Effect of Temperature, Light, Nutrients and Dehardening on Abscisic Acid Induced Cold Hardiness in Bromus inermis Leyss Suspension Cultured Cells

The effects of culture conditions on abscisic acid (ABA)-induced freezing tolerance were determined in smooth bromegrass Bromus inermis Leyss cv. Manchar) cell suspension cultures. Bromegrass cultures initiated with 2 g fr wt of cells achieved maximum freezing tolerances (greater than −32°C) at 25 to 30°C in the presence of 75 to 100 μM ABA. High levels of freezing tolerance induced by ABA were correlated with high growth rates at 25 and 30°C. In control cells, incubation at 10°C induced optimum levels of hardiness with minimal growth. Prolonged exposure (6 weeks) of cells to 3°C, with or without ABA, increased freezing tolerance only by several degrees. Exogenous ABA concentrations greater than 100 μM were not inhibitory to growth. Repeated exposure to ABA, however, retarded growth and made the cells tolerant to temperatures below −40°C. Removal of ABA from the medium resulted in dehardening of the cells both at 25 and 3°C. Nitrogen had a marginal effect on ABA-induced hardening at 25°C, but inhibited age-dependent hardening of control cell cultures. Light had no effect on the freezing tolerance of cultured cells. Addition of 10% sucrose, 30 min prior to freezing, to bromegrass cells treated with ABA for 4 days increased freezing tolerance more than 15°C. These observations are discussed in relation to the contrasting behaviour of the low temperature and photoperiod dependent cold acclimation of plants