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

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Abstract

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.

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Abbreviations

2,4-D :

2,4-dichlorophenoxyacetic acid

BA:

6-benzyladenine

bar :

bialaphos-resistance gene

Carb:

carbenicillin

Cef:

cefotaxime

mgfp5-ER :

modified green fluorescent protein gene

OxO:

oxalate oxidase

PCR:

polymerase chain reaction

PPT:

phosphinothricin

SDS:

sodium dodecyl sulfate

SSC:

sodium chloride sodium citrate

TE:

Tris–EDTA

Tric:

tricarcillin

WPM:

McCown’s woody plant medium salts

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Acknowledgments

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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Authors and Affiliations

  1. Faculty of Forest and Natural Resources Management, State University of New York, College of Environmental Science and Forestry, Syracuse, NY, 13210, USA

    Linda D. Polin, Ronald E. Rothrock, Deborah L. Diehl & Charles A. Maynard

  2. Faculty of Environmental and Forest Biology, State University of New York, College of Environmental Science and Forestry, Syracuse, NY, 13210, USA

    Mutsumi Nishii, Andrew E. Newhouse & William A. Powell

  3. Daniel B. Warnell School of Forest Resources, University of Georgia, Athens, Georgia, 30602, USA

    C. Joseph Nairn

  4. School of Forest Resources, Department of Horticulture and Huck Institute for Life Sciences, Pennsylvania State University, University Park, PA, 16802, USA

    Haiying Liang

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  1. Linda D. Polin
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  2. Haiying Liang
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  3. Ronald E. Rothrock
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  4. Mutsumi Nishii
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  5. Deborah L. Diehl
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  7. C. Joseph Nairn
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  8. William A. Powell
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  9. Charles A. Maynard
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Correspondence to Charles A. Maynard.

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Polin, L.D., Liang, H., Rothrock, R.E. et al. Agrobacterium-mediated transformation of American chestnut (Castanea dentata (Marsh.) Borkh.) somatic embryos. Plant Cell Tiss Organ Cult 84, 69–79 (2006). https://doi.org/10.1007/s11240-005-9002-1

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  • DOI: https://doi.org/10.1007/s11240-005-9002-1

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