The effect of different plant hormones on in vitro regeneration of tomato (Lycopersicon esculentum Mill.) two cultivars Castle Rock and Super strain B, obtained from hypocotyls and cotyledons of cultured in vitro seedlings was examined. The optimal concentrations of plant growth regulators were the MS medium containing 3 mg L-1 of zeatin and 0.1 mg L-1 of NAA of cotyledon and hypocotyl explants of Super strain B cultivar. While, the best regeneration medium was the MS medium containing 1 mg L-1 ZEA, 0.5 mg L-1 BAP and 0.1 mg L-1 IAA of cotyledon and hypocotyl explants of Castle Rock cultivar. Agrobacterium‐mediated genetic transformation system has been expanded of tomato cultivar Super strain B. Transformation efficiency of hypocotyl explants was studied with Agrobacterium tumefaciens strain GV3101 harboring binary vector pICBV19, containing gus and bar genes. Transformed tomato shoots were obtained from hypocotyls explants on MS medium contained with 3 mg L-1 ZEA and 0.1 mg L-1 NAA. The gene expression of gus and bar genes was tested in the putatively transformed tomato plantlets through GUS histochemical and leaf painting assays. The plasmid pBI121 containing an insect resistance gene (cry1Ab) and kanamycin gene (nptII) as a plant selectable marker were introduced into these explants. Putatively transformed plantlets were examined by PCR and southern blot analysis. The expression of cry1Ab gene was tested through insect bioassay. For insect bioassay, experiments were conducted to determine the mortality percentage of Cry1Ab toxin protein against Tuta absoluta (Meyrick). The results indicated that Cry1Ab toxin protein expressed in transformed tomato plants showed 100% instars larval mortality was obtained after feeding for 4–5 days.