Study of the Factors Affecting Agrobacterium Mediated Gene Transformation in Tomato (lycopersicon Esculentum Mill.) Cv. Riogrande Using Rice Chitinase (cht-3) Gene

The factors affecting the Agrobacterium mediated gene transformation of tomato cv. Riogrande are reported in detail. Shoot tips (explants) were transformed with pBI333 carrying rice chitinase (cht-3) gene. Approximately 49% transformation frequency was achieved. The optimum conditions for successful transformation were: infecting the explants with the Agrobacterium suspension for two minutes, co-cultivation period for two days, 50 μM of acetosyringone in the cocultivation medium, preselection for seven days and 500 mg/l cefaxine in the preselection. The transgenic plants were selected on the medium containing 50 mg/l hygromycin. The presence of hygromycin and chitinase gene in the hygromycin resistant (T0) transgenic plants was evaluated by Polymerase Chain Reaction (PCR) analysis. A fragment of 500bp size for the hygromycin gene was amplified from the 100% hyg resistant plants while 250 bp long fragment for the cht-3 gene was amplified from the 88% of the transgenic plants only.