Mechanisms regulating differential freezing tolerance of suspension cell cultures derived from contrasting alfalfa genotypes

A major factor limiting persistence of alfalfa (Medicago sativa L.) in the northern US is poor winter hardiness. Our hypothesis is that suspension cell cultures derived from dormant, winter-hardy alfalfa cultivars would cold acclimate and survive sub-zero temperatures better than cell cultures derived from non-dormant, non-hardy cultivars. Our objectives were (1) to determine if genetic differences in winter hardiness between dormant and non-dormant alfalfa were retained by suspension cells derived from these contrasting cultivars; and (2) to determine the physiological and biochemical bases for differences in freezing tolerance of suspension cells. Cell suspensions derived from '5262' (fall dormant) and '5929' (fall non-dormant) were cold hardened at 2 degrees C for 14 days. Cells were frozen in a cooling bath and cell survival determined by measuring 2, 3, 5-triphenyltetrazolium chloride (TTC) reduction. Cold acclimation improved cell survival of both cultivars to -5 degrees C when compared to unacclimated cells. Only acclimated cells of 5262 survived temperatures of -10 degrees C to -25 degrees C. The freezing tolerance of cold-acclimated 5262 cells was associated with high sugar and starch concentrations, lower alpha-amylase activities and slightly lower cell protein levels when compared to 5929. No differences in polypeptide composition were evident when comparing acclimated and unacclimated cells of 5929, but polypeptide composition did change with acclimation of 5262 cells. As expected, expression of RootCAR1 in 5262 cells increased with cold acclimation, but high levels of RootCAR1 transcript were unexpectantly found in both cold acclimated and unacclimated 5929 cells. With the exception of the RootCAR1 expression, many of the physiological responses of these alfalfa cell lines to cold acclimation were similar to those that have been reported for field-grown plants.