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.
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).
RamanaRao
Mangu Venkata
MV
Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India.
Veluthambi
Karuppannan
K
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
D004269:DNA, Bacterial; C036483:T-DNA; D015534:Trans-Activators