Transformation Of Ri Plasmid Transformation Plants Of Triploid Populus Tomentosa By RolB And RolC Gene

Ri plasmid contain rol genes that can induce formation of adventitious roots, transformation of Ri plasmid or each or several rol genes to plants improved rooting ability of plants. However, some plants exhibited a series of undesirable variations, including plant dwarf, leaves wrinkle, internodes shorten, branches increase, which influenced plant growth ang development significantly. In this work, for improving these undesirable variations, high frequency regeneration and transformation systems of Ri plasmid 30148 strain transgenic plants of triploid Populus tomentosa that worked by Liu xingju were established based on study of the factors affecting poplar leaf explants regeneration efficiency and gene transfer efficiency, rolB and rolC gene were introduced into Ri plasmid transgenic plants of triploid Populus tomentosa. The shape characteristic, PCR detection and histochemical GUS assay of transgenic plants were researched, shape and Growth variation and leaf anatomic structure were also studied. The main research work and results were as follows:1. Regeneration system for Ri plasmid transformed triploid P. tomentosa leaf explants was established, the experiment results showed that the suitable medium compositions for adventitious buds regeneration was MS+6-BA1.0mg.L^-1L+NAA0.05mg.L^-1 and suitable medium compositions for adventitious roots regeneration was MS+NAA0.05mg.L^-1. Based on this suitable medium, the effect of dark cultivation on induction of adventitious buds from leaves explant was studied. It was found that dark cultivation for 5 days would be favorable for induction of adventitious buds.2. Kanamycin sensitive test showed that the critical kanamycin sensitive concentration for inducing shoots and roots of Ri plasmid transgenic plants of triploid P. tomentosa was 30mg.L^-1. Effects of Cefotaxime(CTX) on induction of adventitious buds from Ri plasmid transgenic plants of triploid P. tomentosa explants and inhibition of Agrobacteria were investigated. The results indicated that 400mg.L^-1 was a suitable concentration to control the propagation of Agrobacteria.3. Several crucial factors influencing the transformation efficiency were studied. It was found that infection of leaf explants for 8min with A. tumefaciens and cocultivation for 2 days after infection would be favorable for the transformation.4. Transgenic plantlets with kanamycin resistance were obtained after respective cocultivation of leaves with both of the A. tumefaciens carrying pCMB-B: GUS and pPCV002-CaMVC and selection cultivation on medium contained Km 50mg.L^-1. The integration of rolB and rolC gene into the poplar genome was identified by shape characteristic and PCR analysis. Leaves, stems and roots from part of 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 and vein of leaves.5. Shape and growth variation of transgenic plantlets were also studied. The result indicated that most of transgenic plantlets with rolB gene exhibited dwarf plant, increased leaf area, increased branch, shortened internodes, deereased rooting rate, root number and growth of stem length and most of transgenic plantlets with rolC gene exhibited horizoned leaf, increased branch, shortened intemodes, increased roofing rate, root number and growth of stem length compared with contrast.6. Leaf anatomic structure and tissue thickness of transgenic plantlets were observed. The result indicated that anatomic structure of transgenic plantlets had not significant difference compared with Ri plasmid transgenic plants. Leaf thickness and mesophyll thickness of most transgenic plants with rolB gene had significant difference compared with Ri plasmid transgenic plants, thickness of palisad tissue and spongy tissue had not significant difference compared with Ri plasmid transgenic plants, thickness of epidermis of 50 percent of transgenic plants with rolB gene had significant difference compared with Ri plasmid transgenic plants. Leaf thickness and mesophyll thickness of most transgenic plants with rolC gene had not significant difference compared with Ri plasmid transgenic plants, thickness of palisad tissue of different clones had different extent variation and thickness of spongy tissue of 75 percent of transgenic plants with rolC gene had significant difference compared with Ri plasmid transgenic plants, thickness of epidermis had not significant difference compared with Ri plasmid transgenic plants.