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Studies on Transforming Antisense Phospholipase D_γ (PLD_γ) Gene into White Clover

Author: ZhaoZhiWen
Tutor: CuiDeCai
School: Shandong Agricultural University
Course: Biochemistry and Molecular Biology
Keywords: White clover Bud Agrobacterium tumefaciens Phospholipase gene antisense Genetic transformation Transgenic plants Salt Drought
CLC: S541.2
Type: Master's thesis
Year: 2005
Downloads: 50
Quote: 1
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Abstract


White clover (Trifolium repens) is one of important legumes. It is an important forage crop, but also in soil improvement, water conservation, and ecological and environmental protection plays an active role. The research topics of white clover as experimental material, with its buds as explants cultured for regeneration, systematic study of hormone concentrations, light conditions, explant density, culture containers and many other factors influence on shoot regeneration bud, establish buds of white clover regeneration system. On this basis, with phospholipase Dγ carrying antisense gene (PLDγ) Agrobacterium tumefaciens-mediated transformation of white clover bud explants, through the transformation method and conditions of exploration, establishment of white clover genetic transformation system. White clover Genetic Transformation System for the use of genetic engineering for improved varieties of white clover plays an important role. The results show that different hormone concentration ratio of white clover growth, buds, roots, etc. have a major impact. Hormone concentration ratio through different experiments to determine the optimum seedling medium (MS 0.15mg / L 6-BA 0.1mg / L NAA 0.1mg / L KT 20mg / L Ad) leaf regeneration medium (MS 0.5mg / L 6-BA 0.1mg / L NAA), and the rooting medium (1/2 MS 0.1mg / L NAA). pH, agar concentration, temperature, humidity and other physical factors also white clover growth, differentiation, rooting and so have varying degrees of impact. Were carried out bacteriostatic antibiotic cefotaxime (Cef) and selective antibiotic kanamycin (Km) susceptibility testing results show that the 300 mg / L of Cef can effectively inhibit Agrobacterium overgrowth. But the white clover Cef very sensitive, Cef affect white clover growth, differentiation, so I chose 150mg / L is the best bacteriostatic antibiotic concentration. 15mg / L Km induce leaf buds as critical screening concentration. In the establishment of white clover buds bud regeneration system based on antibiotic screening through the steps to create and optimize the Agrobacterium-mediated genetic transformation system of white clover. The general process is as follows: the culture of Agrobacterium to logarithmic phase (OD600 = 0.5) with an equal volume of 5% sucrose solution (containing a certain concentration of the surfactant) solution diluted dip, dip white clover sprouts 8 minutes, the After dip bud explants placed with a sucrose solution containing the above two layers of filter paper in petri dishes in the dark for 1 days and then were cultured in the light for 3 days. The leaves washed 3 times in sterile distilled water, blot filter surface

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CLC: > Agricultural Sciences > Crop > Fodder crops,pasture > Perennial legume > Clover
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