Until now, maintenance of Podospora anserina strains was tedious, requiring yearly sexual crosses, spore isolation and germination, verification of the genotype and transfer of mycelium to stock tubes maintained at 4 C. We have circumvented these difficulties by storage of ascospores at -80 C. Sexual crosses were made on synthetic medium, either by confrontation or spermatization, to reach high fertility. When perithecia began to shoot ascospores, plates containing agar/salt medium were put upside down over plates containing perithecia. Ascospores were recovered after 24 h by scraping the surface of the agar with a sterile spatula. More than 10(4) asci were recovered from each plate. They were transferred from the spatula to a 1.2 ml Nalgene cryovial containing 0.3 ml sterile storage medium (3 parts 50% glycerol: 7 parts liquid corn meal medium). The vials were vortexed 30 sec and placed at -80 C.
After two years storage, the germination rate of ascospores was only slightly lower than that of freshly isolated ones and several cycles of freeze-thawing did not decrease it significantly (Table 1). This method has been successfully applied to wild-type strains of Podospora anserina and to mutant strains displaying ascospores of wild-type phenotype. We are now testing with strains which have more fragile ascospores. For methods and media used in growing and crossing Podospora anserina, see Esser, K. 1969. Neurospora Newsletter 15:27-31.
Table 1. Survival of wild-type ascospores after -80 C storage
Time of storage strain* 2 months 14 months 24 months 24 months+ _________________________________________________________________ s 20/20 60/60 52/87 71/92 S 8/8 nd 77/83 nd A 17/17 79/80 nd 72/82* : s, S and A are three wild-type strains of Podospora