Volume 16 Part 1 Article 12:Progress in Agaricus bisporus Transformation: Agrobacterium Methodologies and Development of Novel Marker Genes

Volume 16 Part 1 Article 12
Year 2004
Title: Progress in Agaricus bisporus Transformation: Agrobacterium Methodologies and Development of Novel Marker Genes
Authors: K. Leach, V. Odon, C. Zhang, H.K. Kim, J. Henderson, P. Warner, M. Challen and T. Elliott

Abstract:

Transformation of the button mushroom, Agaricus bisporus, has recently become possible due to the use of Agrobacterium-mediated transformation combined with mushroom gill tissue and the Escherichia coli hph marker gene encoding hygromycin resistance. Factors such as the strain of Agrobacterium tumefaciens, co-cultivation conditions, host strain, type of tissue and the use of various promoters are known to effect rates of transformation. Using gill tissue from a number of commercial A. bisporus strains, we have shown that different Agrobacterium strains and the use of vacuum infiltration or sonication treatments enhanced the recovery of transformants. The effect of the virulence inducing phenolic compound, acetosyringone, on the number of transgene copies integrated into the A. bisporus genome also has been investigated. Hygromycin-resistant transformants were confirmed using PCR and Southern analyses. Transcripts were confirmed using RT-PCR in both mycelium and mushroom tissues. To provide alternatives to prokaryotic marker genes, there remains a need for alternative selectable markers for mushroom transformation. Progress in the development of selectable markers from two mushroom genes (resistance to carboxin and sulfonamide) is described. Southern blotting is normally used to confirm transgene copy number. Using the model mushroom, Coprinus cinereus, we have used real-time quantitative PCR to determine transgene copy number.

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