Masanori Sato, Shigeki Inaba, Akira Nakagiri, Yoshinori Matsuo
This study evaluated the long-term cryopreservation efficacy of two previously developed protocols-the Modified Perlite Protocol (MPP) and the Vermiculite Protocol (VP)-using 65 ectomycorrhizal basidiomycete culture strains stored for five years in the vapor phase of a liquid nitrogen tank. Survival rates were assessed based on recovery ratios, with both MPP and VP showing high viability: 64 of 65 strains showed complete recovery under each protocol. Colony morphology remained stable across all methods, showing no visible phenotypic changes due to cryopreservation. Regarding post-thaw mycelial growth potential measured by colony diameter, VP was superior or equivalent to MPP in 49 out of 51 analyzed strains immediately post-thaw, yielding significantly greater growth in 19 of these strains. Further statistical analysis among three protocols (HPP, MPP, and VP) in a subset of 14 strains showed that VP generally produced the largest mean colony diameters immediately post-thaw in most strains and was significantly larger than MPP in six strains, although strain-dependent exceptions were observed. These findings suggest that both MPP and VP are suitable for the long-term preservation of cryosensitive basidiomycete cultures, with VP offering enhanced immediate post-thaw mycelial growth potential.