| Poplar(Populus) not only is an important economic tree species, but also has important use in maintenance of natural environment and balance of ecosystem because of wide distributing and good adaptability. Poplar has many advantages, such as short growth periods , smaller genome, easier to propagate and operate and the natural host of Agrobacterium tumefaciens , so it is the model plants for the studies of tree molecular biology and forest genetic engineering .Weeds seriously affect the quality and quantity of poplar in the cultivating of seedlings. Herbicide resistance Bar gene is the widely used as target gene and selective marker gene. The establishment on poplar regeneration and genetic transformation of herbicide resistance Bar gene offers the foundation in the theory and technology for the process of industrialization breeding and poplar gene engineering. In the experiment, the excellent species, Poplar 84K(P. alba×P. glandulosa), was used to study the effect of several factors on the establishment of poplar 84K regeneration system, which established the steady, repetitive, easier to propagate, high transformation frequency, plant transformation passive system and offered the theory foundation about improving regeneration frequency and transformation frequency. The constructed expression vector was transferred into Agrobacterium tumefaciens to construct project bacterium. On the base of it, genetic transformation system by Agrobacterium tumefaciens was studied, which made the foundation for cultivating herbicide resistant poplar, and offered research material and academic foundation about the molecular biology matters such as genetic transformation, gene construction and expression of poplar and forest plants. It had important effect on academic and realistic. The main results were as following: 1. The multiplication of axillary shoots was directly established on the paper. The best explantlet was tender stem for one year , and the best medium of axillary shoots induction was MS+6-BA 2.0mg/L+ NAA0.5mg/L. It was the prerequisite work for obtaining test-tube plants and adventitious buds, and offered the foundation of keeping the excellent genetic character of poplar 84K. 2. The regeneration system of adventitious buds direct differentiation from lamina without callus was established. In the course of buds differentiation from lamina directly, the best explant was what come from the test-tube plants under subculture 3 times, the best treatment was light/dark14/10h/d without dark culture before it, and the best medium of adventitious buds differentiation from lamina on first time wasMS+6-BA1.0mg/L+NAA0.5mg/L, when the buds grew to 1cm, the adventitious buds were transferred to MS+KT1.0mg/L+NAA0.1mg/L was the best medium of bud. It offered an favourable plant transformation passive system and a steady foundation of genetic transformation. 3. In the research of rootage abducting, the 1/2MS halved in mass element concentration , 7g/L agro concentration , 20g/L sucrose concentration were the best rootage concentration of poplar 84K. 1/2MS without any hormones and 1/2MS+NAA0.2mg/L+ IBA0.5mg/L was the best medium of rooting, in which the rootage rate and survival rate were the highest. It offered the base of rootage in regeneration of transgenic plants. 4. The constructed expression vector pBI121-Bar was transferred into Agrobacterium tumefaciens LBA4404 to construct project bacterium by triparental mating, which was the prerequisite work for gene transformation by Agrobacterium tumefaciens. 5. In the course of gene transformation by Agrobacterium tumefaciens method, the best bacteria inhibitor, selective reagent were confirmed. Effective concentration of cef restrain Agrobacterium tumefaciens was 400mg/L .The proper concentration was 2mg/L PPT in screening resistant bud after transformation and 8mg/L PPT in screening rooting from resistant bud. 6. Several factors affecting transformation of poplar 84K by Agrobacterium tumefaciens method were studied, and a best pathway was established. At first, the leaf di... |
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