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Chestnut, American (Castanea dentata (Marsh.) Borkh.)

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Agrobacterium Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1224))

Abstract

The key to successful transformation of American chestnut is having the correct combination of explant tissue, selectable markers, a very robust DNA delivery system, and a reliable regeneration system. The most important components of this transformation protocol for American chestnut are the following: starting out with rapidly dividing somatic embryos, treating the embryos gently throughout the Agrobacterium inoculation and cocultivation steps, doing the cocultivation step in desiccation plates, and finally transferring the embryos into temporary-immersion bioreactors for selection. None of these departures from standard Agrobacterium transformation protocols is sufficient by itself to achieve transgenic American chestnut, but each component makes a difference, resulting in a highly robust protocol.

The average transformation efficiency that can be expected using the described protocol is approximately 170 stable embryogenic transformation events per gram of somatic embryo tissue, a considerable improvement over the 20 transformation events per gram we reported in 2006 (Maynard et al. American chestnut (Castanea dentata (Marsh.) Borkh.) Agrobacterium protocols, 2nd ed., 2006). We have regenerated nearly 100 of these events, containing 23 different gene constructs, into whole plants. As of the fall of 2013, we had a total of 1,275 transgenic chestnut trees planted at eight locations in New York State and one in Virginia. Based on a combination of field-trial inoculations, greenhouse small-stem inoculations, and detached-leaf assays, we have identified three transgenes that produce stronger resistance to chestnut blight than non-transgenic American chestnut. Depending on the transgene and the event, this resistance can be either intermediate between American chestnut and Chinese chestnut, approximately equal to or even higher than the resistance naturally found in Chinese chestnut.

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Acknowledgments

Financial support was provided by the Forest Health Initiative, the Monsanto Fund, the American Chestnut Foundation (New York chapter and National), USDA-Biotechnology Risk Assessment Grant program (BRAG), the Consortium for Plant Biotechnology Research (CPBR), and ArborGen LLC.

Photography: Greg Boyd Fig. 1; Linda McGuigan Figs. 2, 3a, b; Andy Newhouse Figs. 3c, d; William Powell Fig. 4.

The following individuals have also contributed biological materials, time, and financial support or, in some other way, have contributed to this publication and to the American Chestnut Research and Restoration Project: Stanley and Arlene Wirsig, Herbert and Jane Darling, Dick Radel, Dale Travis, Bryan Burhans, John Dougherty, Joyce Fry, Mary Lou Rath, Dawn Parks, Maud Hinchee, James Donowick, John Ellis, Scott Merkle, Zizhuo Xing, Sharon and Seth LaPierre, Mike Satchwell, Haiying Liang, Katie Damico, and Kristen Russell.

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Authors and Affiliations

  1. Department of Forest and Natural Resources Management, SUNY College of Environmental Science and Forestry, 1 Forestry Dr., Syracuse, NY, 13210, USA

    Charles A. Maynard Ph.D. & Linda D. McGuigan

  2. Department of Environmental and Forest Biology, SUNY College of Environmental Science and Forestry, 1 Forestry Drive, Syracuse, NY, 13210, USA

    Allison D. Oakes, Andrew E. Newhouse, Lilibeth C. Northern, Kathleen M. Baier & William A. Powell

  3. Therapeutic Proteins International, LLC, Chicago, IL, USA

    Bo Zhang

  4. Horn Performance and Environmental Science, Northwestern University, 633 Clark Street, Evanston, IL, 60208, USA

    Allison M. Chartrand

  5. The American Chestnut Research and Restoration Project, SUNY College of Environmental Science and Forestry, 1 Forestry Drive, Syracuse, NY, 13210, USA

    Logan R. Will

Authors
  1. Charles A. Maynard Ph.D.
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  2. Linda D. McGuigan
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  3. Allison D. Oakes
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  4. Bo Zhang
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  5. Andrew E. Newhouse
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  6. Lilibeth C. Northern
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  7. Allison M. Chartrand
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  8. Logan R. Will
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  9. Kathleen M. Baier
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  10. William A. Powell
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Corresponding author

Correspondence to Charles A. Maynard Ph.D. .

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Editors and Affiliations

  1. Plant Sciences Institute, and Department of Agronomy, Center for Plant Transformation, Iowa State University, Ames, Iowa, USA

    Kan Wang

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Maynard, C.A. et al. (2015). Chestnut, American (Castanea dentata (Marsh.) Borkh.). In: Wang, K. (eds) Agrobacterium Protocols. Methods in Molecular Biology, vol 1224. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-1658-0_13

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  • DOI: https://doi.org/10.1007/978-1-4939-1658-0_13

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  • Publisher Name: Springer, New York, NY

  • Print ISBN: 978-1-4939-1657-3

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