This protocol has the basic elements of the protocol described by Sukumaran and Weiser (1972) and later modified by Ristic and Ashworth (1993). This protocol has been further modified and used effectively to determine tolerance of many mutants in our lab (Ishitani et al, 1998; Guo et al, 2002). Many mutations or transgenic modifications may affect the ability of the plant to cold acclimate instead of, or in addition to, any effect on the basal level of cold tolerance. Therefore, it is often of use to compare both cold-acclimated (see Protocol III for cold acclimation procedure) and non-acclimated plants of each genotype under study.