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The Study on Transformation of Dual Genes Lyz-GFP in Medicago Sativa

Author: HanBin
Tutor: MaZuoLing
School: Gansu Agricultural University
Course: Grassland
Keywords: Alfalfa Regeneration System Lysozyme Lyz gene Green fluorescent protein GFP gene Agrobacterium-mediated
CLC: S541.9
Type: Master's thesis
Year: 2010
Downloads: 40
Quote: 1
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Abstract

Alfalfa (Medicago sativa L.) is a leguminous perennial herb, is one of the world's most important forage crops, alfalfa production practices often encounter some pathogenic microorganisms infect and cause alfalfa diseases, diseases caused by severe alfalfa cut and devastating injury, a great deal of production and economic losses. Resistance gene into alfalfa cultivars in the short term, you can directly improved disease resistance of alfalfa. In this thesis, to Gannon Medicago Varia and Gannon alfalfa as the tested materials, a number of influencing factors in the regeneration process, to explore and master the regeneration conditions, identified two kinds of alfalfa plant regeneration good medium; while taking advantage of Agrobacterium-mediated resistance genes marked by green fluorescent protein (GFP) gene - lysozyme (LYZ) gene into the two varieties of alfalfa, systematic study of gene conversion conditions, the establishment of appropriate conversion system, and further by fluorescence microscopy and PCR analysis confirmed Lyz-GFP gene has been successfully transferred to the two kinds of alfalfa. The main contents and results are as follows: 1. Establishment of Regeneration System. To Gannon and Gannon alfalfa tested materials, callus induction, differentiation and rooting research. The results showed that: the hypocotyl Medicago Varia, No. 1 Gannon Gannon alfalfa regeneration explants; optimal induction medium was MS 2 mg / L 2,4-D 0.5 mg / L kt 2.5% sucrose, 0.7% agar, induction time of 30 d; the best bud differentiation medium was MS 0.3 mg / L NAA 0.7% agar 2.0% sucrose; Best rooting medium MS 0.3 mg / L NAA 0.7% 2.0% sucrose agar. Gannon callus induction and differentiation rates were 100% and 28%, Gannon best callus induction and differentiation rates were 100% and 25%. Agrobacterium-mediated genetic transformation and the establishment of the transformation system. Cultured for 7 to 10 days of two alfalfa varieties vaccine hypocotyl explants with Agrobacterium strain as Mediator, Lyz-GFP gene conversion, optimize gene transformation system: Kan screening concentration of 75 mg / L, screening methods for to delay selection; the inhibitory hormone choose CEF better, a concentration of 300 mg / L; explants pre-cultured and co-cultured with 3 d; used to transform invasion dye method and time shaker shake 10 min, the suitable bacterial concentration of OD600 = 0.4 to 0.5. The final four Gannon resistant seedlings and 6 Gannon resistant seedlings, 2 Gannon 1 and 4 Gannon 3 alfalfa expression of green fluorescent conversion rates were 50% and 67%, respectively. 3 transformed callus fluorescence detection and PCR identification. Will get transformed callus and plant fluorescence detection and PCR amplification. By fluorescence detection, Gannon 1, 2 and 4, Gannon alfalfa are the expression of green fluorescence. PCR identification results show that Gannon Medicago Varia and Gannon 3 alfalfa alfalfa callus fragment size of 750 bp bands, the double test results prove the exogenous gene LYZ-GFP has been successfully transferred to Gan agricultural Varia alfalfa and alfalfa callus.

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