Mushroom (Agaricus bisporus)

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.