Transformation Of Potato With Insecticidal Gene Pta (Pinellia Ternata Agglutinin)

Pest is one of major reasons that decreases the world crop production. Pest control has been largely dominated by chemical pesticides at present. But many new problems related to the use of these chemicals were reported such as environment pollution and ecosystem lose its balance and so on. Potato (Solanum tuberosum L.) is the fourth important food crops which has been cultured world-wide for a long period. Potato degeneration was also caused by pest usually results in a serious loss of both tuber yield and quality, which has been a main hinder in potato production for long time. It is very difficult to get a new variety with resistance to pests by traditional breeding method in short time because of lacking insect-resistant potato germplasm. Introducing into to potato the pta gene that encoding Pinellia ternata lectin (Pinellia ternata agglutinin; PTA),a toxic protein to Homoptera, by genetic transformation will produce new germplasm that may show resistance to some kinds of pests. In this study, transgenic potato plants with insect- resistant gene pta was obtained by via Agrobacterium tumerfaciens transfromation. There is an active significance for development of potato industry by breeding of new varieties resistant to insects. The following is the results of the study:1.Transform pBIpta plasmid to Agrobacterium tumefaciensReading fram of pBIpta gne was transformed to Ti plasmid of Agrobacterium tumefaciens through the way of direct transformation and recombinants were screend by the medium added with Kanamycin. Restriction analysis and electrophoresis confirmed that the pBIpta plasmid was transformed to Agrobacterium tumefaciens.2.Optimize the in vitro culture system of high capacity and fast regeneration system of potatoBased on the MS medium, a series of medium were devised and selected by effect. The optimal medium for callus induction from stem is 6-BA 2.5 mg/L+2,4-D 0.1 mg/L for longshu3, xindaping and weishu1. The medium of inducing regeneration from stem is MS+ 6-BA 2.5 mg/L+NAA 0.2 mg/L+GA3 5.0 mg/L for longshu3 and xindaping. The medium of inducing callus and regeneration from stem of longshu6 explants is MS+6-BA 4.0mg/L+NAA 0.05 mg/L and MS+ 6-BA 2.5 mg/L+GA3 5.0 mg/L. The medium of inducing regeneration from Weishu1 stem is also MS+ 6-BA 2.5 mg/L+GA3 5.0 mg/L. The optimal regeneration medium for tube chip of four potatoes is MS+ZT 2 mg/L+IAA 1 mg/L. The inducing regeneration rate of weishu1 reached as high as 96.25%. That tuber slices of Longshu3 have strong ability of regeneration is 38.46%. The in vitro culture system of high capacity and fast regeneration of explants was successive developed in this experiment, which provide the base to transforming more genes of interest into potato.3. Study on factors influencing Agrobucterium-mediated potato transformationDuring transforming explants of potato via Agrobacterium transformation, five factors, Agrobacterium concentration, pre-culture time, co-culture time, genotype of potato and explants were set up to study the effect of Agrobacterium-mediated potato transformation. Regarding the frequency of Kan resistant callus cultureas standard,significant differences existed between genotype of potato and between explants. When two days pre-cultured stem and three days pre-cultured tube chip were mixed with cell suspension of Agrobacterium in 5-8 min, the highest transformation frequency was obtained. The factors influencing Agrobacterium-mediated potato transformation were studied systematically to establish a high efficient and stable potato gene transferring system.4. Assays of transgenic plantsThrough stringent selection of the infected explants on the medium added with Kanamycin, eliminated some explants which without resistance to Kanamycin.The results of molecular biological assays with PCR and Southern blot hybridization of the plants, regenerated from the Kanamycin resistant explants, confirmed that the pta gene was integrated into genome of potatoes.