Stability Of Integration And Expression Of Foreign Gene In Transgenic Birch And Analysis Of Genetic Variation

In this study the occurrence of cytological variation in transgenic birch plants produced by infecting calli with Agrobacterium tumefaciens has been studied. And genetic variation between transgenic plant population and control population has been analyzed by RAPD.The comparison of Polymorphism index produced in previous analyses of transgenic plants and transgenic birch obtained by infecting calli with Agrobacterium tumefaciens has also been analyzed. This study also analysed the influence of different integration patterns on the expression of exogenous gene.1. Genomic DNA was extracted from mature birch leaves in which polysaccharide is very rich by five different methods of SDS, improved SDS, CTAB, CTAB (with high salt), and improved CTAB. The DNA was analyzed by agarose gel electrophoresis, concentration test, rations of D₂₆₀/D₂₈₀ and digestion of restriction enzyme. The results show that the DNA sample obtained by improved CTAB method had high concentration DNA and low protein and polysaccharide content. So the improved CTAB method is the best method for DNA extraction from mature birch leaves.2. The occurrence of somaclonal variation in transgenic birch plants produced by infecting calli with Agrobacterium tumefaciens has been verified by cytological and RAPD analysis. The cytological analysis of transgenic plants showed that the frequency of non-diplod is 78.5%, and the non-transgenic control is 15.3%. In this study 14 arbitrary primers were used to analyze genetic variation between transgenic plant population and control population. 90 loci were detected in total.. In transgenic plant population the percentage of polymorphic loci was 63.33%. The percentage of polymorphic loci was 15.6% in control population. The genetic distance was 0.6502 and the genetic identify was 0.5219 between the transgenic plant population and control population. The results showed that genetic variation in transgenic plant population was much higher than that in control population. The RAPD result indicated the transgenic plants' PI was 31.67. The comparison of data produced in previous analyses of transgenic poplar obtained by infecting calli with Agrobacterium tumefaciens (PI is 57.82) showed that transgenic birch is characterized by fewer genomic changes.3. Among 30 transgenic plants bt gene integration was stabile in three years. And the expression of gus gene was stabile among 28 out of these plants in three years.4. In order to determine the copy number single restriction enzyme HpaI was used to digest the genome DNA. Southern blot analysis of the transgenic birch plants indicates that the Bt gene has been integrated into the genome of the birch. Among the 21 transgenic birch plants, the copy number varies from one to eight. The results of RT-PCR analysis show that bt gene silence in translation level occurs not only in single copy but also in multi-copy plants. So no absolute correlation was found between the numbers of transgene copies and the gene expression. The results of RT-PCR analysis also show that the expression of bt gene at mRNAlevel is different at different developmental stages. Gus gene and npt II was 100%. Bt gene, gus gene and npt II gene was co-expression in six from ten clone.