Transformation of Alfalfa (Medicago Sativa L.) by Agrobacterium ...

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Cancer Research

[Abstract]

Abstract: Adverse environmental stresses have bad effects on growth of plant, so it is very important to study improving alfalfa for tolerance to abiotic stesses. Cultivated alfalfa(Medicago sativa L.), perennial legume crop, is wildly planted in the world and is one of the most frequently studied species. The development of plant gene engineering creates an important approach for alfalfa stress-resistant breeding. In this research, to increase superoxide-scavenging capacity and thereby to improve stresses tolerance of transgenic alfalfa (M. sativa L.) plant, the stress-resistant gene Dscbr from Dunaliella salina, encoding one of early light-induced proteins, was transferred to alfalfa by Agrobacterium-mediated transformation.First, we established the regeneration system of somatic embryogenesis for alfalfa genetic transformation. The cultivar Zhongmu No.1 with high regenerability was selected as the plant material. Callus was induced from leaf-derived explant on UM medium containing 2.0 mg L-1 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.25 mg L-1 KT. We achieved regenerated plants via somatic embryogenesis, embryos germination and roots development. Second, to enhance transformation frequency, we optimized manipulation system with Km-resistant calli assays and the histological detection of GUS activity, which GUS gene comes from plant expression vector pCAMBIA2301G. After pro-cultivated for 4-5 days, the wounded leaves were immersed in the bacterial suspension (OD600=0.3–0.8) for 20 minutes and then co-cultivated on UM medium covered with a piece of sterile filter paper for 7 days before washing. Third, the expression vector pCAMBIA2301G containing the Dscbr gene was introduced into Agrobacterium tumefaciens EHA105 by freeze-thaw method. After 120 days, we obtained 52 regenerated kanamycin-resistant plants with 15 mg L-1 kanamycin selective reagent. Finally, the putative transgenic plants were screened by PCR and RT-PCR analysis. A rapid-trifle method for alfalfa total DNA extraction was <WP=10>developed in this study. PCR analysis showed that 7.7% tested plants, i.e. four transgenic plants, produced the target band. RT-PCR analysis showed that in one individual plant, Dscbr cDNA was transcribed into its mRNA. This project established some methods for using trangene technique to increase stress-tolerance of alfalfa.

Title: Transformation of Alfalfa (Medicago Sativa L.) by Agrobacterium-mediated Process with Dscbr Gene from Dunaliella Salina

Category: Cancer Cell

Filename: Transformation of Alfalfa (Medicago Sativa L.) by Agrobacterium-mediated Process with Dscbr Gene from Dunaliella Salina.pdf

Pages: 168

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