Plant Regeneration Of Pear Rootstock Yunnan Quince (Cydonia Oblonga Mill.) And Genetic Transformation Of OHF333 (Pyrus Communis)

Pear is one of the most important fruit trees in China. The breeding of dwarfing rootstock with excellent traits has important practical significance for pear production. The genetic engineering is an important way to expand the germplasm base of pear, while the stable regeneration system should be established first. Therefore, in this study, the dwarfing rootstocks of pear, Yunnan quince(Cydonia oblonga Mill.) and OHF333(Pyrus communis) were used as experiment materials, the in vitro regeneration system was established from leaves and TCL of Yunnan quince, PbMYB9 which was involved in anthocyanin biosynthesis was successfully integrated into the OHF333 genome via Agrobacterium tumefaciens mediated transformation. The main conclusion as follows:1. Establishment of the regeneration system from leaves of Yunnan quinceSeveral factors affecting the frequency of in vitro leaves regeneration of Yunnan quince were investigated in this study, including different concentrations of NAA in combination with TDZ, dark culture period and explant types. The results showed that the highest regeneration frequency(88.9%) and the mean shoot number per leaf(7.97) were observed when the apical leaf were cultured on MS medium containing NAA 0.5 mg/L and TDZ 1.0 mg/L. This study also suggested a 20 days dark incubation was necessary for the optimum shoot regeneration. The 85.6% rooting rate could be produced from shoots cultured on half-strength MS medium supplemented with 0.3 mg/L NAA after 5 days dark treatment.2. Establishment of the regeneration system from TCL of Yunnan quinceThe stems of Yunnan quince, which were used as explants, were cut into TCL(1~2 mm) and placed on medium containing different concentrations of TDZ and NAA to select the optimal auxin concentration that can induce the adventitious shoot. The results showed that the optimal medium for TCL regeneration of Yunnan quince was MS + 0.5mg/L TDZ, the highest regeneration frequency was 48.3%, the average number of regeneration shoot was 1.89; the polyploid detection of adventitious shoot showed that there were no genetic variation of regenerate adventitious shoots.3. Genetic transformation of OHF333Using Agrobacterium tumefaciens mediated transformation as method and the leaves of OHF333 as explants, the resistant callus were obtained through infection of in vitro leaves for 10 min, co-cultivation for 2d, delay cultivation and selective culture. The proliferation of resistance callus was conducted and PCR analysis of resistance callus showed that the exogenous gene PbMYB9 had been successfully integrated into the OHF333 genome.