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

Abstract

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-SacR genes 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.

Authors:
 [1];  [2];  [2];  [2];  [2];  [3];  [4];  [5];  [2]
  1. Virginia Tech, Blacksburg, VA (United States). Dept. of Crop and Soil Environmental Science; Chinese Academy of Tropical Agricultural Sciences/Key Laboratory of Crop Gene Resources and Germplasm Enhancement in Southern China, Tropical Crops Genetic Resources Institute, Ministry of Agriculture, (China)
  2. Virginia Tech, Blacksburg, VA (United States). Dept. of Horticulture
  3. Virginia Tech, Blacksburg, VA (United States). Dept. of Crop and Soil Environmental Science
  4. Univ. of North Carolina, Chapel Hill, NC (United States). Dept. of Biology
  5. USDA-ARS-Appalachian Fruit Research Station, Kearneysville, West Virginia (United States)
Publication Date:
Research Org.:
Michigan State Univ., East Lansing, MI (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1628188
Grant/Contract Number:  
SC0008338
Resource Type:
Accepted Manuscript
Journal Name:
Frontiers in Molecular Biosciences
Additional Journal Information:
Journal Volume: 3; Journal ID: ISSN 2296-889X
Publisher:
Frontiers Media SA
Country of Publication:
United States
Language:
English
Subject:
Biochemistry & Molecular Biology

Citation Formats

Liu, Yiming, Miao, Jiamin, Traore, Sy, Kong, Danyu, Liu, Yi, Zhang, Xunzhong, Nimchuk, Zachary L., Liu, Zongrang, and Zhao, Bingyu. SacB-SacR Gene Cassette As the Negative Selection Marker to Suppress Agrobacterium Overgrowth in Agrobacterium-Mediated Plant Transformation. United States: N. p., 2016. Web. doi:10.3389/fmolb.2016.00070.
Liu, Yiming, Miao, Jiamin, Traore, Sy, Kong, Danyu, Liu, Yi, Zhang, Xunzhong, Nimchuk, Zachary L., Liu, Zongrang, & Zhao, Bingyu. SacB-SacR Gene Cassette As the Negative Selection Marker to Suppress Agrobacterium Overgrowth in Agrobacterium-Mediated Plant Transformation. United States. doi:10.3389/fmolb.2016.00070.
Liu, Yiming, Miao, Jiamin, Traore, Sy, Kong, Danyu, Liu, Yi, Zhang, Xunzhong, Nimchuk, Zachary L., Liu, Zongrang, and Zhao, Bingyu. Wed . "SacB-SacR Gene Cassette As the Negative Selection Marker to Suppress Agrobacterium Overgrowth in Agrobacterium-Mediated Plant Transformation". United States. doi:10.3389/fmolb.2016.00070. https://www.osti.gov/servlets/purl/1628188.
@article{osti_1628188,
title = {SacB-SacR Gene Cassette As the Negative Selection Marker to Suppress Agrobacterium Overgrowth in Agrobacterium-Mediated Plant Transformation},
author = {Liu, Yiming and Miao, Jiamin and Traore, Sy and Kong, Danyu and Liu, Yi and Zhang, Xunzhong and Nimchuk, Zachary L. and Liu, Zongrang and Zhao, Bingyu},
abstractNote = {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-SacR genes 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.},
doi = {10.3389/fmolb.2016.00070},
journal = {Frontiers in Molecular Biosciences},
number = ,
volume = 3,
place = {United States},
year = {2016},
month = {10}
}

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