Viability of fastidious Phytophthora following different cryopreservation treatments.

Living stock cultures with constant phenotypes and genotypes are required for a wide range of research and industrial applications; however, long-term, stable preservation of fastidious Phytophthora strains has been challenging. In this study, we systematically evaluated different cryopreservation treatments to identify and clarify freezing, thawing, and other conditions appropriate for long-term maintenance. Optimal preservation conditions were largely strain-specific, with robust strains remaining fully viable and the fastidious yielding lower recovery under all test conditions. Nevertheless, several procedures were shown to be generally applicable for effective cryopreservation of most Phytophthora organisms. Fastidious strains retained higher viability following the -1 °C min(-1) freezing protocol (Mr Frosty's) than either of two widely used programmed freezing procedures. Revival was higher when frozen mycelium plugs were thawed at 37 °C for 2 min or 25 °C for 5 min, while lower viability was apparent for fastidious strains thawed at 55 °C for 1.5 min. Among 15 cryoprotective solutions assessed, 5 % dimethyl sulfoxide produced the highest viability for all fastidious strains. The effect of prefreeze and postfreeze treatments on revival was mild, if any, and strain-dependent. This study has generated reliable, practical, long-term preservation solutions applicable to a majority of Phytophthora species. It also has revealed a need for in-depth physiological and morphological investigations to further enhance the preservation methods for fastidious strains.

Effect of cryopreservation protocols on the phenotypic stability of yeast.

Eight cryopreservation protocols were assessed for their effects on the viability and phenotypic stability of the yeast Saccharomyces cerevisiae during a five-year study. It is found that viability and phenotypic features have remained largely unchanged when the yeast was preserved in glycerol, dimethyl sulphoxide, or sucrose at -80 degrees C or in liquid nitrogen. When sorbitol was used as a cryoprotectant, yeast cells frozen and stored at -80 degrees C manifested great decreases in viability after six months in storage and concomitantly large fluctuations in the rate of the trpl auxotrophic reversion. This phenotypic reversion was stable passage after passage. Such a degree of phenotypic fluctuations, however, was not observed for yeast cells preserved in the same sorbitol solution that went through a controlled freezing program and were subsequently stored in liquid nitrogen. These results indicate that some combinations of cryoprotective agent, freezing program, and storage temperature disturb biomaterials more profoundly during cryopreservation and imply a genetic basis of this phenotypic change.