The Optmization Of Regeneration Of Betula Platyphalla And The Transformation Of 4CL,UGPA,UGPase To Improve Its Quality

In order to improve the wood qualities of the birch, Betula platyphylla for paper-making,UDP-glucose pyrophosrylase (UDPase),a key enzyme producing UDP-glucose in the synthesis of sucrose and cellulose and 4-coumarate: CoA ligase ,a key enzyme of phenylpropanoid metabolism related to lignin forming, were transfer into the birth by bioengineering technology and were expected to increase the cellulose content and decrease the lignin content in the wood of the birch in this study.To establish the high-frequency regeneration system of the birch leaf, the factors such as the combination and concentration of different hormones and the types of the basic medium were studied. The regeneration condition was also optimized.1. Murashige and Skoog medium supplemented with 3%(w/v)sucrose,0.58% agar, 5.0 mg/l BA . 0.5-1.0 mg/l KT or with the plant homones 3.0-5.0 mg/l BA, 0.5mg/l NAA can give the highest bud-regenerating rate, over 90%. The average number of differentiated buds was at least six per leaf.2. The optimum medium for the root generation of birch seedlings was 1/2 MS medium supplemented with 2%(w/v)sucrose, 0.58%agar, 0.3-0.5 mg/l IBA or 0.2 mg/l NAA and the corresponding root-generating rate was up to 80%.3. The optimal subculture medium,Nagata and Takebe medium contained 2% (w/v)sucrose.0.58% agar , 0.2mg/l BA, 0.1 mg/l IAA was determined and the corresponding propagation coefficient was 5-8 per plantlet.To test the transformation system, the genes of UGPA and 4CL from Amorpha Fruticosa and UGPase from Acetobacter Xylinum were transferred into the molecular biological model plant, tobacco (Nictiana tabacum 89) by trans-infection with the Agrobacierium tumefaciens strain LBA4404. The target genes integrated into the genomic DNA of the birch were detected and confirmed by PCR and Southern Blotting analysis,ect.The selection condition of the transformation system of the birch was determined. 7mg/L of hygromycin and 10mg/L of kanamycin were used in the bud regeneration medium to select the resistant buds. 400mg/L of cefotaxine was used to kill the Agrobacteria after infection. Transformation rate can be increased by 3 times when 100 u mol/L of acetosyringonewas added into the bud-regenerating medium. The finaltransformation rate was 6.5% by selected with the last concentration of kanamycin50mg/l, hygromycin 15mg/l.