Establishment Of High Efficient Regeneration System And Insect-resistant Transformation Of Populus Deltoides×P. Nigra Cv. Chile

In this study, young leaves disc of Populus deltoides×P.nigra cv. Chile were used as experiment materials to regeneration system research. Several factors such as basic culture medium, auxin and cytokinin were studied which are the effects in regeneration. On the basic of the above result, adapting to Agrobacterium-mediated to introduce insect-resistance gene of Cry3Aa gene.Established the high frequency regeneration system:the optimal medium for differentiation was MS+6-BA0.25mg/L+NAA0.09mg/L+Agar5g/L+Sugar30g/L,99.07%differentiation frequency may obtained; the suitable medium for inducing the callus with stems was1/2MS+6-BA0.1mg/L+NAA0.1mg/L+IAA0.1mg/L+Agar5g/L+Sugar30g/L, there will grow many regeneration buds from the callus when put them on the differentiated medium; the best medium for rooting was1/2MS+IBA0.05mg/L+IAA0.1mg/L+Agar5g/L+Sugar30g/L, with100%rooting rate, which can offered enough materials for next transformation work.Determined the selective pressure of transformation:30mg/L of Kanamycin was suitable in transformation.Optimized the system of transformation:five main factors affecting Agrobacterium-mediated transformation were studied, such as pre-culture time, infection time, co-culture time, co-culture temperature and AS concentration. Drawing the best transform condition: pre-culture3d; infecte20min in the bacterial concentration with OD600=0.4; add AS300umol/L in bacterium liquid before infecting; co-culture for3d in dark with the temperature of22～25℃.Detected and obtained transgenic plants:PCR analysis showed there were2positive plants in the68resistant plants. The result of this research demonstrated primarily that the Cry3Aa gene was introduced into genome of Populus deltoides×P. nigra cv. Chile.Transplanted and observed the transgenic and CK plants:the present growth status showed that transgenic plant was growed slowed than CK one obviously. Although the preliminary result which obtained by both PCR detection and growth condition observation showed that the51and43ones were positive plants, but we can not make sure their insect-resistant completely.If Conditional, Southern blottiong could be done to confirm the43and51ones are Positive plants before insect bioassay test. But there had no enough time to do Southern blottiong, so the meterials have been kept for Southern blottiong. At the same time, do insect-resistant transformation, insect bioassay test must be done, but there were too few meterials to perform the test, so it is necessary for multiplying them and do insect bioassay test to determine their insect-resistant.