Abstract: To optimize the tomato regeneration system and screen out the optimal one, and at the same time, the ProDH interfering expression vector was genetically transformed to tomato, and regenerated plants were obtain by antibiotics screening. The test materials were hypocotyls and cotyledons of tomato variety‘Naiyun 2000? and‘O-33-1?, the basic medium was MS which respectively contained 1.0, 2.0, 3.0 mg/L 6-BA and 0.1, 0.2, 0.3 mg/L IAA. The appropriate concentration combinations were selected. The results showed that the optimal callus culture medium was MS + 2 mg/L 6-BA + 0.2 mg/L IAA, and the optimal differentiation medium was MS + 2.0 mg/L 6-BA + 0.1 mg/L IAA. The optimal rooting medium was MS + 0.2 mg/L IAA; in the optimum medium, the emergence rate, differentiation rate and rooting rate of cotyledon were higher than that of hypocotyls by 25%, 42% and 20% respectively, so the cotyledon was appropriate for explants. Meanwhile, there was also a difference between the two varieties, and‘O-33-1? had a higher regeneration rate than that of ‘Naiyun2000?. Using the optimized regeneration system, the ProDH interference expression vector was genetically transformed to‘O-33-1? to obtain resistant plants.