Long-term cryopreservation of Lentinus crinitus strains by wheat grain technique

Lentinus crinitus (Basidiomycota: Polyporales) is a saprophytic fungus with biotechnological importance described more than 20 years ago. However, there are few studies on the long-term preservation of this basidiomycete. Cryopreservation is a long-term storage technique that reduces the metabolic activity of microorganisms, but its success depends on the adjustment of the freezing process, the cryoprotectants, and the protective substrates for each species. This study aimed to assess the mycelial viability and genetic stability of L. crinitus strains cryopreserved at -86 °C for two years by the wheat grain technique using different cryoprotectants and freezing methods. Three strains of L. crinitus (U9-1, U13-5, and U15-12) were subjected to different concentrations and types of cryoprotectants (dimethyl sulfoxide, glycerol, glucose, and sucrose), freezing methods such as immediate freezing from 25 to -86 °C and progressing freezing from 25 to -86 °C in a freezing container with isopropyl alcohol to control the rate of cell freezing at -1 °C min^-1, protective substrate (wheat grain and 2% malt extract agar), and cryopreservation period (1, 6, 12, and 24 months). After thawing, samples were evaluated for mycelial viability, time to mycelial recovery, mycelial stability, and genetic stability of the fungus. All techniques achieved effective cryopreservation at -86 °C, mainly with the wheat grain technique. All cryoprotectants (3.5% glycerol, 1.5% dimethyl sulfoxide, 25% sucrose, and 5% glucose), freezing methods (immediate and gradual), and protective substrate (wheat grain and malt extract agar) were effective for cryopreservation of the three L.crinitus strains in an ultra-low temperature freezer for two years. Mycelial viability, mycelial stability, and genetic stability of the fungus were not affected after two-year cryopreservation, evidencing the robustness of the long-term cryopreservation technique and the fungus.