Mushroom (Agaricus bisporus)

  • C. Peter Romaine
  • Carl Schlagnhaufer
Part of the Methods in Molecular Biology book series (MIMB, volume 344)

Abstract

We have devised an easy and effective genetic transformation method for the preeminent edible mushroom, Agaricus bisporus. Our method exploits the T-DNA transfer mechanism in Agrobacterium tumefaciens and relies on the reproductive fruiting body as the recipient tissue. The use of fruiting body explants, particularly the gill, provided highfrequency transformation, overcoming the inefficacy of Agrobacterium-based methods targeting fungal spores or vegetative mycelium. The protocol entails incubation of A. tumefaciens for 3 h with acetosyringone, a signaling molecule that launches the gene transfer mechanism, co-cultivation of the induced bacterium and gill explants for 3 d, and selection for transformants based on an inherited resistance to the antibiotic hygromycin. Between 7 and 28 d on the selection medium, upwards of 95% of the gill explants generate hygromycin-resistant colonies. About 75% of the mushroom transformants show a singlecopy of the hygromycin-resistant gene integrated at random sites in the genome.

Key Words

Agaricus bisporus button mushroom filamentous fungi Basidiomycete Agrobacterium tumefaciens genetic transformation Agrobacterium-mediated transformation hygromycin 

References

  1. 1.
    Bundock, P., Dulk-Ras A.D., Beijersbergen A., and Hooykaas, P.J.J., (1995) Transkingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J. 14, 3206–3214.PubMedGoogle Scholar
  2. 2.
    de Groot, M.J.A., Bundock, P., Hooykaas, P.J.J. and Beijersbergen, A.G.M. (1998) Agrobacterium-mediated genetic transformation of filamentous fungi. Nat. Biotechnol. 16, 839–842.PubMedCrossRefGoogle Scholar
  3. 3.
    Tzfira, T. and Citovsky, V. (2003) The Agrobacterium-plant cell interaction—taking biology lessons from a bug. Plant Physiol. 133, 943–947.PubMedCrossRefGoogle Scholar
  4. 4.
    Kunik, T., Tzfira, T., Kapulnik, Y., Gafni, Y., Dingwall, C., and Citovsky, V. (2001) Genetic transformation of HeLa cells by Agrobacterium. Proc. Natl. Acad. Sci. USA 98, 1871–1876.PubMedCrossRefGoogle Scholar
  5. 5.
    Chen, X., Stone, M., Schlagnhaufer, C., and Romaine, C.P. (2000) A fruiting body tissue method for efficient Agrobacterium-mediated transformation of Agaricus bisporus. Appl. Environ. Microbiol. 66, 4510–4513.PubMedCrossRefGoogle Scholar
  6. 6.
    Challen M., Gregorg, K., Sreenivasaprasad, S., et al. (2000) Transformation technologies for mushrooms. Mushr. Sci. 15, 165–172.Google Scholar
  7. 7.
    Leach, K., Odon, V., Zhang, C., et al. (2004) Progress in Agaricus bisporus transformation: Agrobacterium methodologies and development of novel marker genes. In: Science and Cultivation of Edible Fungi, and Medicinal Fungi (Romaine, C.P., Keil, C.B., Rinker, D.L., and Royse, D.J., eds.), The Pennsylvania State University, University Park, PA, pp. 93–102.Google Scholar
  8. 8.
    Mikosch, T.S., Lavrijssen, B., Sonnenberg, A.S.M., and van Griensven, L.J. L.D. (2001) Transformation of the cultivated mushroom Agaricus bisporus using TDNA from Agrobacterium tumefaciens. Curr. Genet. 39, 35–39.PubMedCrossRefGoogle Scholar
  9. 9.
    Foster, G.D., Burns, C., Bailey, A., Challen, M., Elliott, T., and Burton, K.S. (2004) Tools for Agaricus and Coprinus transformation and analysis of gene expression. In: Science and Cultivation of Edible Fungi and Medicinal Fungi (Romaine, C.P., Keil, C.B., Rinker, D.L., and Royse, D.J., eds.), The Pennsylvania State University, University Park, PA, pp. 59–66.Google Scholar
  10. 10.
    Velcko, A.J., Kerrigan, R.W., MacDonald, L.A., Wach, M.P., Schlagnhaufer, C., and Romaine, C.P. (2004) Expression of novel genes in Agaricus bisporus using an Agrobacterium-mediated transformation technique. In: Science and Cultivation of Edible Fungi and Medicinal Fungi (Romaine, C.P., Keil, C.B., Rinker, D.L., and Royse, D.J., eds.), The Pennsylvania State University, University Park, PA, pp. 591–598.Google Scholar
  11. 11.
    Zhang, C., Odon, V., Kim, H.K., et al. (2004) Mushrooms for molecular pharming. In: Science and Cultivation of Edible Fungi and Medicinal Fungi, (Romaine, C.P., Keil, C.B., Rinker, D.L., and Royse, D.J., eds.), The Pennsylvania State University, University Park, PA, pp. 611–618.Google Scholar
  12. 12.
    Zolan, M.E. and Pukkila, P.J. (1986) Inheritance of DNA methylation in Coprinus cinereus. Mol. Cell. Biol. 6, 195–200PubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2006

Authors and Affiliations

  • C. Peter Romaine
    • 1
  • Carl Schlagnhaufer
    • 1
  1. 1.Department of Plant PathologyThe Pennsylvania State UniversityUniversity Park

Personalised recommendations