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.
Highlights
Agrobacterium-mediated genetic transformation is one of the most popular techniques used for the generation of transgenic plants (Gelvin, 2000; Tzfira and Citovsky, 2006)
We demonstrated that the mutant strain GV2260 maintains its capacity of transforming plant cells, and its growth can be efficiently inhibited by regular tobacco tissue culture medium supplemented with 3% sucrose
To generate a mutant Agrobacterium strain that is sensitive to sucrose, a suicide vector pLVC18L carrying the SacB-SacR gene cassette was integrated into the recA gene locus in the genome of A. tumefaciens strain GV2260 through marker-exchange mutagenesis
Summary
Agrobacterium-mediated genetic transformation is one of the most popular techniques used for the generation of transgenic plants (Gelvin, 2000; Tzfira and Citovsky, 2006). The infected Agrobacterium cells are eliminated or suppressed by using various antibiotics, and the transgenic plant cells are selected by using antibiotics or other chemicals (Jones et al, 2005; Tsuda et al, 2012). To eliminate or inhibit the Agrobacterium overgrowth, different antibiotics such as carbenicillin, TimentinTM, Augement, Clavamox, and Cefotaxime are used during the plant tissue culture selection steps (Bhau and Wakhlu, 2001; Tereso et al, 2006; Zang et al, 2009; Li and Qu, 2011; Ren et al, 2012). A more reliable and cost-effective method to inhibit the overgrowth of Agrobacterium tumefaciens is highly desirable
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