Comparison of viability tests for assessing cross-adaptation to freezing, heat and salt stresses induced by abscisic acid in bromegrass ( Bromus inermis Leyss) suspension cultured cells

Abstract

Several viability assays were compared to determine the most sensitive and appropriate method for estimating the freezing, heat and salt tolerance of Bromus inermis Leyss cells cultured with or without 75 μM abscisic acid (ABA) for 4–7 days at 25°C. The sensitivity and reliability of individual viability tests depended on the type of stress applied and degree of injury. Regrowth, amino acid (AA) leakage and fluorescein diacetate (FDA) staining all gave comparable estimates of freezing tolerance. Triphenyl-tetrazolium chloride (TTC) reduction assays slightly overestimated freezing tolerance, but was most convenient. Polypeptide leakage from freeze-thawed cells, as determined by SDS-polyacrylamide electrophoresis (SDS-PAGE), revealed that protein leakage only occurred in ABA-treated cells frozen to −21°C or colder in contrast to control cells which leaked proteins following freezing to −5°C. The most sensitive test for assessing heat tolerance was regrowth, followed by FDA staining, when TTC. TTC tests overestimated heat tolerance compared with the other tests. The degree of overestimation was greater for heat tolerance estimates than for freezing tolerance estimates. It was partially improved by washing the cells prior to TTC assays. AA leakage tests were not appropriate for assessing heat tolerance, due to the erroneously high values of A 280 readings obtained. For estimating salt tolerance, TTC assays were most convenient and were in close agreement with regrowth measurements whereas FDA staining tended to overestimate it. In general, while regrowth was most sensitive and reliable, TTC was most convenient. All viability assays consistently showed that ABA induced cross-adaptation to freezing, heat and salt stresses in bromegrass cells without a prior exposure to any of these stresses.