| Poplar is one of the important sylvicultural and commercial tree;it is very popular throughout the world, especially in china. Populus euramericana 108 is a kind of hybrid strain from populus nigra and popolus deltoids. It is the main strain which has been popularized for its high values such as growing fast, high resistance, long fibre etc.by the State Forestry Administration. With the most widely cultivation of Populus euramericana 108, the Arising of Plant Diseases and Pests were more and more frequent. The research of gene transformation on poplar by gene engineering technique is a effective way for the selection of new species of poplar with tolerance, insect-resistant and disease-resistant.In this study, leaf and stem of Populus euramericana 108 were used as experiment material, through the screening to the basic culture medium, the adjustment of bormone kind and proportion, choice of the culture condition, the medium and the culture condition of Populus euramericana 108 regeneration system were optimized.On the basis of the establishment of high frequency regeneration system, adapting to Agrobacterium-mediated to conduct transformation of insect-resistance and disease-resistance genes of TCS gene. Through exploring the factors affecting the genetic transformation, the transgenic system, which selects hpt as The Selectable Marker Genes, was established and the resistant shoot were attained for the first time. The resistant shoots were identified by PCR and PCR-Southern analysis. The results showed that TCS gene had been integrated into Populus euramericana 108 genome.The main conclusion of this experiment was as following:Through the screening to through the screening to the basic culture medium, the adjustment of hormone kind and proportion and choice of the culture condition, the optimal medium and the culture condition were attained.the optimal medium for differentiation from leaf derived from in vitro cultured was MS+6 — BA 0.6 mg·L-1+ NAA 0.2 mg·L-1 the optimal medium for Callus induction from stem and Leafstalk was WPM+2, 4 -D 1.5 mg·L-1 + KT 0.75 mg·L-1.The optimal medium for Root Induction was WPM+0.4 mg/L IBA. They were cultured under thecontrolled condition of 25±1℃ with 2000 lx of successive light. Differentiation rate, Callus induction rate and and Root Induction rate individual was up to 100%.The appropriate concentration of Hyg for selection culture was determined. The differentiation of leaf explants was inhabited on differentiation medium containing 2mg/L Hyg;all shoots that were transferred to rooting medium containing 0-2mg/L Hyg were inhibited completely to root.The effect of cefotaxime and Carbenicillin in different concentration was evaluated oninhibiting Agrobacterium LBA4404 growth, the leaf differentiation and root. It showed that Agrobacterium LBA4404 cultured on solid differentiation medium containing 600mg/L cefotaxime or 400 mg-L' Carbenicillin was killed;in the concentration the effect of cefotaxime to leaf explants was observed than higher that of Carbenicillin and the effect of cefotaxime to shoot rooting was observed than less that of Carbenicillin. So the proper inhibit Agrobacterium LBA4404 concentration was 400mg/L Carbenicillin in leaf differentiation and 600mg/L cefotaxime in shoot rooting.At last, through exploring the factors affecting the genetic transformation, the transgenic system, which selects hpt as The Selectable Marker Genes, was established. Adapting to Agrobacterium-mediated to transform the leaf explants, 18 resistant shoots were obtained and transformation frequency was 4.3%. The resistant shoots were identified by PCR and PCR-Southern analysis. The results showed that TCS gene had been integrated into Popuhis euramericana 108 genome. |
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