Restraining Polyphenol Oxidase Of 'Granny Smith' Apple By Double-stranded RNA

APPO was used as the purpose gene, the transformation of 'Granny Smith' apple by Agrobacterium-mediated was studied. By optimizing the transformation system of 'Granny Smith' apple, a high-efficient transformation system was established, the transgenic plants were obtained. The experiment research results as follows:1 Establishment of tissue culture in 'Granny Smith' appleFactors influencing the explant browning in preliminary culture in vitro of 'Granny Smith' apple were studied. VC, active carbon, PVP, adumbral explants and laying low temperature restrained the browning of shoots of 'Granny Smith' apple. The results show: Culturing at low temperature is a necessary condition for avoiding browning.2 Adventitious shoot regeneration from leaves of 'Granny Smith' apple in vitroThe effects of different concentrations of BA, TDZ, dark incubation time, leaf burl and AgNO₃ on the shoot regeneration of 'Granny Smith' apple in vitro were studied. The results showed that the optimum medium were MS + TDZ2.0mg/L + NAA0.3mg/L + Sucrose30 g/L+Agar 6.0g/L or MS+ BA 4mg/L+ NAA0.2mg/L+ Sucrose30g/L + Agar 6.0g/L. The optimum dark incubation time was 20 days.3 Development of a high-efficient transformation system of 'Granny Smith' appleTo develop procedures for the efficient production of apple transgenic plants, we analyzed the principle factors affecting genetic transformation efficiency. In the transformation system of 'Granny Smith' apple, the available concentration of Cb was 250mg/L. The efficient time for explants dipping into Agrobacterium suspension was 5min. Co-cultivation time was 3-4d. It has beneficial effects on genetic transformation efficiency to no append growth hormone in co-culture medium in steps. Resulting in 93.8% Km resistant calli and 14.1% Km resistant shoots.4. Assays of transgenic applepYF7704 was transformed into apple (Malus domestica) cv. 'Granny Smith' via Agrobacteriumtumefaciens mediated leaf disc transformation. With the selection of Kanamycin and GUS detection assays, 4 plants were detected positive to GUS staining. Four GUS positive plants were confirmed that the foreign gene had been integrated into the genome of apple plants by PCR amplification.