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CNS Young (insect resistance, Populus carried deltoides of Ⅰ -63 Ⅰ -69) is assigned from the black poplar clones Ⅰ -63 × Ⅰ -69 hybrid seedling offspring breeding improved strains, but there is not strong defects. At present, although the use of insect-resistant gene (Bt gene) transformed Populus nigra, the male white poplar, Populus deltoides, Europe and the United States Yang received reports of success, but a variety of factors by the receptor system transformation conditions , the obtained transgenic Young insect resistance is not high, has not been transferred to the insect-resistant genes Yang reported for afforestation production. Translational research in poplar insect-resistant genes, a receptor system is a key link in that transformation success. The experiments on the basis of the preliminary work of the Laboratory, successfully established CNS No. 8 Yang Directly to Differentiate Buds detached leaves of efficient receptor system, provides a great convenience for the the deltoides regeneration and transformation. Obtained by Agrobacterium-mediated transformation of our laboratory Zhongjia the 8th Yang to turn Btcry1A transgenic plants were identified, screened six new lines with a certain insect resistance. My room had Differentiate Buds CNS 8 Young leaves induced callus regeneration system. On this basis, continue to affect leaf differentiation factors: basic medium, growth regulators, antibiotics, and other research, the establishment of the CNS Yang leaves directly differentiate adventitious buds gene transformation receptor system. The study results show that: the best Leaf differentiation of adventitious buds medium: (1/2N) MS NAA0.02mg / the L BA0.2mg / L sucrose 25g / L agar 6.5 g / L, average differentiation rate of adventitious buds up to 85 % or more. Several antibiotics Agrobacterium tumefaciens strain LBA4404 antibacterial test and impact study of the differentiation of adventitious buds Zhongjia No. 8 Young leaves, that a the best bacteriostatic antibiotic cefotaxime (cef), the amount of 150-200mg / L, Agrobacterium correction antibacterial rate of 81.8% and 100%, leaf differentiation of adventitious buds frequency of up to 73.3%. Select the antibiotics kanamycin (Km) dosage of 5-60mg / L completely inhibited adventitious buds occurred; Km ≥ 60 mg / L, bud survival rate of 0. Therefore, Km Sprouts used at a concentration of 30mg / L appropriate. Rooting screening Km concentration of 15mg / L is more appropriate. The experiment also found that streptomycin (str) has a similar effect of auxin the poplar leaves differentiation adventitious buds. Str (20 mg / L), NAA (0.02 mg / L) and BA (0.2 mg / L), with adventitious bud differentiation rate of 100%. In addition, a certain concentration of streptomycin affect parts of the leaves budding on medium containing streptomycin, some leaves, petioles punishment of adventitious buds or the petiole leaf margin while Differentiate Buds, and control of adventitious buds only from the leaf margin occur. Obtained on of our laboratory transformed BtCry1A gene 17 Zhongjia No. 8 Young Km ~ r plants Molecular Detection and Identification of insect resistance. By molecular detection, 17 Km ~ r plants were screened genetically BtCry1A gene PCR positive plants 6; the ELISA test BtCry1A gene in six PCR positive plants have to express the magnitude of Bt toxic protein content of 3.88-2.36ppb, average For 3.32ppb. That the BtCry1A gene was integrated into the CNS Yang genome and expressed protein. Measured by MTT assay results showed that the CNS No. 8 Young PCR positive plants protein processing Trichoplusia ni cells a higher mortality rate, and the pathological changes similar to the Bt toxin protein processing Trichoplusia ni cells lesions, further evidence BtCry1A gene expression in transgenic plants, and transgenic plants on insect cells in vitro cytotoxicity. The indoor insect resistance test results showed that the transgenic lines developed significantly inhibited larvae growth anachoreta, even a direct result of the death of the larvae. Larvae reared to 10 days, the larvae fed transgenic lines relative mortality were 75.99%, 36% and 55.99%, respectively, at this time, the control untransfected survival gene leaves fed larvae all pupate, and three test strains of leaf feeding survival pupation rate was 33.3%, 18.8%, 54.5%, delayed the surviving larvae action and did not pupate, smaller, fed to 12 days still can not pupate. The larvae of anachoreta transformation leaves, the pathological changes of the intestinal epithelial cells typical of Bt toxins in Lepidoptera pathological features. Therefore, this study screened six Zhongjia Yang to turn BtCrylA gene plants for insect-resistant plants of Lepidoptera have some resistance, but further studies are needed to test and apply it to practice.
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