| In this study, we selected LH04-8 poplar as the experiment material. Through shifting the basic medium, the related growth regulator type and concentration, as well as the appropriate choice of culture conditions, we optimized the regeneration system of LH04-8 poplar and provided a stable receptor system for the latter part of the genetic transformation test. Meanwhile, we explored the effects of different factors on the insect-resistant genes Cry1C+Cry9C conversion efficiency of LH04-8 poplar through agrobacterium-mediated method and obtained resistant plants. The main results were as follows:1.Establishment of high frequency regeneration system of Populus LH04-8 Clones(1) We used secondary disinfection method to carry out the sterilization experiments for Populus LH04-8 stems, petioles and leaves:with 70% ethanol for 20s, then 0.1% HgCl2 for disinfection, the specific processing time were described as follows.Directly picked explants in the field:Stems for 10min, petiole for 8min, leaves for 6min; Explants obtained from the Hydroponic plants:Stems for 6min, petiole for 5min, leaves for 3min.(2) On the basis of screening basic medium, we explored the influence that different combinations and concentrations of plant growth regulators to the Populus LH04-8 plantlets in vitro regeneration, obtained a highly efficient and stable regeneration system. The research showed that the suitable medium for the blade differentiation was composed as MS+4.0mg/LBA+0.2mg/LNAA+0.005mg/LTDZ, the differentiation rate was 86.66%, and single leaf buds were more differentiated.2. We used LH04-8 poplar leaves of regenerated plants as part of its insect-resistant transgenic receptor material, and the efficient and stable genetic transformation system was established.(1) In this test, we selected Cefotaxime as the bacteriostatic antibiotics of Agrobacterium EHA105. The suitable concentration of Cefotaxime and kanamycin for the leaf differentiation was respectively 200mg/L and 20mg/L; In the rooting culture stages, the suitable concentration of Cef and Kan was 100mg/L and 10mg/L respectively.(2) Through the method of Agrobacterium-mediated, we carried out the transformation for insect gene Cry1C+Cry9C. By the aid of discussing the effect of the factors just as the pre-incubation time、infection conditions and the others to the genetic transformation frequency of Populus LH04, we have found that the ultimately conversion conditions were just as follows:Firstly, made the leaves inoculated in the differentiation medium pre-cultured for 3d, then removed the blade, soaked in bacterial which the OD600 value was 0.8 for 10min, then transferred into medium to co-culture for 3d, and Kanamycin selection for 15d was benefit for the induction of the resistant shoots. In this transformation system, the induction rate of Kan resistant shoots was up to 17.78 percent.(3) Resistant plants in medium containing antibiotics could differentiate and generate roots normally. We have obtained seven Kan-resistant plants for PCR testing, and the results showed that there were four DNA samples showed positive, which preliminarily certified that the target gene Cry1C+Cry9C had been imported into Populus LH04-8 genome.(4) A PCR-positive transgenic plant of LH04-8 poplar was survived by transplanting. |
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