Trehalose as cryoprotectant for the freeze preservation of carrot and tobacco cells

Suspension cultures of carrot (Daucus carota, line C1), tobacco (Nicotiana tabacum, line TX1), and Nicotiana plumbaginifolia (line NP) were frozen under controlled conditions with trehalose as the sole cryoprotectant. Maximal post-thaw viability (71-74%), measured by phenosafranin dye exclusion, was obtained with the C1 cells following a 24hour pretreatment with 5 or 10% trehalose and with 40% trehalose as the cryoprotectant during freezing. TX1 cells pretreated for 24 hours with 10% trehalose and cryoprotected with 40% trehalose during freezing showed 47% viability following thawing as determined by phenosafranin dye exclusion. The NP cells required a 3 to 6 day pretreatment with 10% trehalose and 40% trehalose as a cryoprotectant at the time of freezing for the recovery of viable cells. Growing cells were recovered when the C1 and NP cells treated as described were plated on agar-solidified medium following thawing.