Studies of free radical-mediated cryoinjury in the unicellular green alga Euglena gracilis using a non-destructive hydroxyl radical assay: A novel approach for developing protistan cryopreservation strategies

The development of cryoconservation methods for the long-term storage of al gal cultures is important for the ex situ preservation of biological divers ity and the maintenance of genetic stability within this group of important organisms. However, as many unicellular algae are recalcitrant to cryogeni c storage, this study aims to evaluate the role of oxidative stress in cryo injury. A non-invasive, non-destructive assay method previously applied to animal cells has been developed to evaluate free radical mediated oxidative stress in Euglena gracilis exposed to different cryopreservation treatment s. The procedure employs dimethyl sulphoxide as a probe for the hydroxyl ra dical. Adopting this approach it was possible to identify those components of the cryopreservation protocol which were the most damaging. These were i dentified as preparative centrifugation and sub-zero freezing treatments. P ost-storage survival in E. gracilis was significantly (P < 0.05) enhanced w hen the chelating agent desferrioxamine was included in the recovery medium whilst methane production was significantly (P < 0.004) reduced, suggestin g that the additive was capable of ameliorating oxidative stress. The poten tial of using novel, exogenous antioxidant treatments developed for medical applications and applying them to enhance cryopreservation tolerance in re calcitrant unicellular algae is discussed.