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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

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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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  1. Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai, 625 021, Tamil Nadu, India

    Mangu Venkata RamanaRao & Karuppannan Veluthambi

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  1. Mangu Venkata RamanaRao
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Correspondence to Karuppannan Veluthambi.

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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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