Study On Agrobacterium Mediated Potato Transformation Of IP-10 Gene

Potato minituber was used as explant in this study.A efficient regeneration system of cultivar Dongnong303 was optimized by testing different hormone combinations.At the same time IP-10 gene was introduced into potato genome with Agrobacterium mediated method. PCR,RT-PCR and GFP fluorescence detection was done to prove that target gene had been conformed into potato genome.In addition,the best conditions of transgene potato microtuber formation was discussed.1.Virus-free potato plantlets was used to do regeneration testing by different IAA,NAA, GA3,IBA,6-BA and ZT combinations.The results showed that the optimal medium for callus induction from stem was MS+IBA 0.1mg/L+6-BA 0.5mg/L and its inductivity was 86%,but germination rate was low;The best differentiation medium for leaves was MS+ZT 10.00mg/L+NAA 0.1 mg/L+GA₃ 0.2mg/L and the shoot induction rate was 150%.On the other hand,the optimal differentiation medium for tuber disc was MS+IAA 1.0mg/L+ZT 3.5 mg/L and the differentiation rate reached 120%.2.Transgenic plants which were obtained by Agrobacterium infection and hygromycin selection were performed to PCR amplification of IP-10 and hygromycin phosphortransferase genes,respectively.Corresponding bands of 276bp and 602bp were obtained.Three transgenic lines were identified by further RT-PCR reaction and protein GFP fluorescence laser scanning confocal detection.3.The effect of phytohormone and sucrose content to transgenic lines of tuber formed test-tube tuber was discussed.Result showed that the most formation was obtained when KT concentration was 100mg/L,B9 concentration was 150mg/L and sucrose was 8%,and MT/Plantlet were 1.4,1.5 and 1.6,respectively.We confirmed KT,B9 and sucrose could be used as reagent for induction of transgenic test tube-tuber.