Agrobacterium Tumefaciens-mediated Transformation Of The Apple With LEAFY Gene

Apple was one of the most popular fruit species in the word. It had a long juvenile phase that delayed its reproductive development in early times. Genetic transformation of apple with the key genes regulating flowering could accelerate its flowering time and bring great benefit to production. In this article, the efficiency regeneration of adventitious bud from apple stems and transformation were studied. The efficient regeneration of M26 stems and Agrobacterium-mediated regeneration and transformation system of M26 rootstock were established. The flower meristem identity gene (LFY) in Arabidopsis was introduced into M26, Gala and Fuji by Agrobacterium strain EHA105 by this transformation system. It was tested by GUS assays that the GUS activity was expressed in the leaf discs of transformed shoots. And it was proved by PCR analysis that the target LFY gene had been integrated into the genome of regenerated plants. Results were as followed:1. The effects of auxin, darkness, part of stem, stem deposited modes, and subculture time on adventitious bud regeneration from apple stem was studied. And the efficient regeneration system was defined. On the MS medium containing BA 4.0mg/L and NAA 0.1mg/L, biology bottom touching medium and 10 days dark culture treatment, regeneration rate of M26 rootstock stems reached 73% and the adventitious shoots per stem reached 4.15.2. The antibiotics had effect on rooting of shoots. When the concentration of Km was 5.0mg/L, the rooting of the shoot was inhibited. And the concentration of Cef500mg/L had some inhibitatoin on rooting. So the optiumal concentration of antibioitics on rooting was Km 5mg/L and Cef 500mg/L.3. In the transformation system of M26, the available concentration of cefotaxime was 500mg/L. The efficient immersing time for explants in Agrobacterium suspension was 3~5min with Agrobacterium OD6oo=0.5. Three days cocultivation 3 days delayed selection and AS20mg/L in the cocultivation medium improved the GUS expression and the regeneration rate of explants.By which, the Agrobacterium-mediated transformation system of apple was developed. On the basic medium MS containing BA 4.0mg/L NAA 0.2mg/L, the leaf was dipped in Agrobacterium suspension for 3~5min, then cocultivated with Agrobacterium for3 days in the dark and the cocultivation medium containing AS 20mg/L. After 3 days on regeneration medium with cefotaxime (500mg/L), leaf was transfered on selection medium containing cefotaxime (500mg/L) and kanamycin (30mg/L). Then kanamycin-resistant shoots were obtained after 7-8 times selective subculture.4. There were significant difference between leaf and stem in transformation. The kanamycin-resistant shoots were difficulty to regenerate from stem. The GUS expression of stem was 13.33%, and GUS activity was low, only in the incision part and bottom.The rate of kanamycin-resistant shoots was 0.2%. It was negative with GUS assays and PCR analysis.5. In this study, by Agrobacterium tumefaciens-mediated transformation system, the transgenic plants with LFY gene of M26, Fuji and Gala apple were obtained. GUS assays, PCR analysis were made. The results proved that the LFY had been transferred into genome of transformed plants.