Long term storage of Pleurotus ostreatus and Trametes versicolor isolates using different cryopreservation techniques and its impact on laccase activity

Ivana Eichlerová, Ladislav Homolka, Michal Tomšovský, Ludmila Lisá

Institute of Microbiology, Academy of Sciences of the Czech Republic (ASCR), v.v.i., Vídeňská 1083, 142 20 Prague 4, Czech Republic.

Abstract

The strain Pleurotus ostreatus Florida f6, its 45 basidiospore-derived isolates (both monokaryons and dikaryons prepared in our laboratory), Trametes versicolor strain CCBAS 614 and 22 other Trametes versicolor isolates obtained from the sporocarps collected in distant localities were successfully preserved for 12 years using perlite and straw cryopreservation protocols. All tested isolates survived a 12-year storage in liquid nitrogen (LN) and their laccase production and Poly B411 decolorization capacity was preserved. Also mycelium extension rate and the types of colony appearance of individual isolates remained unchanged. This is the first report on using the perlite protocol and our modified straw protocol for the cryopreservation and storage of basidiomycetes for more than 10 years and we firstly confirmed successful cryopreservation of individually differing isolates derived from one parental strain. Different cryopreservation techniques were also tested for the storage of the culture liquid with extracellular laccase produced by Trametes versicolor strain CCBAS 614. The influence of the cryopreservant and the cryopreservation techniques on laccase activity presented in culture liquid were studied during the short time (24 hours) and the long time (6 month) storage period. The results showed that 10 % glycerol was the most suitable cryopreservant. The absence of the cryopreservant did not cause high loss of laccase activity in the samples; the presence of DMSO (5 or 10%) in LN-stored samples caused mostly a decrease of laccase activity. For the preservation of laccase activity in the liquid culture the storage in the freezer at -80°C is more convenient than the storage in liquid nitrogen.

Keywords: Cryopreservation; Perlite protocol; Straw protocol; Pleurotus ostreatus; Trametes versicolor; Laccase.