Cryoprotectant removal temperature as a factor in the survival of frozen rice and sugarcane cells

Removal of cryoprotective additives through use of a room temperature (22 degrees C) washing step, instead of 0 degrees C, was found to improve the recovery of sugarcane suspension culture and rice callus tissues. Cultured cells were cryoprotected by gradual addition of a mixture of polyethylene glycol, glucose, and DMSO (PGD) to a final concentration of 10%-8%-10%, w/v, respectively, added at either 0 or 22 degrees C. After a programmed slow freezing of the cells, they were thawed rapidly and the cryoprotectants were gradually diluted and washed out using a 22 or 0 degree C washing medium. Viability of suspension cultured sugarcane cells protected with PGD was greatly diminished when a cold washing solution was used, whether the cells had been frozen (-23 degrees C) or not. Two mutant lines of rice callus when frozen to -196 degrees C in PGD and thawed showed less growth than unfrozen cells, but their growth was improved by washing the thawed cells with a 22 degrees C solution. With all cultures tested, the addition of PGD at 0 degrees C and post-thaw washing out at 22 degrees C gave improved survival. Particularly with the rice lines, optimizing the addition and washing procedures allowed culture survival of liquid nitrogen freezing not otherwise attained.