Cryopreservation of embryogenic suspension cultures of Cyclamen persicum mill.

We have developed a simple protocol for the cryopreservation of embryogenic suspension cultures of Cyclamen persicum. Embryogenic suspension cultures in the linear growth phase 7-10 days after subculture were used for cryopreservation. Of the different cryoprotectants tested during a 2-day pre-culture, 0.6 M sucrose resulted in the highest re-growth rates of 75%. An additional pre-treatment with 0.6 M sucrose and 10% DMSO (dimethylsulfoxide) for 1 h also positively affected re-growth. Microscopic studies on viability revealed that only few small embryogenic cells survived cryopreservation, while vacuolated single cells died. Experiments in which the duration of the pre-culture period--i.e. the length of time the embryogenic suspension cells were exposed to 0.6 M sucrose--was varied showed that 2-4 days was the most optimal exposure time to 0.6 M sucrose. Callus re-grown after cryopreservation showed growth rates similar to that of unfrozen callus and regenerated even higher numbers of somatic embryos than unfrozen callus.