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| Optimizing of Agrobacterium tumefaciens-Mediated Genetic Transformation Sytem in Fusarium pseudograminearum |
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Abstract Crown rot caused by Fusarium pseudograminearum is one of the main devastating diseases in wheat (Triticum aestivum) production. Studying the interaction mechanism between F. pseudograminearum and wheat varieties is the most economical and effective measures to control of wheat crown rot. The objectives of this study are to optimize the Agrobacterium tumefaciens-mediated transformation (ATMT) technology system of F. pseudograminearum, and obtain the transformant strains of F. pseudograminearum (WZ-8A) successfully transformed with with the green fluorescent protein (GFP). Firstly, the transformation conditions were optimized, and then the GFP gene was transformed into the conidia of F. pseudograminearum strain WZ-8A using the A. tumefaciens strain carrying plasmid pCAM-GFP-Hyg. After transformation, transformants randomly collected were screened and identified through the analysis of the hygromycin B (hyg) resistance, as well as using the fluorescence microscopy techniques. A pathogenicity detection was subsequently conducted. The results showed that a satisfactory transformation with a efficiency of 42 transformants per 1×106 spores could be achieved in a sytem of 1×106 spores per milliliter of F. pseudograminearum spore suspension which were co-cultured with Agrobacterium cells under the culture in the presence of co-culture medium containing CaCl2 at 2.6×10-2 g/L at 28 ℃ for 2 d. The transformants was stable when grown on hygromycin B-free PDA medium for 5 generations. The transformants were found to be hyg-positive by PCR amplification and Southern blot analysis, and to be GFP-positive through the detection using fluorescence microscopy, compared with the wild-type strain of WZ-8A. The subsequent test showed that the transformants really did not lose the pathogenicity. It thus be concluded that the T-DNA bearing GFP gene is successfully inserted into the genome of F. pseudograminearum with the optimized system mediated by A. tumefaciens. The optimized genetic transformation sysytem mediated by A. tumefaciens in F. pseudograminearum could be used for the studies on the pathogenic mechanisms as well as the resistance mechanisms in wheat cultivars.
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Received: 24 January 2017
Published: 01 November 2017
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