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High throughput Agrobacterium tumefaciens-mediated germline transformation of mechanically isolated meristem explants of cotton (Gossypium hirsutum L.)

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Abstract

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Agrobacterium tumefaciens mediates high frequency of germline transformation of cotton meristem explants. The meristem transformation system we developed is rapid, high throughput and genotype-flexible.

Abstract

We have developed a high throughput cotton transformation system based on direct Agrobacterium inoculation of mechanically isolated meristem explants of cotton (Gossypium hirsutum L.). The explants were inoculated with a disarmed A. tumefaciens strain, AB33 harboring a 2 T-DNA binary vector pMON114908. This vector contained a gene of interest, an intron-disrupted β-glucuronidase gene in one T-DNA, and a selectable marker gene, aadA in the other T-DNA. Critical factors, such as method of co-culture, culture temperature during selection, composition of selection medium, and selection scheme were found to influence transformation frequency. The cycle time from initial inoculation to the transplanting of transgenic plants to soil was 7–8 weeks. Stable integration of transgenes and their transmission to progeny were confirmed by molecular and genetic analyses. Transgenes segregated in the expected Mendelian fashion in the T1 generation for most of the transgenic events. It was possible to recover marker-free events in the T1 generation when utilizing a binary vector that contained the selectable marker and gene of interest expression cassettes on independent T-DNAs. The procedure presented here has been used to regenerate thousands of independent transgenic events from multiple varieties with numerous constructs, and we believe it represents a major step forward in cotton transformation technology.

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Acknowledgments

We would like to thank C. Roth for genotype similarity data, M. Amacker and D. Kelm for explant production, K. Lockwood, G. Bean, S. Schroeder for Agrobacterium preparation, R. Byington, D. Warner, M. Irwin for media preparation, T. Emborg, J. Esser, J. Brewer, K. Janssen, L. Frank for molecular characterization, J. Fraederichs, T. Van-Ert, M. Dickens, B. Reel for plant care in greenhouse, E. Williams, and B. Zeng for statistical analyses, D. Duncan for valuable advice and discussion, D. Somers, D. Boyes and C. Armstrong for leadership and critically reading the manuscript.

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Author notes

  1. Xudong Ye & Yuechun Wan

    Present address: Monsanto Company, 700 Chesterfield Parkway, St. Louis, MO, 63017, USA

  2. Miriam Paris

    Present address: Monsanto Company, 800 North Lindbergh Blvd, St Louis, MO, 63167, USA

Authors and Affiliations

  1. Monsanto Company, Agracetus Campus, 8520 University Green, P. O. Box 620999, Middleton, WI, 53562, USA

    Yurong Chen, Anatoly Rivlin, Andrea Lange, Xudong Ye, Zarir Vaghchhipawala, Elizabeth Eisinger, Erik Dersch, Miriam Paris, Brian Martinell & Yuechun Wan

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  1. Yurong Chen
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  2. Anatoly Rivlin
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Correspondence to Yurong Chen.

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Communicated by K. Wang.

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Chen, Y., Rivlin, A., Lange, A. et al. High throughput Agrobacterium tumefaciens-mediated germline transformation of mechanically isolated meristem explants of cotton (Gossypium hirsutum L.). Plant Cell Rep 33, 153–164 (2014). https://doi.org/10.1007/s00299-013-1519-x

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  • DOI: https://doi.org/10.1007/s00299-013-1519-x

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