Anatomical study of freezing injury in hardy and nonhardy alfalfa varieties treated with cytosine and guanine

Artifically hardened Vernal (hardy) and Caliverde (nonhardy) varieties of alfalfa, Medicago sativa L., were frozen to −8 and −15 °C at 1 °C/min to study the relationship between cold hardiness and freezing damage. Prior to hardening, foliar applications of 10 −3 m cytosine and guanine were applied. Regardless of the variety or chemical treatment, tissues usually froze at approximately −3 °C. Ice crystal formation was most pronounced in bud and leaf tissue. Microscopic examination revealed separation of the epidermis from the rest of the tissues apparently resulting from an abundance of ice crystals in these tissues. Tissues frozen to −15 °C showed greater damage than those frozen to −8 °C and Vernal showed considerably less damage than Caliverde. While cytosine treatment appeared to reduce histological damage in both varieties, guanine had little or no effect.