Study On Insect-Resistant Gene Transformation Of Populus Alba Var.Pyramidalis

Poplar is one of the important forest trees for timber with fast growth. The research of gene transformation on poplar by gene engineering technique is a effective way for the selection of new species of poplar with insect-resistant, disease-resistant and so on.The plant regeneration from leaf disc of populus alba var.pyramidalis and the effects of various factors on its receptor system of genetic transformation were described in this paper. Several factors were studied which are the effects of auxin and cytokinin, method of inoculation and antibiotics on the differentiation of adventition buds from leaf disc. The results showed that auxin was not the major factor, but the different kinds of cytokinin had an significant different effect on the differentiation rate. The differentiation rate was significantly improved by use of very low concentration of TDZ. In addition, the leaf sensitivity to antibiotic Cef and Km was studied and the results indicated that the time of adding Km in the medium significantly affected the leaf sensitivity with Km. An effective protocol in optimized conditions which were 1/2MS + TDZ 0.005mg/L + BA 0.25mg/L + IAA 0.5 mg/L for differentiation of poplar (Populus alba var.pyramidalis) was developed. The frequency of differentiation reached to 100% and the number of mean regeneration shoots reached to 6.18 in this protocol.Based on this established receptor system of genetic transformation, several factors affecting transformation of the poplar (Populus alba var.pyramidalis) were studied by infecting with Agrobacterium Tumefaciens LBA4404, such as pre-culture time, infection time, co-culture time, different AS adding method, AS concentration in the co-culture medium, bacterium liquid preparation method, explants statue. An effective protocol in optimized conditions which were eight hours for pre-culture, fifteen minutes for infection, five days for co-culture and dosage 80 umol/L AS in co-culture medium for transformation of poplar (Populus alba var.pyramidalis) was developed and the frequency of transformation increased from 6% to 38.1% in this protocol.Modified cowpea trypsin inhibitor (CpTI) gene was transformed into poplar, and 96 regenerated km-resistant plantlets were obtained. PCR analysis on 80 selected regenerated km-resistant plantlets showed that CpTI gene was integrated into the genome of 38 regenerated clones and the PCR positive frequency reached 47.5%. Dot-Southern showed that 6 of 17 selected clones which were positive in PCR were positive and the rate of dot-southern reached 35.3%. Insect bioassay indicated that transgenic plants of X46 and X50 were extremely resistant to the larvae of Apocheimia cinerarius Erschoff, whereas the untransformed controlplants were sensitive to them. It appeared that a decreased leaf consumption by larvae, a lower larvae weigh and inhibition to larvae development.Also the result showed that the plantlet transformed with Bt gene have not yet gotten by the method of infecting with Agrobacterium Tumefaciens. It seemed that the significant difference between Bt and CpTI bacterium.