title: Selectable marker elimination in the T0 generation by Agrobacterium-mediated co-transformation involving Mungbean yellow mosaic virus TrAP as a non-conditional negative selectable marker and bar for transient positive selection.
abstract: Transient selection involving the bar gene and non-conditional negative selection against stable T-DNA integration through the use of the Mungbean yellow mosaic virus (MYMV) transcriptional activator protein gene (TrAP) were used in a novel co-transformation strategy to generate selectable marker gene (SMG)-eliminated transgenic tobacco plants in the T(0) generation itself. Two compatible binary plasmids, pCam-bar-TrAP-gus harbouring bar as an SMG and the MYMV TrAP gene as a non-conditional negative selectable marker, and pGA472 with the nptII gene as an unselected experimental gene of interest (GOI) were placed in the Agrobacterium tumefaciens strain EHA105 and used for co-transformation. Transient selection with 5 mg l(-1) phosphinothricin (PPT) for 2-4 weeks and subsequent establishment in a PPT-minus medium yielded 114 plants from 200 leaf discs. The unselected nptII gene was detected by Southern blot analysis in 13 plants, revealing a co-transformation efficiency of 11.5%. Five of these plants harboured only the nptII gene (GOI) and not the bar gene (SMG). Thus, SMG elimination was achieved in the T(0) generation itself in 4.4% (5/114) of plants, which were transiently selected for 2-4 weeks on PPT. MYMV TrAP, a non-conditional negative selectable marker, effectively reduced the recovery of plants with stable integration of the SMG (bar).
authors.affiliation: Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India.
authors.forename: Mangu Venkata
authors.initials: MV
authors.lastname: RamanaRao
authors.forename: Karuppannan
authors.initials: K
authors.lastname: Veluthambi
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D015139:Blotting, Southern; D004269:DNA, Bacterial; D018506:Gene Expression Regulation, Plant; D005814:Genes, Viral; D005818:Genetic Engineering / Q000379:methods*; D009029:Mosaic Viruses / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics; D014026:Nicotiana / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D004269:DNA, Bacterial; C036483:T-DNA; D015534:Trans-Activators