Cryopreservation of Digitalis lanata cell cultures

Suspension cultures of Digitalis lanata strain I were grown in a medium containing 3% mannitol. For cryopreservation cell suspensions were treated with a mixture of sucrose-glycerol (20%/20 V%), cooled slowly (about 1 degrees C/min) till -100 degrees C and then were transferred to liquid nitrogen. After storage in liquid nitrogen the cells were thawed rapidly in a water bath of 40 degrees C and spread on the surface of a solidified nutrient medium. After 7 days of regrowth the cells were suspended in liquid nutrient medium for further cultivation. About 50% of the cells survived freezing and thawing. However, also the apparently surviving cells showed signs of injury (membrane vesicles outside the plasmalemma, dilated ER cisternae and separation of the nuclear membranes). The cultures derived from the surviving cells had the same growth rate and biochemical activity relative to the transformation of cardenolides, e.g., digitoxin, as the parent cultures. The frequency distribution of the nuclear DNA content in the cell cultures was the same before and after cryopreservation. These results indicate that there is no selection of a special cell type during freezing and thawing.