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High-efficiency phosphinothricin-based selection for alfalfa transformation

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Abstract

Despite the significant advantages of using herbicide resistance for selection of genetically engineered plants, alfalfa transformation has relied primarily on selection for antibiotic resistance. In the few studies reporting the use of resistance to the herbicide phosphinothricin (PPT), transformation efficiencies were low. The present investigation describes a PPT-based selection system for alfalfa transformation that uses the phosphinothricin acetyl-transferase (pat) gene as a selectable marker and 5.0 mg l−1 of bialaphos as the selective agent. The method achieves transformation efficiencies, measured as the percentage of explants giving rise to one or more transformed plantlets, greater than 50%. These plantlets accumulated detectable amounts of PAT at levels varying from 2 to 1367 pg μg−1 total protein. Transformed plants transferred to soil in the greenhouse were phenotypically normal and exhibited resistance to bialaphos leaf painting at 5 g l−1 and applications of PPT equivalent to field-level use (0.5 kg ha−1).

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Abbreviations

CsVMV:

Cassava vein mosaic virus

GA:

Ammonium glufosinate

GS:

Glutamine synthetase

PAT:

Phosphinothricin acetyl-transferase

PPT:

Phosphinothricin

SAS:

SAS/STAT™ statistical software

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Acknowledgements

The technical assistance of Penny Slack and Michelle Daigneault, and the continued encouragement and guidance of our collaborators Drs. Patricia Shewen and Reggie Lo, are gratefully acknowledged. We thank Drs. S. Austin-Phillips and D. Samac for sharing materials and information. We are also appreciative of the significant editorial contributions of an anonymous reviewer. Financial support was provided by Dow AgroSciences Canada Inc, the National Science and Engineering Research Council of Canada, and the Ontario Ministry of Agriculture, Food and Rural Affairs. This work was in partial fulfillment of requirements for the M.Sc. degree of AM.

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Author notes

  1. Andrew Montague

    Present address: Canadian Food Inspection Agency, 59 Camelot Drive, Ottawa, ON, Canada, K1A 0Y9

Authors and Affiliations

  1. Department of Plant Agriculture, University of Guelph, Guelph, ON, Canada, N1G 2W1

    Andrew Montague, Asma Ziauddin, Raymond Lee & Judith Strommer

  2. Dow AgroSciences, 9330 Zionsville Road, Indianapolis, IN, 46268, USA

    W. Michael Ainley

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  1. Andrew Montague
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  2. Asma Ziauddin
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  3. Raymond Lee
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  4. W. Michael Ainley
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  5. Judith Strommer
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Correspondence to Judith Strommer.

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Montague, A., Ziauddin, A., Lee, R. et al. High-efficiency phosphinothricin-based selection for alfalfa transformation. Plant Cell Tiss Organ Cult 91, 29–36 (2007). https://doi.org/10.1007/s11240-007-9274-8

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  • DOI: https://doi.org/10.1007/s11240-007-9274-8

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