A high efficient Agrobacterium tumefaciens-mediated transformation system for kiwifruit
2003
Li, Ming | Huang, Zhenguang | Han, Lixing | Zhao, Gai-Rong | Li, Yu-Hong | Yao, Jialong
In the past six years, we have developed a high efficient, reproducible Agrobacterium tumefaciens-mediated gene transfer system in kiwifruit. Factors that were important to high frequency of gene transfer included the health of leaf explants, the genotypes of Actinidia, the strain of Agrobacterium, the days of co-cultivation of leaf explants with Agrobacterium, and the leaf explants surface orientation ab- or adaxial to the medium. Placing the leaf explants on moist filter paper and the use of acetosyringone during co-cultivation gave significantly increased frequencies of gene transfer. For the time being, in our experiments gene transfer frequency of leaf explants forming kanamycin-resistant callus were over 30% for Actinidia deliciosa, and over 60% for Actinidia chinensis. Using this optimized transformation system, it was very easy to obtain large transgenic plants of kiwifruit. In the past few years, large kanamycin-resistant plants of the genetically transformed kiwifruit were obtained at very high frequency from the optimized system in the experiments using leaf explants co-cultured with Agrobacterium tumefaciens strain A281, which contained the neomycin phosphotransferase II gene (nptII), and the antisense ACC synthetase and oxidase genes or ipt gene. KanR, PCR and Southern analysis of the regenerated plants confirmed the transgenic nature.
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