Study On Transferring Of Coat Protein Gene Of Potato Virus A (PVA CP) Into Potato (Solanum Tuberosum L.)

A regeneration system available for potato transformation was established considering by severial factors, such as the potato genetype, the explant type, the concentration of different phytohormones and bactericides, the time of co-culture. In this study, PVA CP gene was transferred into cvs. Dongnong303 and Dadi NO.1, main two local cultivars, with Agrobacterium mediated-method. The explants included the stem segments of plantlets in vitro, discs of microtubers and minitubers. The transgenic plants were tested by PCR and PCR-Southern, and the PVA CP gene was confirmed to be integrated in the genome of potato. The main results were showed as following:1. Establishment of the regeneration system for potato transformationThe stem optimal medium for callus culture was MS+ 0.1mg/L 2,4-D+ 2.5mg/L BA, and the best medium for differentiation was MS+ 5mg/L GA3+ 2.5mg/L BA. And the oplimal regeneration medium for tuber disc was MS+ 3.5mg/L ZT + 0.88mg/L IAA. Under the same conditions, we compared the stem regeneration frequency of the Dongnong303, Atlantic, Kexin No.4 and Kexin No. 13, and found that the highest one is Atlantic, which is 75.00%. The differentiation frequency of Dongnong303 was higher than that of Dadi No.1.2. Establishment of high frequency transformation system of potato2.1 The optimal time of pre-culture was 2 days and the optimal time of co-culture was 3 days for stem explant. The optimal time of co-culture was 2 days for tuber explant.2.2 Different explant types needed different respomdence for transformation. For the stems explant, the optimal concentration of Agrobacterium was OD600 0.5, the optimal time of infection was 10 minutes. The frequency of callus resistant to Kanamycin was highest under this condition, reaching 78.50%. For the tuber discs, the optimal concentration of Agrobacterium was OD600 0.5, the optimal time of infection was 5 minutes. The frequency of shoot resistant to Kanamycin was highest under this condition, reaching 53.40%2.3 In the comparative experiment of several bactericides, the best one was Cefotaxime sodium made in USA, and it didn't inhibit the explant regeneration. Its concentration was 150mg/L for the tuber discs and 200 mg/L for the stem segments.2.4 Kanamycin was used as the selective agent. Different explant types needed different selective concentration. The concentration was 50mg/L for the stem segments and 100mg/L for the tuber discs at the stage of shoot and root formation.3. Test of transgenic plantsTwenty-two regeneration potato plants were attained, in which 15 were confirmed by the test of PCR and PCR-Southern blotting to be transgenic plants with PVA CP gene being integrated into potato genomes.