Study On Genetic Transformation Of Populus Tomentosa With RolB Gene And Improving Its Rooting Ability

Populus tomentosa is a very important afforest tree species with fast-growing and high wood quality in the north of China, which is widely used as the woody sources of fiber for the pulp and paper industry, and timber for the architecture and furniture industry. Unlike most poplar species, which does not root easily from hardwood cuttings in the propagation. Normal rooting percentages are very low and it makes high costs in the mass propagation. The difficulty of rooting in hardwood cuttings has seriously hindered the popularization of this excellent tree species by using vegetative propagation technique. Genetically improving the rooting ability of P. tomentosa has attracted many reseachers' attention. Recent biotechnology progress provides us a new approach to improve the rooting capacity of the woody species that are difficult to root by genetic engineering.In this work, high frequency regeneration and transformation systems were established based on study of the factors affecting poplar leaf explants regeneration efficiency and gene transfer efficiency. rolB gene was introduced into P. tomentosa through the improved protocols. The molecular deletion and rooting ability of transgenic plants were also studied. The main research work and results were as follows:1. Expression frame GH3 promoter-rolB gene-nos terminator was isolated from plasmid vector pUC-CH3-rolB-nos digested with EcoRI, which was then cloned into the binary vector pBin19 to construct the plant expression vector pBin-GH3-rolB-nos. The new vector was further transferred into Agrobacterium tumefaciens LBA4404 by freeze-thaw method and used for transformation experiment of poplar.2. Regeneration system for poplar leaf explants was established by orthogonal design, the experiment results showed that the optimal medium compositions for adventitious buds regeneration was MS+6-BA1.0mg/L+ZT0.3mg/L+NAA0.3mg/L. Based on this optimal medium, the regeneration ability of different explant materials from clones, tissue and organs was studied. It was found that plant genotype had significant influence on the regeneration efficiency, among six P. tomentosa clones, the regenerations ability of cloneL2 and L5 is the highest, leaf regeneration frequency of adventitious buds reached 98.3% and 91.7%, respectively. The adventitious bud differentiation rate of different explants types was leaf (95.1%) > stem segment (66.3%)> root segment (61.5%).3. Kanamycin sensitive test showed that the critical kanamycin sensitive concentration for inducing shoots and roots of P. tomentosa was 20mg/L and 25mg/L, respectively. Effects of Cefotaxime (Cef) and Carbenicillin (Cb) on regeneration of adventitious buds and roots from P. tomentosa explants were investigated. The results indicated that regeneration of adventitious buds and roots was suppressed when Cef or Cb concentration was over 400mg/L, and their ability of regeneration would decrease as the antibiotics concentration increased in the culture medium. Cef was more effective than Cb in killing Agrobacteria after transformation and 200~400mg/L was suggested as the suitable concentration to control the propagation of Agrobacteria.4. Several crucial factors influencing the transformation efficiency were studied. It was found that pre-culture of explants for 2 days before infection with A. tumefaciens, cocultivation for 2~4 days after infection would be favorable for the transformation. Cocultivation medium supplemented with 200uMacetosyringone was able to increase the transformation frequency. In this experiment, it was also found that the infection ability of nopaline strain C58 was remarkably higher than that of octopine strain LBA4404.5. Transgenic plantlets with kanamycin resistance were obtained after coculture of leaves with the disarmed strain A. tumefaciens carrying rolB gene. The integration of rolB gene into the poplar genome was identified by PCR and Southern blotting analysis. Leaves, stems and roots from the binary vector pCMB-B:GUS transformed plants were analyzed for gus gene expression. Histochemical GUS assay demonstrated that rolB-GUS gene was mainly expressed in the root tip (meristems) and the vascular tissue of stem.6. Rooting ability was tested on the medium using both intact shoots and stem segments. The results showed that integration of the rolB gene increased auxin sensitivity and rooting ability compared with untransformated control shoots.