METHOD: Chemical Assay CONDITIONS: Growth Chamber - 14h light at 18-22C or 2-4C METHOD NARR: Five-week-old plants were subjected to 2-10 days of cold acclimation. Cycloheximide (CHI) was added to the culture medium. In additionlled methionine was added to the medium at approx. 3 hr. intervals. Cold hardiness was measured by an electrical conductivity test. The freezing temperature that caused a 50% loss of total leachate was designated as the kilcid (ABA) was extracted and purified and immunoassay was performed. Protein concentration was measured by a BCA protein assay kit. The incorporation rate of radioactivity in soluble protein fraction per gram fresh weight leafULTS NARR: The 1-day pretreatment with CHI resulted in less hardy plantlets. The 6hr-pretreatment with CHI resulted in full blockage of cold hardiness development. Return to CHI-free medium allowed hardiness to -8.3C. Initiation or 3 days of cold acclimation allowed plantlets to remain hardy at -8.3C. AUTHOR EXPLANATION: Cycloheximide is an inhibitor of cytoplasmic protein synthesis. Increase in free ABA during cold acclimation can be blocked i Theory 1: CHI blocks increase in ABA which is required as a trigger for synthesis of new proteins involved in cold acclimation. Theory 2: CHI blocks synthesis of cold acclimation proteins directly. Previous work sA increase is regulated by newly synthesized proteins in response to low temperatures, and the subsequent development of cold hardiness depends on ABA-mediated gene expression.