Study On The Potato Transformation With Leaf Senescence-inhibition Gene P_SAG12-IPT

Cytokinin(CTK)has a far-ranging biology signification in plant.It can delaying senescence of leaf and improving stress resistance of plant. At present, the direct synthesize approach and tRNA were two recognized approach of cytokinin synthesize, and direct synthesize approach is the primary synthesize approach, and ipt is a key synthetase in the process. The PSAG12-ipt is a chimeric gene, ipt gene come from the Agrobacterium tumefaciens plasmid ipt gene and PSAG12 of specific senescence-inhibition promoter of Arabidopsis thaliana can delay senescence of tobacco's leaf and increase yield. The PSAG12-ipt gene were transformed into potato in order to investigate mechanism and function of transgenic potato plant, and tried to find one new route that can increase yield of potato, or create germ plasm resource of high yield in potato. The transgenic host system of potato was established, and the genetic transformation system was studied systemic, the results showed that stem cutting was better than leaf for its high callus induction rates and strong ability to differentiation and regeneration. The optimal genetic transformation system of potato was that stem and leaf were pre-cultured on the MS medium with NAA0.25mg/L, 2,4-D0.25mg/L for 2 days, OD600 of the Agrobacterium tumefaciens is 0.2~0.5, infection Agrobacterium tumefaciens for 8 min, co-culture 3 days on MS medium under 25℃in darkness, and used 50mg/L and 75mg/L Kan concentration as the grads of callus induction and rooting; in addition to appending appropriate concentration Na2SO3 can restrain effectively stem break and can promote rooting and enhance induction of callus. The transgenic plant of Kanamycin-resistant were examined by dual-PCR with specific primers, in which some transgenic plants showed positive signals, indication preliminary that PSAG12-ipt gene was transformed into the genome of potato. Morphological observation of transgenic plants showed that "Atlantic-6", "Atlantic-28"and "Atlantic-32"were growing normal, but delay the senescence course more than control, the examination indicated that the ipt gene had expressed successed under PSAG12 promoter in three transgenic plants, and promoted the synthesize approach of Cytokinin(CTK)in leaf of potato. The Chlorophyll , Cytokinin(CTK), Superoxide dismutase(SOD) and Malonaldehyde(MDA)of leaf in different development period were examined, the results showed that Cytokinin( CTK ) was consanguineous correlation with Chlorophyll, Cytokinin(CTK), Superoxide dismutase(SOD) and Malonaldehyde(MDA), which the contents of Chlolophyll, activity of Cytokinin(CTK) and Superoxide dismutase(SOD) in transgenic potato plant were higher than control, however the Malonaldehyde(MDA) was lower than control.