The Genetic Transformation Of Bt And CBF1 Genes In Potato Via Agrobacterium-mediation

A regeneration system available for potato transformation was established considering byseverial factors, such as the kind and concentration of different phytohormones and bactericides,the concentration of sucrose and agar, the time of co-culture, the effect of selection. In this study,Bt-CryV and CBF1 genes were transferred into Dongnong303, a main local cultivars, withAgrobacterium-mediated method. The explants were microtuber discs. The transgenic plantswere tested by PCR and PCR-Southern, and they were confirmed to be integrated in the genomeof potato. The main results were showed as following:1 The oplimal regeneration medium for tuber disc was MS+3.5 mg/L ZT+1.0 mg/L IAA.The highest rate of inducing shoot is 89.85%. The best physiological estate of tuber explant fortransformation is three month ofresting stage. The concentration of sucrose and agar in basic culture medium is 30 g/L and0.7%.2 The optimal conditions of Agrobacterium-mediated method had no difference in theintention of gene. Both of CryV gene and CBF1 gene, the optimal concentration ofAgrobacterium was OD₆₀₀ 0.5, the optimal time of infection was 5 minutes. In this condition,the rate of tuber disc with CryV gene was 61.90%, the rate of tuber disc with CBF1 gene was68.33%, both of them, the rate of pollution were low.3 Co-culture time had great influence on activity of gene. The optimal co-culture time ofDongnong303 with CryV gene was 60 hours and that of CBF1 gene was 36 hours.4 In the comparative experiment of several bactericides, the effective concentration withCefotaxime sodium made in China: for CryV gene was 400mg/L, for CBF1 gene was 500mg/L.The best one was Cefotaxime sodium made in USA, and its effective concentration was150mg/L for both CryV and CBF1 genes, which didn't have negative effect to the growth ofexplants.5 The manner of selection was confirmed seven days later. Kanamycin was used as theselective agent. The concentration was 100mg/L for the tuber discs at the stage of shoot and rootformation.6 Transformants were attained for CryV gene in this experiment, in which 21 plants wetepassed rootage (Kan100mg/L), and in which 10 transformants were confirmed by the test ofPCR and PCR-Southern blotting to be transgenic plants. 96 transformants were attained forCBF1 gene in this experiment, in which 31 plants wete passed rootage (Kan100mg/L), and inwhich 15 transformants were confirmed by the test of PCR and PCR-Southern blotting to betransgenic plants. Both CryV gene and CBF1 genes were integrated into potato genomes.