CRYOPRESERVATION OF EMBRYOGENIC SUSPENSION CULTURES OF CYCLAMEN PERSICUM MILL.

Abstract

We have developed a protocol for the cryopreservation of embryogenic suspension cultures of Cyclamen persicum, which does not involve the need of sophisticated equipment. With the aim of achieving this simple protocol for cryopreservation, different cryoprotectants in the pre-culture and pre-treatment periods on cell growth and re-growth after cryopreservation were tested. For cryopreservation experiments embryogenic suspension cultures in the linear growth phase 7-10 days after subculture were used. Of the different cryoprotectants tested during a 2-day pre-culture, 0.6 M sucrose resulted in the highest re-growth rate of 75% based on the number of Petri dishes plated after thawing. Additionally, a pre-treatment with 0.6 M sucrose and 10% DMSO (dimethylsulfoxide) for 1 h immediately prior to freezing, also positively affected re-growth. Experiments in which the optimal pre-culture duration was varied showed that 2-4 days was the best 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 control callus. Microscopic studies on viability using FDA (fluorescein diacetate) staining revealed that only few small embryogenic cells survived cryopreservation, while vacuolated single cells died.