Spawn Cryopreservation of Agaricus Bisporus and A. Subrufescens Strains

One of the main problems for conservation of mushroom germplasm is that the traditional method of subculturing facilitates aging and contamination of cultures. The conservation of strains at ultra-low temperature in liquid nitrogen (-196 °C) is a widely used method, however, this method become expensive in collections with large number of strains and requires highly specialized personnel. The aim of this study was to evaluate the viability of spawn of A. bisporus and A. subrufescens strains frozen at different temperatures (-20 and -80 oC). Two strains of A. subrufescens and one strain of A. bisporus were studied using spawn prepared in sorghum seeds. Fully-incubated sorghum seeds were placed in polycarbonate vials with a sterile glycerol solution (10% v/v) (G) or without glycerol (WG) and frozen at -20 or -80 oC. After 3, 6 and 12 months the samples were thawed (30 °C, 10 min) and the percentage of sample recovery was evaluated. Mycelial growth was also evaluated in potato dextrose agar (PDA) or a medium with glucose and compost extract (C) recording mycelial diameter after 7 days of incubation at 25 °C. The results showed that in the samples frozen at -20 °C, the recovery was minimal (2 to 4%). However, the samples frozen at -80 °C showed a recovery of 100% from 1 to 6 days after thawing. The recovered samples had a significantly higher growth in the medium C. In all cases the recovered samples produced normal basidiomata when cultivated on compost substrate.