Study on genetic transformation of tomato (Lycopersicon esculentum Mill.) with an insect resistance gene cryIAb by Agrobacterium tumefaciens
Keywords:Agrobacterium tumefaciens, Bacillus thuringiensis, Lycopersicon essculentum, genetic transformation.
Damage caused by insects to crops is a major problem for the agricultural economy in the tropics. The main objective of this study was to transfer the gene cryIAb into tomato by using an Agrobacterium-mediated transformation method. Kanamycin was used as the transformant selectable agent. Agrobacterium tumefaciens strain EHA105, containing plasmidCAMBIA2301cryIAb that consists of kanamycin resistance gene, gusA gene and cry1Ab gene encoding for delta endotoxin of Bacillus thuringiensis, was used. Conditions such as bacterial concentration and concentration of acetosyringone were studied. For genetic transformation, the cotylendons were infected and co-cultivated with the Agrobacterium tumefaciens. After the 2 day co-cultivation, the cotylendons were washed by the cefotaxime solution and transferred to the MS medium containing IAA (0.5 mg/l), BA (2 mg/l) and kanamycin (100 mg/l) for selection. After a few weeks, putative transformed explants, able to grow on the kanamycin-containing medium, were transferred to MS medium for root development. Expression of GusA was detected by the X-glu substrate in the GUS assay. The presence of nptII and cry1Ab in the putative transformed tomatoes was confirmed by polymerase chain reaction analysis. Transgenic tomatoes were able to produce Bt endotoxin in the leaf tissue, revealed by the Envirologix Quickstix strip test.