Freezing Tolerance Mechanisms of Suspension Cells Derived from Alfalfa Cultivars Differing in Fall Dormancy.
N.E. Kalengamaliro, J.A. Gana, S.M. Cunningham, and J.J. Volenec Department of Agronomy, Purdue University, West Lafayette, IN 47907-1150

A major factor limiting persistence of alfalfa in the Northern US is poor winter hardiness. Cold acclimation increases tolerance of fall dormant alfalfa to subsequent freezing temperatures. Our objective was to determine if suspension cells derived from cultivars exhibiting contrasting fall dormancy reaction also differed in freezing tolerance. Suspension cells derived from Pioneer 5262 (fall dormant, winter hardy) and Pioneer 5929 (nondormant, nonhardy) were acclimated at 2^oC for 14 d, then frozen for 30 min at 0, -5, -10, -15, -20, and -25^oC. Cells were slowly thawed and cell viability determined using 2,3,5-triphenyl tetrazolium chloride (TTC). Acclimated cells of 5262 survived to -25^oC whereas cells of 5929 died at temperatures below -5^oC (Fig. 1).

Figure 1. Freezing tolerance of alfalfa cells measured using TTC reduction (proportional to respiratory activity). 5262 is a fall dormant, winter hardy cultivar, whereas 5929 is a nondormant, nonhardy cultivar.

Sugar concentrations increased 31-fold during cold acclimation of 5262 when compared to cold acclimated 5929 cells. Starch concentrations of acclimated 5262 cells also were greater than those of acclimated 5929 cells. High levels of a-amylase activity occurred in cells derived from 5929 irrespective of temperature. In contrast, cold acclimation at 2^oC reduced a-amylase activity of 5262 cells to 1/4th that observed at 27^oC, and may have permitted starch to accumulate in cells grown at 2^oC. Cold acclimation reduced soluble protein concentrations of 5262 cells, while it had no effect on protein concentrations of 5929 cells. Several polypeptides disappeared during cold acclimation of 5262 cells, whereas no change in protein composition was observed during cold acclimation of 5929 cells. Cold acclimation and freezing tolerance of these cell lines mimicked winter hardiness responses of the cultivars from which the cells were derived.