Plant Regeneration In Vitro And Primary Study On Genetic Transformation Of Acer Negundo L.

In this study, tissue culture technique and regeneration system for Acer negundo L. were first established. Moreover, the insect-resistant genes were first transformed into Acer negundo via Agrobecterium-mediaXed transformation and needle-sticking method transformation in the aceraceous plants. Positive calli and plants were obtained by PCR amplification and PCR-Southern analysis. The main contents and results are listed as follows:1. The establishment of regeneration system by the route of adventitious buds of Acer negundo(1) A regeneration protocol through axillary buds has been developed for Acer negundo. Stalk nodus and tip shoot explants were placed on MS medium supplemented with different hormone or different concentration and then compared their ratio of proliferation and differentiation. The best medium and hormone combinations for tip shoot and stalk nodus were established. The result showed that MS medium containing 0.1 mg/L 6-BA and 0.01 mg/L NAA suited proliferation culture for stalk nodus and tip shoot. The best differentiation medium for them were separately MS solid medium with 0.1 mg/L TDZ and 0.01 mg/L 2,4-D and MS solid medium supplemented with 0.01 mg/L TDZ. The best rooting medium was MS medium with 0.1 mg/L NAA. The rooting ratio was 89%.(2) Nodal segments were separately cultured MS liquid medium and solid medium supplemented with different TDZ concentration. The effect of differentiation in liquid medium was better than in solid medium and adventitious shoot produced earlier on the medium with the same TDZ concentration.2. The establishment of regeneration system by the route of callus from stem et al. of Acer negundoA tissue culture system for Acer negundo by the route of inducing calli were primarily established through screening out the best media and hormone combinations for different explants (stem segment, leaf, cotyledon, petiole). The calli induced from different explants had a little difference in color and texture. Their capability of induced calli and differentiation were different and stem segment was the strongest and petiole was poor. The best medium for differentiation of stem segment was MS basal medium supplemented with 0.0005 mg/L TDZ and 0.01 mg/L NAA. The ratio of differentiation was 22.8 %. The else explants did not regenerate.3. The study of anatomy of calli induced from stem et al.The inside tissue of calli from different color and texture was observed. Moreover, the histological events during adventitious bud development in stalk nodus have been examined. The results showed the arrange mode and size of different origin calli were different slightly. Tint brown or tin calli were embryogenic callis in majority. The research would establish basis for identification embryogenic calli from color and texture. Meristematic activity in thesuperficial layers led to bud primordial formation.4. The primary study on genetic transformation of Acer negundo(1) The concentration of kanamycin was confirmed for screening out transformed explants from different explants. It was fitting that leaves , stem and stalk nodus , calli were selected by 15mg/L, 25mg/L, 15mg/L of kanamycin separately. The effects of different concentration of bacterium solution and infecting times on the transformant ratio were studied. As a result, the bacterium liquid diluted with the same volume LB liquid medium infected explants for 10 minutes would have the higher transformant ratio.(2) The Toxin gene and Bt gene were first transformed into Acer negundo by Agrobacterium-mediated method and needle-sticking method. Transgenic calli and transgenic plants were obtained by PCR and PCR-Southern hybridization analysis.