Freezing studies of plant cells point to membranes at cell surfaces and in cell organelles as being the primary site of freezing injury (5,7,8,11,12). The correlation of increases in phospholipid content in tree cells with extreme hardening has also focused attention on the involvement of membranes in the adaptation of the plant cell to freezing (13,14).While evidence exists showing that injury to the plasma membrane occurs when the whole plant cell is damaged by the dehydrative stresses of extracellular freezing (12), evidence pointing to plant mitochondria as also the targets in such cell injury is based largely on observations made on the freezing of the isolated organelle (3,4,6,7). Neither the important functional examination of the level of mitochondrial impairment sustained in situ during extracellular freezing of the plant cell nor whether the mitochondria, the cell, and other cellular membranes experience similar degrees of injury at freezing temperatures which are lethal to the cell has been reported.In this communication, we present evidence for the relative insensitivity of plant mitochondria in situ. to damage under extracellular freezing regimes which are clearly lethal to the cell.MATERIALS AND METHODS Plant Material. Coleoptiles (15-20 mm in length) were isolated from winter rye (Secale cereale L. cv. Puma) seedlings grown in the dark for 65 hr at 24 C (nonhardened) and from seedlings grown in the dark for 4 weeks at 2 C (hardened).Freezing Procedure. Freezing experiments were performed on the excised coleoptiles as follows. One-g batches of coleop- tiles were placed in polyethylene bags (10 x 15 cm) with moist cotton and sprayed with a fine mist of water before the bags were sealed with a 200 w bar sealer. The bags were first equilibrated at 0 C for 1 hr in a programmed freezer, the temperature was then allowed to drop at 1 C/hr to -20 C and thereafter at 3 C/hr until the desired temperature was attained. Cell survival after freezing was estimated by vital staining of the cells with neutral red or from observation of protoplasmic streaming. Survival of the isolated coleoptiles as a function of the freezing temperature was correlated with the survival of whole seedlings which had been subjected to identical freezing conditions and replanted on vermiculite flats as described by de la Roche et al. (2). Fast freezing of the coleoptiles was performed by immersing the polyethylene bags containing the coleoptiles into a beaker containing ethylene glycol cooled to -20 C. A thermometer placed in the polyethylene bag indicated that approximately 30 sec were required to reach -20 C. Freezing of the coleoptile tissues at different freezing rates was also examined on a microscope freezing stage. At rates of freezing comparable to the fast freezing described above, complete intracellular freezing was observed whereas only extracellular freezing was noted when the slower freezing rate was employed. Cell survival after extracellular freezing was dependent on the degree of hardening and the temperature to which ...