Glyphosate selection of gene amplification in suspension cultures of 3 plant species

Authors

  • Jack M. Widholm,

    Corresponding author
    1. University of Illinois, Department of Crop Sciences, Edward R. Madigan Laboratory, 1201 W. Gregory Dr., Urbana, IL 61801, USA
      * Corresponding author, e-mail: widholm@uiuc.edu
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  • A. R. Chinnala,

    1. University of Illinois, Department of Crop Sciences, Edward R. Madigan Laboratory, 1201 W. Gregory Dr., Urbana, IL 61801, USA
    2. Present address: Regional Institute of Education, Pushkar Road, Ajmer 3005004, India
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  • Jeom-Ho Ryu,

    1. University of Illinois, Department of Crop Sciences, Edward R. Madigan Laboratory, 1201 W. Gregory Dr., Urbana, IL 61801, USA
    2. Present address: Chonbuk National University, Department of Agronomy, Chonju 560-756, Korea
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  • Hee-Sook Song,

    1. University of Illinois, Department of Crop Sciences, Edward R. Madigan Laboratory, 1201 W. Gregory Dr., Urbana, IL 61801, USA
    2. Present address: BASF Plant Science, P.O. Box 13528, 26 Davis Drive, Research Triangle Park, NC 27709-3528, USA
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  • Therese Eggett,

    1. University of Illinois, Department of Crop Sciences, Edward R. Madigan Laboratory, 1201 W. Gregory Dr., Urbana, IL 61801, USA
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  • Jeffrey E. Brotherton

    1. University of Illinois, Department of Crop Sciences, Edward R. Madigan Laboratory, 1201 W. Gregory Dr., Urbana, IL 61801, USA
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* Corresponding author, e-mail: widholm@uiuc.edu

Abstract

Stepwise selection was carried out with increasing glyphosate concentrations to produce suspension cultures of Medicago sativa L. (alfalfa), Glycine max L. (Merr.) (soybean) and Nicotiana tabacum L. (tobacco) (two lines) that were at least 100-fold more resistant than the original culture as measured by the I50. The selection process required from 8 to 11 transfers to fresh medium over a total period from 161 to 312 days. The alfalfa and soybean lines contained 62- and 21-fold higher activity levels of the glyphosate target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), respectively. The tobacco lines had EPSPS enzyme activity levels more than 800-times higher than the original cultures. The EPSPS gene copy number and mRNA were increased in all of the lines as measured by southern and northern hybridization, respectively. Thus, as has been found before with most glyphosate-resistant suspension cultures, the resistance is caused by high EPSPS enzyme activity due to EPSPS gene amplification. Alfalfa and soybean EPSPS gene amplification and the very high EPSPS enzyme activity increases found in the tobacco cultures have not been reported before. These studies show that EPSPS gene amplification can occur in many plant species to confer glyphosate tolerance.

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