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Environmental and Stress Physiology

Regeneration of Freezing-Tolerant Spring Wheat (Triticum aestivum L.) Plants from Cryoselected Callus ¹

National Research Council of Canada, Plant Biotechnology Institute, Saskatoon, Saskatchewan, Canada S7N 0W9, Department des Sciences Biologiques, Université de Québec à Montréal, Montreal, Québec, Canada H3C 3P8, Department of Horticulture, Oregon State University, Corvallis, Oregon 97331

A cryoselection protocol has been developed that provides freezing-tolerant callus that, in turn, can regenerate plants with enhanced cold hardiness. Tolerant calli were selected from spring wheat (Triticum aestivum L.) callus by immersion in liquid nitrogen without addition of cryoprotectants. Less than 15% of the calli survived the initial challenge, whereas 30 to 40% of previously selected calli survived subsequent exposure. Seed progeny from five of 11 regenerant (R2) lines tested exhibited significantly enhanced tolerance to freezing at –12°C. Thus, cryoselection appears to involve at least in part, selection for genetic rather than epigenetic variants. Analysis of one callus line indicated that cryoselection did not induce significant alterations in lipid composition, adenylate energy charge, or freezing point. An increase in the soluble sugar component was detected. Changes were also detected in the protein complement of microsomal membrane and soluble protein extracts of cryoselected callus. In all, seven unique proteins ranging from 79 to 149 kilodaltons were identified. The results demonstrate that freezing tolerant callus can be isolated from a heterogeneous population by cryoselection, and factors that contribute to hardiness at the callus level are biologically stable and can contribute to tolerance at the whole plant level.