Abstract
Cryopreservation using liquid nitrogen is among the most useful and efficient methods for long-term storage of basidiomycetes fungal strains. However, there are still a limited number of reports on its efficiency in terms of the stability of intrinsic habits of the fungal strains. This study aims to understand the effects of frozen storage using liquid nitrogen on mycelial growth as well as the mycorrhization ability of Tricholoma matsutake strains. The mycelia of four strains of T. matsutake were preincubated on particles of autoclaved vermiculite immersed in nutrient liquid media in cryovials. The cryovials were filled with a cryoprotectant (to a final concentration of 5% DMSO and 10% trehalose) and then frozen to −80°C at a cooling speed of −1°C/min using a freezing container or a program freezer. They were then stored in gaseous liquid nitrogen at approximately −180°C in a locator cryogenic storage system for up to 3 years for one strain (Y1, identical to IFO33136) and 2 years for the other three strains. Following frozen storage, the regenerated mycelia were subjected to tests to investigate the hyphal extension rate on culture plates and mycorrhization abilities with red pine seedlings in vitro. All strains were viable after frozen storage with high survival rate, and formed ectomycorrhizal roots with the pine seedlings. The results indicate that frozen storage using liquid nitrogen can be an efficient method for the long-term stable preservation of T. matsutake and does not compromise both their mycelial growth and ectomycorrhization abilities.
