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SacB-SacR Gene Cassette As the Negative Selection Marker to Suppress Agrobacterium Overgrowth in Agrobacterium-Mediated Plant Transformation

文献类型： 外文期刊

作者： Liu, Yiming 1 ; Miao, Jiamin 3 ; Traore, Sy 3 ; Kong, Danyu 3 ; Liu, Yi 3 ; Zhang, Xunzhong 1 ; Nimchu, Zachary L. 4 ; Li 1 ;

作者机构： 1.Vrginia Tech, Dept Crop & Soil Environm Sci, Blacksburg, VA USA

2.Chinese Acad Trop Agr Sci, Key Lab Crop Gene Resources & Germplasm Enhanceme, Trop Crops Genet Resources Inst, Minist Agr, Danzhou, Peoples R China

3.Virginia Tech, Dept Hort, Blacksburg, VA 24061 USA

4.Univ N Carolina, Dept Biol, Chapel Hill, NC 27515 USA

5.USDA ARS Appalachian Fruit Res Stn, Keameysville, WV USA

关键词： plant transformation; Agrobacterium overgrowth; CRISPR-Cas9; Nicotiana benthamiana; SacB-SacR gene cassette

期刊名称：FRONTIERS IN MOLECULAR BIOSCIENCES （ 影响因子：5.246； 五年影响因子：5.389 ）

ISSN：

年卷期： 2016 年 3 卷

页码：

收录情况： SCI

摘要： Agrobacterium overgrowth is a common problem in Agrobacterium-mediated plant transformation. To suppress the Agrobacterium overgrowth, various antibiotics have been used during plant tissue culture steps. The antibiotics are expensive and may adversely affect plant cell differentiation and reduce plant transformation efficiency. The SacB-SacR proteins are toxic to most Agrobacterium tumefaciens strains when they are grown on culture medium supplemented with sucrose. Therefore, SacB-SacRgenes can be used as negative selection markers to suppress the overgrowth of A. tumefaciens in the plant tissue culture process. We generated a mutant A. tumefaciens strain GV2260 (recA-SacB/R) that has the SacB-SacR cassette inserted into the bacterial genome at the recA gene locus. The mutant Agrobacterium strain is sensitive to sucrose but maintains its ability to transform plant cells in both transient and stable transformation assays. We demonstrated that the mutant strain GV2260 (recA-SacB/R) can be inhibited by sucrose that reduces the overgrowth of Agrobacterium and therefore improves the plant transformation efficiency. We employed GV2260 (recA-SacB/R) to generate stable transgenic N. benthamiana plants expressing a CRISPR-Cas9 for knocking out a WRKY transcription factor.

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