Selectable marker elimination in the T0 generation by Agrobacterium-mediated co-transformation involving Mungbean yellow mosaic virusTrAP 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 T0 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 5mgl−1 phosphinothricin (PPT) for 2–4weeks 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 T0 generation itself in 4.4% (5/114) of plants, which were transiently selected for 2–4weeks on PPT. MYMV TrAP, a non-conditional negative selectable marker, effectively reduced the recovery of plants with stable integration of the
SMG (bar).

KeywordsCompatible binary vectors-Co-transformation-Marker-free transgenic plants-Non-conditional negative selectable marker-Transient selection