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 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 T0 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).
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Abbreviations
- BAP:
-
6-Benzylaminopurine
- bar:
-
Bialaphos resistance gene
- GOI:
-
Gene of interest
- gus :
-
β-Glucuronidase gene
- IAA:
-
Indole-3-acetic acid
- MS:
-
Murashige and Skoog
- MYMV:
-
Mungbean yellow mosaic virus
- NAA:
-
1-Naphthaleneacetic acid
- nptII:
-
Neomycin phosphotransferase II gene
- NSM:
-
Negative selectable marker
- PEM:
-
Plant establishment medium
- PPT:
-
Phosphinothricin
- SIM:
-
Shoot induction medium
- SMG:
-
Selectable marker gene
- TrAP :
-
Transcriptional activator protein
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Acknowledgments
We thank Dr. S.B. Gelvin, Purdue University, for providing the Agrobacterium tumefaciens strain EHA105, pGA472 and Nicotiana tabacum L. cv Wisconsin 38 plants, and Dr. Richard A. Jefferson, Canberra, Australia for pCAMBIA3301. M.V. RamanaRao is thankful to the Department of Biotechnology (DBT), Government of India for the Postdoctoral Fellowship granted to him.
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Communicated by H. Ebinuma.
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RamanaRao, M.V., Veluthambi, K. 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. Plant Cell Rep 29, 473–483 (2010). https://doi.org/10.1007/s00299-010-0836-6
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DOI: https://doi.org/10.1007/s00299-010-0836-6