Genetic Transformation Techniques Of Nicotiana Tabacum And Poplar Mediated By Agrobacterium

To establish an efficient genetic transformation system for Agrobacterium-mediated series deletion fragments of Xylem-specific promoters JCesAP,Tobacco seedlings and triploid aspen tissue culture plantletsas are used as test material.The plant expression vectors,which were with the deletion fragments of the xylem-specific promoter before constructed,were used to convert into tobacco and poplar by Agrobacterium-mediated method.The resistant plants were detected by PCR and report gene activity.The main results are as follows:?1?In the process of genetic transformation of tobacco,it was established that the suitable concentration of bacterial liquid was OD₆₀₀?0.4⁰.6,and the general infection time was 8¹0min,and co-cultivation was appropriate for 2 days.The optimum differentiation medium was MS+2.0mg/L 6-BA+0.1mg/L IBA,the inhibitory concentration of CTX was 50mg/L,the suitable concentration in the screening process of Kana was 20mg/L,and the concentration of rooting of Hyg was 5mg/L.?2?The instant transformation of tobacco was carried out by injection method,GUS staining?pBI121?and GFP green fluorescence detection?pCAMBIA1302?were performed on tobacco leaves,and the results showed that the transgenic plants with different deletion fragments and GUS reported genes could observe blue in different parts.The green fluorescence can be observed in different parts of transgenic plants which carry different missing fragments and GFP reporting gene.This indicates that the missing fragments of the promoter series have promoter activity.Using Agrobacterium tumefaciens-mediated leaf disc transformation method,the pBI121 and pCAMBIA1302 plant expression vectors which fused with promoter deletion fragments were transformed into tobacco.Through PCR detection and analysis of gene activity,the results showed that different target genes had been integrated into 24 transgenic plants with stable expression in tobacco genome,and the conversion rate was 27%.?3?In the process of genetic transformation of Populus tomentosa,it was determined that the best time for infection of Poplar explants was 3min,the most suitable bacterial concentration was OD₆₀₀?0.4,the best differentiation medium MS+2.0 mg/L 6-BA+0.1mg/L IBA was established,and the bacteriostatic concentration of CTX in the screening and rooting process was 50mg/L,the suitable concentration of Kana was 20mg/L.?4?The method of instantaneous transformation was used to analyze the leaf of poplar by GUS staining,the results showed that the transgenic plants with different deletion fragments and GUS report gene could observe blue in the corresponding site,which indicated that the deletion fragments of the promoter series had promoter activity.The genetic transformation of Poplar was carried out by using the pBI121 plant expression vector mediated by Agrobacterium tumefaciens.After PCR molecule-detection and GUS staining,the results showed that the ZU2,ZU3,ZU4,L2 missing fragments had been transferred to Poplar,and the plants obtained by L3 and L4 genes were false-positive plants and did not convert to Poplar.A total of 17 transgenic plants with stable expression were obtained,and the conversion rate reached 53%.