtained from the leaf discs. The npt-II gene (Kanamycin resistance gene) serves as a selectable marker system in plants. The regenerated plantlets grown on selection media was subjected to primary screening by isolating the genomic DNA by CTAB (Cetyl Trimethyl Ammonium Buffer) method and the transformation was confirmed by the presence of amplified fragments of WGA gene by PCR analysis.