SacB-SacRGene Cassette As the Negative Selection Marker to SuppressAgrobacteriumOvergrowth inAgrobacterium-Mediated Plant Transformation.
Agrobacteriumovergrowth is a common problem inAgrobacterium-mediated plant transformation. To suppress theAgrobacteriumovergrowth, 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 mostAgrobacterium tumefaciensstrains when they are grown on culture medium supplemented with sucrose. Therefore,SacB-SacRgenes can be used as negative selection markers to suppress the overgrowth ofA. tumefaciensin the plant tissue culture process. We generated a mutantA. tumefaciensstrain GV2260 (recA-SacB/R) that has theSacB-SacRcassette inserted into the bacterial genome at therecAgene locus. The mutantAgrobacteriumstrain 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 ofAgrobacteriumand therefore improves the plant transformation efficiency. We employed GV2260 (recA-SacB/R) to generate stable transgenicN. benthamianaplants expressing a CRISPR-Cas9for knocking out a WRKY transcription factor.
Department of Crop and Soil Environmental Science, Virginia TechBlacksburg, VA, USA; 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 AgricultureDanzhou, China.
Yiming
Y
Liu
Department of Horticulture, Virginia Tech Blacksburg, VA, USA.
Jiamin
J
Miao
Department of Horticulture, Virginia Tech Blacksburg, VA, USA.
Sy
S
Traore
Department of Horticulture, Virginia Tech Blacksburg, VA, USA.
Danyu
D
Kong
Department of Horticulture, Virginia Tech Blacksburg, VA, USA.
Yi
Y
Liu
Department of Crop and Soil Environmental Science, Virginia Tech Blacksburg, VA, USA.
Xunzhong
X
Zhang
Department of Biology, University of North Carolina at Chapel Hill Chapel Hill, NC, USA.
Zachary L
ZL
Nimchuk
USDA-ARS-Appalachian Fruit Research Station Kearneysville, WV, USA.
Zongrang
Z
Liu
Department of Horticulture, Virginia Tech Blacksburg, VA, USA.
Bingyu
B
Zhao
Agrobacterium overgrowth; CRISPR-Cas9; Nicotiana benthamiana; SacB-SacR gene cassette; plant transformation