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Sexually mature transgenic American chestnut trees via embryogenic suspension-based transformation

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Abstract

The availability of a system for direct transfer of anti-fungal candidate genes into American chestnut (Castanea dentata), devastated by a fungal blight in the last century, would offer an alternative or supplemental approach to conventional breeding for production of chestnut trees resistant to the blight fungus and other pathogens. By taking advantage of the strong ability of embryogenic American chestnut cultures to proliferate in suspension, a high-throughput Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into the tree was established. Proembryogenic masses (PEMs) were co-cultivated with A. tumefaciens strain AGL1 harboring the plasmid pCAMBIA 2301, followed by stringent selection with 50 or 100 mg/l Geneticin. A protocol employing size-fractionation to enrich for small PEMs to use as target material and selection in suspension culture was applied to rapidly produce transgenic events with an average efficiency of four independent transformation events per 50 mg of target tissue and minimal escapes. Mature somatic embryos, representing 18 transgenic events and derived from multiple American chestnut target genotypes, were germinated and over 100 transgenic somatic seedlings were produced and acclimatized to greenhouse conditions. Multiple vigorous transgenic somatic seedlings produced functional staminate flowers within 3 years following regeneration.

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Abbreviations

EDM:

Embryo development medium

GM:

Germination medium

IMM:

Induction/maintenance medium

PCR:

Polymerase chain reaction

PEM:

Proembryogenic mass

SM:

Selection medium

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Acknowledgments

The research reported here was supported by ArborGen LLC and the Institute of Forest Biotechnology. The authors would like to thank Paul Montello for help with graphics, John Bond for photography and Steve Pettis for greenhouse assistance. We would also like to thank Tom Kubisiak and Charles Burdine of the USDA Southern Research Station, Wayne Parrott and Barbara Artelt for technical assistance. We thank Fred Hebard of The American Chestnut Foundation and Gary and Lucille Griffin of the American Chestnut Cooperators Foundation for supplying us with American chestnut material.

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Authors and Affiliations

  1. Warnell School of Forestry and Natural Resources, University of Georgia, Athens, GA, 30602, USA

    Gisele M. Andrade, Campbell J. Nairn, Huong T. Le & Scott A. Merkle

Authors
  1. Gisele M. Andrade
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  2. Campbell J. Nairn
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  3. Huong T. Le
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  4. Scott A. Merkle
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Correspondence to Scott A. Merkle.

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Communicated by K. Kamo.

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Andrade, G.M., Nairn, C.J., Le, H.T. et al. Sexually mature transgenic American chestnut trees via embryogenic suspension-based transformation. Plant Cell Rep 28, 1385–1397 (2009). https://doi.org/10.1007/s00299-009-0738-7

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  • DOI: https://doi.org/10.1007/s00299-009-0738-7

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