Transfer NAC-43 In The Wheat Of Immature Embryos By Gene Particle Bombardment And Preliminarily Study The Insertion Site

Wheat is one of the important food crops, It is one of the important methods to improve the quality of wheat by Using plant genetic engineering to geneticly improve the wheat.The Important goal of genetic improvement of wheat are enhancing the ability of wheat to insect resistance and stress tolerance, increasing wheat production, improving wheat quality. In this study, using eight kinds of Common Southwest Wheat as materials induce The immature embryo into and different the callus and Finally regenerat into plants. We also Compare to the ability of the induction and differentiation and regeneration between species,in order to screen the bast genotypes what have the bast ability of The Tissue culture. On this basis,using the Callus which come from Improved strains of southwestern wheat-Mianyang 11 and chuannong23-as experimental material, Using particle bombardment to Transduce PBI121-NAC-43 into the wheat and get the transgenic plants. After getting the transgenic plants, we also study the insertion sites which has the target genes in transgenic plants by using TAIL-PCR technique and certainly optimize this technology. The main results obtained are as follows:1.Through studing 8 different genotypes of wheat about immature embryos of callus induction,differentiation and regeneration, We find that it is not differentbetween the majority of immature embryos of wheat genotypes callus induction capacity but The Regeneration and Sprouting are On the contrary. using eembryo-isolated method(EI),we induce callus from immature wheat embryos and culture for differentiation and regeneration, Finally,we selecte Mianyang 11 and chuanong 23 as the excellent southwest of wheat genotypes which have the high rate of callus, good callus quality, high capacity of differentiation and high rate of seedling.2. Using PMD19-T-NAC-43 as template, we clone NAC-43 gene and constructe NAC-43 gene to a new expression vector-PBI121. Then we transforme the PBI121-NAC-43 and PAHC20 which contains the bar gene into mianyangll and chuanong23 to get transgenic wheat.3. According to the left and right borders'known sequence of vector PBI121-NAC-43 (npt and gus gene), we severally design 3 nested primers which are varying distances from the border, totaly of 6 specific Primers, and design 3 degenerate primers according to the conserved amino acid sequences of wheat protein. Then we Combinate the degenerate primers and specific primers in various and make the TAIL-PCR reaction. Also we make the Comparative study of the dosage-ratio of the two primers and the template dilution of two or three times in the reaction. Thus we initially optimized TAIL-PCR technology, and study the target gene insertion sites of transgenic plants.