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| Alfalfa (Medicago sativa) Transformation with Salt-tolerant Muti-Gene Mediated by Agrobacterium tumefaciens and Molecular Identification |
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Abstract Abstract The salt overly sensitive (SOS) signal-transduction pathway is a control ion balance signal pathway. This pathway plays a key role in discharge Na+ and ion homeostasis on cellular lever and improves plant salt resistance. It has some realistic significance on enhanced salt tolerance in alfalfa (Medicago sativa), which transformed with muti-gene mediated by Arabidopsis thaliana SOS1, SOS2, SOS3, SOS3-like calcium binding protein 8 (SCaBP8) and bialaphos resistance (Bar) 5 genes of polygenic plant expression vector pSOS. The Agrobacterium-mediated transformation method was used, "Golden Empress" cotyledonary nodes of alfalfa were selected as explants. The best concentration of glufosinate herbicide was identified as 0.6 mg/L, the best concentration of bacteriostat cefotazime was identified as 300 mg/L. 25 independent sources of transformed plants were obtained through screening after the herbicides selection. These plants applied PCR amplification and molecular detection, which showed that there were 6 transformants with the special bands. The positive rate was 24%. Four of positive transgenic plants were randomly selected Southern hybridization identification, and the results showed that there were 3 hybridization signal, and copy number was 1 or 2. The result of RT-PCR analysis demonstrated that the same as expected electrophoretic bands size of Bar gene (463 bp) and SOS1 gene (700 bp) expressed in transgenic plants. It was proved initially that multiple exogenous salt-tolerant genes have been integrated into the transgenic plant genome and expressed at the transcriptional level. After treated with 250 mmol/L NaCl stress, the growth of wild type plants and transgenic plants were inhibited, but the plant height of the transgenic plants were significantly higher than the wild type, the leaf area of the transgenic P1 strain and P2 strain were obviously higher than the control group, the fresh weight of 3 strains were significantly higher than controls, leave chlorophyⅡ content of the transgenic P2 strain and P4 strain were apparently higher than the control group, which demonstrated overexpression of SOS pathway genes could improve the salt tolerance in transgenic alfalfa. The successful construct of transgenic alfalfa provides theoretical basis for selection for saline-alkali soil planting the salt-tolerant alfalfa varieties and alfalfa salt tolerance identification and salt tolerance mechanism research.
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Received: 24 October 2016
Published: 20 July 2017
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