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).

Less is more: strategies to remove marker genes from transgenic plants

“…Negative selection markers are used to optimize transformation efficiency; opposite to positive SMGs, they kill the transformed cells. This positive–negative selection method usually places a negative SMG next to a positive SMG in the construct [42-47]. The positive SMG is used for selection of the T 0 transgenic plants, and the negative SMG is then used to remove plants still harboring the ‘negative SMG’ cassette from the subsequent T 1 segregation population, and greatly reduce the transgenic plants for researchers to screen for the GOI-only transgenic plants (Figure 2).…”

“…Bacterial cytochrome P450 SUI converts non-toxic pro-herbicide R4702 into cyto-toxic herbicide R4702 [50,55,56]. Most recently, a transcriptional activator protein gene ( TrAP ) from Mungbean yellow mosaic virus (MYMV) was also used as a negative SMG in a positive–negative selection system to generate SMG-free tobacco plants [47]. …”

“…With a short selection phase, those cells may survive on the select medium with the transient expression of the SMG. While performing co-transformation using a positive–negative marker selection, Ramana Rao and Veluthambi [47] recovered 4.4% (5/114 plants) of GOI-only, SMG-free, transgenic plants in the T 0 generation with a ‘ transient ’ (2–4 weeks) period of positive selection. Dutt et al used a transient positive-selection method to obtain SMG-free, GOI-only, transgenic grapevine from T 0 generation [46].…”

“…Finally, co-transformation-based SMG removal is typically applied on sexually propagated plants whose genomes undergo recombination and segregation, not to vegetatively propagated plants. However, a recent approach using transient positive selection coupled with long negative selection recovered a few SMG-free GOI-only transgenic plants at the T 0 generation for vegetatively propagated plants, but it is not an efficient system [47]. …”

Co-expression of VpROMT gene from Chinese wild Vitis pseudoreticulata with VpSTS in tobacco plants and its effects on the accumulation of pterostilbene

“…To investigate the synthesis of pterostilbene, the two genes were over-expressed in tobacco by co-transformation. Numerous researches about co-transformation were successfully reported (Daley et al 1998;Sripriya et al 2008;RamanaRao and Veluthambi 2010). The highest co-transformation efficiency was achieved with an equal mixture of the two Agrobacterium strains (Depicker et al 1985;Komari et al 1996).…”