Abstract
The key to successful transformation of American chestnut is having the correct combination of explant tissue, selectable and scorable markers, and a reliable regeneration system. Rapidly dividing somatic embryos, growing as proembryogenic masses, are a suitable tissue; the bar gene is a suitable selectable marker in conjunction with 1.0 to 10 mg/L phosphirothricin (PPT); and the mgfp5-ER gene is an effective nondestructive scorable marker. We have also found that the more gently the somatic embryos are treated during the inoculation and co-cultivation steps, the higher the transformation efficiency. The average transformation efficiency that can be expected using the described protocol is approx 20 stable and embryogenic transformation events/g of somatic embryo tissue. Cell line and batch-to-batch deviations both upward and downward should be expected. Finally, somatic embryos can be induced to form shoots, which can then be micropropagated and acclimatized.
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Griffin, G.J. (1986) Chestnut blight and its control. Hortic. Rev. 8, 291–336.
Murrill, W.A. (1906) A serious chestnut disease. J. N.Y. Bot. Gard. 7, 143–153.
Diller, J.D. and Clapper, R.B. (1965) A progress report on attempts to bring back the chestnut tree in the eastern United States, 1954–1964. J. For. 63, 186–188.
Gonzales, M.L., Vieitez, A.M., and Vieitez, E. (1985) Somatic embryogenesis from chestnut cotyledon tissue cultured in vitro. Sci. Hortic. 27, 97–103.
Piagnani, C. and Eccher, T. (1990) Somatic embryogenesis in chestnut. Acta Hortic. 280, 159–161.
Vieitez, F.J., San-Jose, M.C., Ballester, A., and Vieitez, A.M. (1990) Somatic embryogenesis in cultured immature zygotic embryos in chestnut. J. Plant Physiol. 136, 253–256.
Merkle, S.A., Wiecko, A.T., and Watson-Pauley, B.A. (1991) Somatic embryogenesis in American chestnut. Can. J. For. Res. 21, 1698–1701.
Maynard, C.A. (1991) Using PCR to confirm Agrobacterium transformation of American chestnut (Castanea dentata). Abstract No. 927, In: Program and Abstracts of the Third International Congress of Plant Molecular Biology: Molecular Biology of Plant Growth and Development, Tucson, Arizona, October 6–11, 1991.
Merkle, S., Carraway, D.T., Watson-Pauley, B.A., and Wilde, H.D. (1992) Somatic embryogenesis and gene transfer in American chestnut. In: Proceedings of the International Chestnut Conference, West Virginia University Press, Morgantown, WV. July 10–14, 1992.
Carraway, D.T., Wilde, H.D., and Merkle, S.A. (1994) Somatic embryogenesis and gene transfer in American chestnut. J. Am. Chest. Found. 8, 29–33.
Vieitez, A.M., Ballester, A., Vieitez, M.L., and Vieitez, E. (1983) In vitro plantlet regeneration of mature chestnut. J. Hortic. Sci. 58, 457–463.
Vieitez, A.M. and Vieitez, M.L. (1983) Castanea sativa plantlets proliferated from axillary buds cultivated in vitro. Sci. Hortic. 18, 343–351.
Chauvin, J.E. and Salesses, G. (1988) Advances in chestnut micropropagation (Castanea sp.). Acta. Hortic. 227, 340–345.
Chevre, A.-M. and Salesses, G. (1987) Choice of explants for chestnut micropropagation. Acta. Hortic. 212, 517–523.
Mullins, K.V. (1987) Micropropagation of chestnut (Castanea sativa Mill.). Acta. Hortic. 212, 525–530.
Piagnani, C. and Eccher, T. (1988) Factors affecting the proliferation and rooting of chestnut in vitro. Acta Hortic. 227, 384–386.
Strullu, D.G., Grellier, B., Marciniak, D., and Letouze, R. (1986) Micropropagation of chestnut and conditions of mycorrhizal synthesis in vitro. New Phytol. 102, 95–101.
Vieitez, A.M., Ballester, A., and Vieitez, E. (1987) Vitrification in chestnut shoots regenerated in vitro. Acta. Hortic. 212, 231–234.
Maynard, C., Satchwell, M., and Rieckermann, H. (1993) Micropropagation of American chestnut (Castanea dentata (Marsh.) Borkh.): rooting and acclimatization. In: Proceedings of the Second Northern Forest Genetics Association Conference. St. Paul, Minnesota., July 29–30, 1993, pp. 161–170.
Carraway, D.T. and Merkle, S.A. (1997) Plantlet regeneration from somatic embryos of American chestnut. Can. J. For. Res. 27, 1805–1812.
Robichaud, R.L., Lessard, V.C., and Merkle, S.A. (2004) Treatments affecting maturation and germination of American chestnut somatic embryos. J. Plant Physiol. 161, 957–969.
Seabra, R. C. and Pais, M. S. (1998) Genetic transformation of European chestnut. Plant Cell Rep. 17, 177–182.
Corredoira, E., Montenegro, D., San-Jose, M.C., Vieitez, A.M., and Ballester, A. (2004). Agrobacterium-mediated transformation of European chestnut embryogenic cultures. Plant Cell Rep. 23, 311–318.
Cheng, M., Hu, T., Layton, J., Liu, C., and Fry, J.E. (2003) Desiccation of plant tissues post-Agrobacterium infection enhances T-DNA delivery and increases stable transformation efficiency in wheat. In Vitro Cell. Dev. Bio. Plant. 39, 595–604.
Hood, E.E., Gelvin, S.B., Melchers, L.S., and Hoekema, A. (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res. 2, 208–218.
Polin, L.D., Liang, H., Rothrock, R., et al. (2006) Agrobacterium-mediated transformation of American Chestnut] [Castanea dentata (Marsh.) Barkh.] somatic embryos. Plant Cell Tiss. Org. Cul. 84, 69–78.
White, J., Chang, S.Y., and Bibb, M.J. (1990) A cassette containing the bar gene of Streptomyces hygroscopicus: A selectable marker for plant transformation. Nucl. Acids Res. 18, 1062.
Garbino, J.E. and Belknap, W.R. (1994) Isolation of a ubiquitin-ribosomal protein gene (ubi3) from potato and expression of its promoter in transgenic plants. Plant Mol. Biol. 24, 119–127.
Haseloff, J. and Siemering, K.R. (1998) The uses of GFP in plants. In: Green Fluorescent Protein: Properties, Applications, and Protocols (Chalfie, M. and Kain, S. eds.), Wiley-Liss, New York, (pp. 191–220).
Dratewka-Kos, E., Rahman, S., Grzekzak, Z.F., Kennedy, T.D., Murray, R.K., and Lane, G. (1989) The polypeptide structure of germin as deduced from cDNA sequencing. J. Biol. Chem. 264, 4896–4900.
Mason, H.S., DeWald, D.B., and Mullet, J.E. (1993) Identification of a methyl jas-monate-responsive domain in the soybean vspB promoter. Plant Cell 5, 241–251.
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Maynard, C.A., Polin, L.D., LaPierre, S.L., Rothrock, R.E., Powell, W.A. (2006). American Chestnut [Castanea dentata (Marsh.) Borkh.]. In: Wang, K. (eds) Agrobacterium Protocols Volume 2. Methods in Molecular Biology, vol 344. Humana Press. https://doi.org/10.1385/1-59745-131-2:239
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DOI: https://doi.org/10.1385/1-59745-131-2:239
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