Study On Agrobacterium-Mediated Transformation Of The Apple With ASACC Gene

An effective regeneration of 'Starkrimson' and agrobacteriura-mediated transformation system were developed in this study. ASACC12 was introduced into 'Starkrimson', and a new transgenetic apple germplasm was obtained. The result were as fallow-.1. The browning of explants was effectively inhibited when active carbon was added at 2g/L on early culture.2. In order to found an effective propagation in vitro system for 'Starkrimson', effect of medium was compared. Result shows that shoots propagated better on alternative medium MS and modified C17 with BA3.0 mg/L and NAA0. 1 mg/L than other mediums.3. Factors that effect percent of regeneration of 'Starkrimson' were studied, including type of basic medium, type and content of hormone, time of darkness treatment, node position of leaf, way of leaf deposition. The regeneration system which had the highest regeneration rate at 90.9% was chosen. The process was that culture leaf separated from second to fourth node was on put MS medium containing TDZ 0.5mg/L and NAA 0.3mg/L, and cultured in darkness for 21 days, then light subculture was continued.4. Factors that influence percentage of transformation including time of preculture, dipping and coculture, type and content of antibiotic, day of delayed selection culture, intension of selection were studied. The effective transformation system of 'Starkrimson' was developed as follow: Leaves precultured in darkness for three days were dipped into agrobacterium suspension for three minutes, then were cocultured in darkness on MS medium with TDZ0.5mg/L and NAA 0.3mg/L for three days, and then cultured in darkness on MS medium with TDZ0.5mg/L NAA0.3mg/L and Cef 250mg/L. Selection culture was carried out also in darkness on MS medium with TDZ0.5mg/L NAA0.3mg/L Kml2.5mg/L till resistance callues were formed. Resistance callues were light subcultured with light for one to two times, then resistance shoots were obtained, and was propagated through continued subculture.5. In this study, we obtained 'Starkrimson' transgenetic plants carrying ASACC gene ASACC12 identified by PCR and PCR-Southern. 'Gala' transgenetic plants detected positive to GUS dyeing were also obtained, and detection experiments were expected to be continued.