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The influence of ionic composition and pH of extractant on the relationship between the extracted proteins and the cold tolerance of Vernal and Arizona Common alfalfa (Medicago sadiva L.) was examined. Five environments were used to induce different tolerance levels.The quantity of protein extracted from plants was influenced by the hardening environment, cultivar, and ionic composition and pH of 29 extractants. Extractants with a pH below 6 generally extracted less protein.The measured cold tolerance of the plants was correlated with the quantity of protein detected in many of the 14 regions of the electrophoresis gel columns regardless of extractant but was most dosely associated with the protein in either region 7 or 8 with nine of ten extractants. The purpose of this research was to examine the influence of ionic nature and pH of extractants on the relationship between extractable proteins and cold tolerance. Sources of variation in cold tolerance were cultivars of alfalfa and hardening environments. MATERIALS AND METHODSPlant Material. Cans with perforated bottoms and a volume of 3.78 liters were filled with a sandy loam soil and seeded with approximately 50 seeds of cold-tolerant Vernal or cold-sensitive Arizona Common alfalfa (Medicago sativa L.). The seedlings were allowed to grow in the greenhouse for 7 months and then were moved outside in April. The plants were allowed to reach a flowering stage before clipping to maintain high food reserve levels. Insects were controlled by spraying with malathion and Sevin.In late September, the containers were randomly assigned to one of five environments: (a) day and night temperatures of 7 and 2 C, respectively, and a photoperiod of 8 hr; (b) day and night temperatures of 16 and 10 C, respectively, and a photoperiod of 12 hr; (c) natural environment in the field at Morgantown, W. Va.; (d) greenhouse conditions maintained for vigorous growth; and (e) day and night temperatures of 27 and 21 C, respectively, with a photoperiod of 16 hr. Light in the growth chambers was supplied by six cool-white, 40-w fluorescent lamps and four 60-w incandescent bulbs/chamber. This lighting system provided a light intensity of approximately 12,900 lux at the plant tops. Relative humidity was regulated at approximately 80% in the chambers. A randomized block design was used with three replications for each environmental regime.After the plants had been subjected to the environments for 44 to 46 days, they were sampled for cold tolerance determinations and protein analyses. Plant roots were trimmed to a length of approximately 2.5 cm and crowns to 5 cm. All dead material and plant leaves were removed from the samples. A 10-g portion of prepared crown and root sample was used to measure cold tolerance using the technique of Dexter et al. (2). The remainder of each sample was immediately frozen in liquid N2, lyophilized, ground in a Wiley mill to pass through a 40-mesh screen, and stored at -20 C in air-tight vials that were enclosed in plastic bags.