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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.

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