Cryopreservation of Cyclamen persicum Mill. Callus

Abstract:

In this paper we studied cryopreservation of Cyclamen persicum Mill. callus to avoid variations produced by sub-culture. The callus in the logarithmic phase after sub-culture were used for experiments. Firstly, the callus were pre-cultured in culture-medium containing 4%, 6% or 8% sucrose for different time periods, transferred to different cryoprotectants to directly cryopreserve or incubated for 2 hours at -20°C, then submersed in liquid nitrogen, lastly thawed rapidly in a waterbath at 37°C, and washed with liquid culture-medium containing the corresponding concentration of sucrose. Cell survival rate was computed after stained by Neutral Red, and SPSS 13.0 software was used for statistical analysis. The results showed that sucrose concentration, pre-culture time, cryoprotectants had various impacts on cell survival rate. We have developed a simple but effective protocol for the cryopreservation of callus of C. persicum. Of the different protocols tested, 4%sucrose, pre-culturing for 3 days, No. 9 cryoprotectant and freezing directly after 30 minutes at 0°C results in the highest cell survival rate.