Plant Cell, Tissue and Organ Culture

, Volume 84, Issue 1, pp 69–79 | Cite as

Agrobacterium-mediated transformation of American chestnut (Castanea dentata (Marsh.) Borkh.) somatic embryos

  • Linda D. Polin
  • Haiying Liang
  • Ronald E. Rothrock
  • Mutsumi Nishii
  • Deborah L. Diehl
  • Andrew E. Newhouse
  • C. Joseph Nairn
  • William A. Powell
  • Charles A. MaynardEmail author


These studies were designed to test if a binary vector containing the gfp, bar and oxalate oxidase genes could transform American chestnut somatic embryos; to see if a desiccation treatment during co-cultivation would affect the transformation frequency of different American chestnut somatic embryo clones; to explore the effects of more rapid desiccation; and to see if the antibiotics used to kill the Agrobacterium were interfering with the regeneration of the somatic embryos. Two days of gradual desiccation was found to significantly enhance transient GFP expression frequency. When this treatment was tested on six American chestnut clones, five were transformed and four of these remained embryogenic. Transformation was confirmed by Southern hybridization. Phenotypically normal transgenic shoots were regenerated and rooted. Vascular tissue specific expression of the oxalate oxidase gene was detected in one transgenic line. Carbenicillin, cefotaxime, and tricarcillin were found to not interfere with the regeneration of transformed embryos.

Key words:

bar clonal variation desiccation gfp oxalate oxidase regeneration 



2,4-dichlorophenoxyacetic acid




bialaphos-resistance gene






modified green fluorescent protein gene


oxalate oxidase


polymerase chain reaction




sodium dodecyl sulfate


sodium chloride sodium citrate






McCown’s woody plant medium salts


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The authors thank Joyce Fry and John Dougherty for their technical support and suggestions. We also thank Scott Merkle and Gisele Andrade from the University of Georgia, H. Dayton Wilde of ArborGen LLC and Fred Hebard of the American Chestnut Foundation for supplying chestnut somatic embryo cultures. We are grateful to Herb Darling as well as Arlene and Stan Wirsig of the New York State Chapter of the American Chestnut Foundation. Finally, the New York State Chapter of the American Chestnut Foundation, the Monsanto Fund, and ArborGen, LLC provided generous financial support for this project.


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Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  • Linda D. Polin
    • 1
  • Haiying Liang
    • 4
  • Ronald E. Rothrock
    • 1
  • Mutsumi Nishii
    • 2
  • Deborah L. Diehl
    • 1
  • Andrew E. Newhouse
    • 2
  • C. Joseph Nairn
    • 3
  • William A. Powell
    • 2
  • Charles A. Maynard
    • 1
    Email author
  1. 1.Faculty of Forest and Natural Resources ManagementState University of New York, College of Environmental Science and ForestrySyracuseUSA
  2. 2.Faculty of Environmental and Forest BiologyState University of New York, College of Environmental Science and ForestrySyracuseUSA
  3. 3.Daniel B. Warnell School of Forest ResourcesUniversity of GeorgiaAthensUSA
  4. 4.School of Forest Resources, Department of Horticulture and Huck Institute for Life SciencesPennsylvania State UniversityUniversity ParkUSA

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