Agrobacterium-mediated Genetic Transformation Of Antisense ACC Synthase And ACC Oxidase In 'Jiro' Persimmon

In this study, Jiro persimmon in vitro plantlet leaves were used for test materials. The first time, Agrobacterium tumefaciens EHA101-mediated ACO and ACS antisense gene transformation into Jiro persimmon was studied.The study include the effects of selection antibiotics Spc and antibacterial agents Cef on direct differentiation of adventitious buds; A genetic transformation system was established using Jiro Persimmon leaves as receptors, such factors that impacted the conversion as pre-culture time, co-culture time, de-culture time, Agrobacterium tumefaciens concentration for infection and AS concentration were detailedly discussed, and the conversion conditions were optimized; Agrobacterium tumefaciens -mediated methods were used to transform ACS and ACO antisense gene into Jiro persimmon; Resistant shoots were regenerated and screened; Transformation was preliminarily conformed by PCR detection. Grafting conditions of Jiro persimmon were also studied. The main findings were as follows:1. Leaf explants were very sensitive to spectinomycin. Adventitious buds differentiation could be completely inhibited in the differentiation medium with 30mg/L spectinomycin; Cefotaxime did not seriously affect leaf differentiation. 300mg/L Cefotaxime would be able to inhibit Agrobacterium tumefaciens growth, and adventitious shoot regeneration rate was 72.4%.2. Leaves pre-cultured for 4d, infected for 10min with the bacteria liquid OD600 value of 0.6, adding 200 mg/L acetosyringone, co-cultured on DKW (1/2N) medium with ZT 1.0 mg/L, TDZ 0.5 mg/L and cef 300 mg/L for 3d, and then de-cultured for 3d, were suitable for transformation. The highest differentiation rate of leaf discs was 66%.3. Through genetic transformation, 320 strains of resistant buds were obtained, of which 38 strains were positive.4. Grafting with stamping a hole about 3~5mm with the grinding forceps tip, and then inserting the bottom epidermis scraped scion into the rootstock hole about 6mm size, obtained the highest grafting survival rate.