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In vitro germination and transient GFP expression of American chestnut (Castanea dentata) pollen

  • Genetic Transformation and Hybridization
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Abstract

The development of the male reproductive structures of American chestnut (Castanea dentata) is described to advance our understanding of its reproductive behavior. This information has been vital in the development of a strategy to collect pollen grains from male catkins suitable for in vitro germination and transformation experiments. Cutting male catkins into small segments and rolling them over a culture plate resulted in evenly dispersed and large amounts of pollen with minimal unwanted accessory floral parts. To optimize pollen viability, the effect of various storage conditions on in vitro germination was examined. Our results showed that initial storage at 4°C for 2 weeks significantly increased percent germination as compared to freshly collected pollen and those stored directly at −20°C or −80°C. This also means that for long-term storage of American chestnut pollen, the catkins should first be kept at 4°C for a couple of weeks and then at −80°C. The use of pollen grains with high viability is necessary for the transformation of American chestnut pollen. To optimize pollen transformation via particle bombardment, the effects of target distance, target pressure, and pollen developmental stage were examined. Statistical analysis showed that bombardment of ungerminated pollen at 1,100 psi resulted in the highest percent transient GFP expression (4.1%).

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Acknowledgements

The authors thank: Drs. William Powell, Charles Maynard, and Haiying Liang for their help in cloning of plasmid DNA, allowing access to the trees at Heiberg, and much other support throughout the project; Drs. Jim Haseloff and John Runions for providing the GFP construct; Dr. Jose Vidal for providing guidance on bombardment; and Dr. Bruce I. Reisch for supporting the project and allowing access to his laboratory. The collaboration between laboratories of BIR and DDF was made possible through the Edna Bailey Sussman Fund awarded to JLR. Funding for this project was provided by a grant from the New York Chapter of the American Chestnut Foundation to DDF.

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Authors and Affiliations

  1. Department of Environmental and Forest Biology, State University of New York College of Environmental Science and Forestry, 241 Illick Hall, 1 Forestry Drive, Syracuse, NY, 13210, USA

    Danilo D. Fernando & Javonna L. Richards

  2. Department of Horticultural Sciences, New York State Agricultural Experiment Station, Cornell University, Geneva, NY, 14456, USA

    Julie R. Kikkert

Authors
  1. Danilo D. Fernando
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  2. Javonna L. Richards
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  3. Julie R. Kikkert
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Correspondence to Danilo D. Fernando.

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Communicated by S.A. Merkle

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Fernando, D.D., Richards, J.L. & Kikkert, J.R. In vitro germination and transient GFP expression of American chestnut (Castanea dentata) pollen. Plant Cell Rep 25, 450–456 (2006). https://doi.org/10.1007/s00299-005-0088-z

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  • DOI: https://doi.org/10.1007/s00299-005-0088-z

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