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Alternative substrates for cryopreservation at -20°C have been little explored for basidiomycetes and could bring new possibilities of lower cost cryopreservation. Nevertheless, freezing temperatures between -15 and -60°C are very challenging because they frequently result in cryoinjuries. The objective of this study was to evaluate substrates associated to cryoprotective agents for Pleurotus ostreatus cryopreservation at -20 or -70°C in order to develop alternative techniques for basidiomycete cryopreservation. P. ostreatus was grown on potato dextrose agar or whole grains of oat, wheat, rice or millet and transferred to cryovials with cryoprotective solution with 1 % dimethyl sulfoxide, 5 % glycerol, 10 % saccharose, 4 % glucose, 6 % polyethylene glycol-6000 or 5 % malt extract. The mycelium in the cryovials were cryopreserved at -20 or -70°C and recovered for evaluation of the mycelial growth viability after 1 and 3 years. Both substrates and cryoprotectants affect the viability of the mycelial growth cryopreserved at -20 or -70°C; wheat grains combined with cryoprotectants such as saccharose or glucose are effective for keeping mycelium viable after cryopreservation at -20°C for 1 or 3 years; for cryopreservation at -70°C after 1 or 3 years, any substrate combined with any cryoprotectant is effective for preserving the mycelium viable, except for millet grains with polyethylene glycol after 3 years; semi-permeable cryoprotective agents such as saccharose and glucose are the most effective for cryopreservation at -20 or -70°C for at least 3 years.

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