Cryopreservation of Alkaloid-Producing Cell Cultures of Periwinkle (Catharanthus roseus)

A procedure for cryogenic storage of alkaloid producing cell lines of periwinkle, Catharanthus roseus (L.) G. Don., has been developed. The procedure differs from established cryopreservation protocols in several aspects. Specifically, 4-day-old suspension subcultures of three cell lines were precultured in nutrient media supplemented with 1 molar sorbitol for 6 to 20 hours. The cells were then incubated in nutrient media with 1 molar sorbitol plus 5% DMSO in an ice bath for 1 hour and, thereafter, were frozen in this solution at a cooling rate of 0.5°C per minute to −40°C prior to immersion in liquid nitrogen (LN). After rapid thawing in a 40°C water bath, the regrowth of LN stored cells was achieved by transferring them without washing onto filter paper discs over nutrient media solidified with agar for a period of 4 to 5 hours. The filter paper discs with the cells were then transferred to fresh media of the same composition for regrowth. The viability immediately after thawing as evaluated by the 2,3,5-triphenyl tetrazolium chloride method was about 60% of controls. Suspension cultures established from LN stored cells retained the capability for alkaloid synthesis and accumulation.