A Simple Protocol for Cryopreservation of Mosses

Maintaining numbers of aseptic cultures of bryophytes by serial transfer not only consumes supplies and labor but can result in loss of vigor or the appearance of other abnormalities in long-term cultures. Cryopreservation of cultures solves the problems associated with long-term culture, and is of particular importance for cultures or mutant cell lines incapable of making spores or where spores are short-lived. Dramatic simplification of a published protocol for moss cryopreservation is accomplished by preconditioning cultures for 3-4 days in medium supplemented with 10-5 M ABA and 100 mM proline. This proline/ABA protocol can be successfully used with Ceratodon purpureus, Funaria hygrometrica, Physcomitrella patens, and two species of Sphagnum; cryopreserved cultures remain viable for a minimum of one year at -800C. Experiments demonstrate a differential response by cell types to cryopreservation, as well as differences between species or for particular selected cell lines within a species. Other experiments suggest that successful cryopreservation is not a simple consequence of brood cells in ABA-treated