Induction of freezing tolerance in alfalfa cell suspension cultures

The effects of ABA, 2,4-D, kinetin and cold exposure on the cold hardiness of Medicago sativa L. cell suspensions were investigated. Cultures treated with 5×10(-5) M ABA at 2°C for 4 weeks in the absence of kinetin showed a 50% survival after freezing to -12.5°C, whereas cultures grown at 25°C under normal conditions tolerated freezing to only -3°C. The optimum ABA treatment of 5×10(-5) M for 4 weeks was effective only in combination with cold exposure. Of six cell lines tested, all showed different degrees of induced cold hardiness. The results suggest that ABA alone cannot induce freezing tolerance on alfalfa cell suspension cultures and that the deletion of kinetin and combination of low temperature and ABA is critical for the induction of cold hardiness in alfalfa cell suspension cultures.