Abstract: Taking the self bred cherry tomato ‘J5’ with good quality as the test material, the effects of the combination of medium type, soaking time and disinfection method on the culture of sterile seedlings were studied by using the method of plant tissue culture; effects of different hormone combinations on callus induction and adventitious bud differentiation; effect of IAA concentration on rooting, in order to provide reference for the establishment of a complete regeneration system.The results showed that the best sterile seedlings were obtained from tomato seeds on 1/2 MS medium after soaking for 24 hours and 5% NaClO sterilization treatment, and the seedlings emerged neatly and grew well.The healing rate of the explants was not significantly different between the combination of zeatin（ZT）+indoleacetic acid（IAA） and 6-benzylaminopurine（6-BA）+IAA（P＜0.05,the same below）,but the germination rate was significantly higher under the combination of 6-BA+IAA than ZT+IAA;the germination rate of cotyledons was higher than that of hypocotyls, and the optimal medium for shoot differentiation was MS+2.0 mg·L^-1 6-BA+0 mg·L^-1 IAA,and the differentiation rate reached 40.00%.The optimal rooting medium was 1/2 MS+0.2 mg·L^-1 IAA,and the rooting time was only 5-6 days.The roots were denser and thicker; the seedlings were refined using the incubator and transplanted into a substrate with a nutrient soil to vermiculite ratio of 3∶1,and the survival rate was up to 100%.In conclusion, the culture of sterile seedlings was influenced by the type of medium, seed soaking time and disinfection method; the choice of cotyledons for explants was appropriate, and 6-BA promotes the differentiation of shoots better; low concentration of IAA helps the rooting of adventitious shoots.