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Use of 4-methylindole or 7-methyl-DL-tryptophan in a transformant selection system based on the feedback-insensitive anthranilate synthase α-subunit of tobacco (ASA2)

  • Genetic Transformation and Hybridization
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Abstract

Effective selectable markers are needed for basic research and commercial applications that do not involve antibiotic or herbicide resistance. A novel selection system based on a feedback-insensitive anthranilate synthase α-subunit of tobacco (ASA2) as selectable marker using either 4-methylindole (4MI) or 7-methyl-DL-tryptophan (7MT) as the selection agent was developed. We found that these two components were able to discriminate better between ASA2 expressing and untransformed lines than the most commonly used analog 5-methyltryptopan (5MT) in the seedling growth inhibition test. We successfully integrated an expression cassette containing an ASA2 cDNA driven by a cauliflower mosaic virus 35S promoter into tobacco leaf discs by A. tumefaciens and selected transgenic plants on medium supplemented with 300 μM of 7MT or 4MI. Due to the expression of the feedback-insensitive ASA2, the transgenic lines produced showed higher free tryptophan (Trp) concentrations than the untransformed WT control. These results demonstrate the feasibility of the selection system with the ASA2 gene in combination with the use of Trp or indole analogs as selective agent.

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Abbreviations

AS:

Anthranilate synthase

BA:

6-Benzyladenine

NAA:

Naphthalene acetic acid

Trp:

Tryptophan

7MT:

7-Methyl-DL-tryptophan

4MI:

4-Methylindole

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Acknowledgments

The authors thank Wei Q. Zhong and Guirong Zhang for their technical assistance and Dr. Anatoliy V. Lygin for tryptophan content measurement. This research was supported in part through The Consortium for Plant Biotechnology Research, Inc., by DOE Prime Agreement No.GO12026–175. This support does not constitute an endorsement by DOE or The Consortium for Plant Biotechnology Research, Inc., of the views expressed in this publication. This study was also supported in part by funds from the Illinois Agricultural Experiment Station, the Biotechnology Research and Development Corp. and Dow AgroSciences LLC.

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  1. Department of Crop Sciences, Edward R. Madigan Laboratory, University of Illinois, 1201 W Gregory Drive, Urbana, IL, 61801, USA

    P. Barone & Jack M. Widholm

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  1. P. Barone
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  2. Jack M. Widholm
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Correspondence to Jack M. Widholm.

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Communicated by A. Altman.

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Barone, P., Widholm, J.M. Use of 4-methylindole or 7-methyl-DL-tryptophan in a transformant selection system based on the feedback-insensitive anthranilate synthase α-subunit of tobacco (ASA2). Plant Cell Rep 27, 509–517 (2008). https://doi.org/10.1007/s00299-007-0480-y

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  • DOI: https://doi.org/10.1007/s00299-007-0480-y

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