Study On The Acceptor Of Genetic Transformation For Populus Deltoides (I-63×I-69) And Examination Of Plants Transferred By BtCry1A Gene

Populus deltoides (I-63×I-69) is the clone of I-63×I-69 hybrid which breed selectively from seed descendant,but been damaged by insect quite seriously. The anti-insect gene,mainly is Bt gene,has been successfully transformed into Populus alba, Populus nigra, Populus tomentosa, Populus simonii, Populus deltoids. However, it is little known about the plantation of transgenic insect-resistant poplars to date, because of many actors,such as acceptor system, transformed condition and so on, the resistance of P. deltoides which transferred the anti-insect gene is not good. In anti-insect gene transforming research, a good acceptor system is the essential link whether the transformation successful or not. The experiment in the foundation of laboratory earlier research,has established the P. deltoides highly effective acceptor system of the leaf direct differentiation the bud, providing the enormous convenience for the P.deltoides regeneration and transformation. In addition, carries on the appraisal the plant which transferred the Btcry1A gene by the Agrobacterium tumefaciens, and obtains six anti-insect new product department.In the earlier experiment, the regeneration system of P.deltoides leaves induction to callus differentiation bud has been establishing. This paper continue to conducting the research about the affective factors of leaf differentiation:the basic culture medium, the growth adjustment matter,the antibiotic and studying the acceptor system of populus leaf direct differentiation the bud. the result indicated that best culture medium is: (1/2N)MS+NAA 0.02mg/L + BA 0.2mg/L + sucrose 25g/L + agar 6.5g/L, while the average differentiation rate to be possible reaching above 85%. Then,several antibiotics were used to eliminate A.tumefaciens LBA4404 during transformation and analyzed to affect the bud differentiation Best bacteriostasis antibiotic is cefotaxime, 150-200mg/L of which in the medium can suppressed approximately 81.8%-100% of A. tumefaciens LBA4404 with the buds differentiation 73.3%. Selective antibiotic kanamycin suppress the bud's survival, the lowest concentration of kanamycin used for selection of transformation was 30mg/L when taking buds as acceptor, and 15mg/L at rooting stage. The experiment also proved that streptomycin has the same effect as NAA on the bud regeneration, combinating of 20mg/L streptomycin and 0.02 mg/L NAA and 0.2 mg/L BA making the differentiation rate up to 100%. Forthemore, streptomycin influence the spots of leaves differentiation buds.The molecular-examination and the anti-insect appraisal are carried on seventeen kanamycin-resistant P. deltoids transformed BtCry1A gene. PCR analysis showed that six of these seventeen kanamycin-resistant rooted plants were BtCry1A gene positive which further confirmed by ELISA analysis, indicated BtCry1A gene has efficiently expressed and the quantity of Bt insecticidal protein is about 3.88-2.36ppb, average of 3.32ppb. MTT analysis showed the protein of PCR positive plant named X1 process Hi5 cell, of which the mortality rate is very high and the pathological change is similar as Bt toxin protein processing directly. That also proved the BtCry1A gene has expressed and transformed plant has strikingly insecticidal to Hi5 cell. Bioassay results that X1 is significantly inhibitoryaction the growth of Clostera anachoreta, even cause the death of insects, relative moranity rate respectively is 75.99%,36% and 55.99% at 10^th when the comparison insect complete pupate, the survival insect which are feeded on transferred plant leaves pupation rate is only 33.3%,18.8%,54.5%, the other are significant smaller and slow-acting and still not to be able to pupate at 12^th . After taking food on transferred plant leaves, mesogaster epithelial cell of Clostera anachoreta has the model of insect's pathology characteristic of Bt toxin function to the Lepidoptera. Therefore, this research screens six P. deltoids transferred the BtCry1A gene which has certain anti-insect activity,waiting for further studies and appling to the practice.