Studies On Improving Rooting Ability And Apical Dominance Of Plant By Transforming Two Genes

Low rooting ability influenced the popularization and application of trees seriously especially some precious. Such as Populus euphratica living in northwest desert areas, Populus alba growing in high and cold area of Tibet grew, Populus tomentosa and populus hopeiensis planted in the North China Plain etc.Genetic engineering offered an important means for improving the rooting ability of the plant. More researches on improving rooting ability by the Ri plasmid of Agrobacterium rhizogenes have been taken at present. But the trans-genetic plants demonstrate serious phenotype variation at the same time. For example apical dominance is weakened, shortening, male sterility, early-maturing, etc. Studies indicated that Ri plasmid contain four rol genes at least. The expression of rolB gene of Ri plasmid can induce large amount of hair rooting alone and the trans-genetic plants with rolB gene have certain weakened apical dominance variation.The gibberellins are the important regulation hormone during plant development and growth. Promoting the apical dominance of plant is the main physiology function of gibberellins. With more and more gibberellins mutants discovered, it has been confirmed in the past ten years that the GA20-oxidize was one of the main feedback regulate center on the gibberellins biosynthesis pathway.In this dissertation plant binary vector containing both rolB and pttGA20ox genes was constructed for the first time. The gene transformation receptor system of the new tobacco genotype W38 was optimized and the experiment of antibiotic sensitiveness to it has been carried on. On the basis of constructed rolB and pttGA20ox gene plant vector by restriction endonuclease and PCR method with high fidelity DNA polymerase of pfu and pyrobest, the binary vector was constructed with both that two genes. The gene transformation of rolB and pttGA20ox with tobacco leaf dishes were conducted by Agrobacterium LBA4404. The transgenic tobacco was identified by molecular biology method such as antibiotics PCR and PCR-Southern blot. And also the rooting ability, apical dominance and phenotype variation of the three trans-genetic tobaccos were analysed in vitro. The feasibility to improving rooting ability and topical dominance with both rolB and pttGA20ox genes at the same time was proved by transforming mode plant tobacco.The result shows that: The leaves were the most suitable receptor of W38 tobacco forgene transformation; The selected bud differentiation culture media MS +2.0mg/L 6-BA+1.0 mg/L NAA was the most effective one for buds differentiation from leaves of tobacco and the average regeneration bud number from the leaves reaches 15.7; The density of the antibiotic for selecting the resisting tobacco bud is best with Kanamycim 75-100 mg/L or G418 50- 80 mg/L; The optical OD value of Agrobacterium density for the most effective tobacco gene transformation was 0.5; Adding AS (Acetosyringone) of 200 umol/L in bud differentiation can make the conversion ratio of the tobacco more than 20%; The 4-7 days postpone on selection of the transgenic bud with Kanamycim antibiotic helps to reduce the appearance of the false positive shoot.The statistics of the three kinds of transgenic tobacco show that: The tobacco transformed with rolB single gene or rolB and pttGA20ox double genes taking root were 3-5 days ahead of the contrasted tobacco and the transformed two genes tobacco and the quantity and length of the rooting were obviously strengthened; The growth state of the tobacco transformed with pttGA20ox single gene or rolB and pttGA20ox double genes were obviously higher than contrasted and transformed tobacco with rolB single gene and the difference state has reached the extremely remarkable level; The phenotype of the apical dominance and root ability of the tobacco transferred with both rolB and pttGA20ox two genes possess the character of the tobacco transferred with individual rolB and pttGA20ox gene at the same time.The experimental result proved: Improving the rooting ability and apical dominance with rolB and...