Freezing Injury in Potato Leaves 1 Received

Time-temperature profiles of freezing leaves from frost-resistant (Solanum acaule Bitt.) and frost-susceptible (Solanum tuberosum L. subsp. tuberosum Hawkes) types of potatoes did not reveal any major differences. The pattern of change in resistance of leaves to low voltage, low frequency current during freezing was different in the frost-resistant and susceptible leaves. In tissue sections from both types of leaves, cells freeze extracellularly at cooling velocities lower than 5 C per minute. Cells from leaves of resistant plants showed a higher osmotic pressure but not a higher water permeability than those from susceptible plants. The extent of injury caused by even very slow freezing was greater than that caused by equivalent isopiestic desiccation, particularly in susceptible leaves. The higher osmotic pressure in cells of leaves from resistant plants can account for the greater desiccation resistance but not for the frost resistance observed. Horticulltural Scienice, University of Minniesota, St. Paul, According to Mazur (6), the critical cooling rate above which intracellular freezing occurs depends mainly on the water permeability of the plasma membrane, the critical velocity being lower for cells with low permeability. The rate of cooling during natural frosts is low (usually a few degrees per hour) and intracellular freezing is believed to occur very rarely if at all in nature (4, 5). During extracellular freezing the protoplasts become desiccated, because at a given temperature the aqueous vapor pressure over ice is less than that over supercooled water, and therefore water moves from the protoplasts to the extracellular ice. Injury from extracellular freezing is generally believed to be caused by the desiccation which results (4-6). Olien (19), however, believes that extracellular ice can cause mechanical damage when crystallization is rapid. He suggests that even when crystallization proceeds slowly and the amount of ice formed is a continuous function of temperature, factors other than simple desiccation may be involved in injury (10). Sufficient work to test these hypotheses has not been done, especially in frost-susceptible plants such as potato. MATERIALS AND METHODS The yield of many crops, such as potato, is seriously reduced by partial and total frost damage to foliage during the growing season. Efforts to develop techniques for selecting resistant genotypes or reducing injury by cultural practices have been seriously hampered by our limited understanding of the critical lethal stress which occurs during freezing. When plant tissues are subjected to subfreezing temperatures, ice initially forms in the extracellular spaces where the water contains less dissolved substances and freezes at a higher temperature. Crystallization often does not proceed into the protoplasts because the plasma membrane acts as a barrier to nucleation (4, 9). At relatively low rates of cooling the protoplasts initially supercool, but soon equilibrate by losing water to the extracellular ice, which has a lower vapor pressure, and thus avoid freezing. At rapid rates of cooling the protoplasts may become substantially supercooled as the plasma membrane restricts the movement of cellular water to extracellular ice. When the system is sufficiently displaced from equilibrium, the supercooled protoplasts may become nucleated either spontaneously or through the membrane resulting in intracellular freezing (4,6). Intracellular freezing is almost lethal to plant and animal cells. Although extracellular freezing kills many tender plant species, numerous hardy perennial plants survive without injury at least at certain times of the year when they are frost hardened. 'Scientific journal series paper No. 7701 of the Minnesota Agricultural Experiment Station. This research was supported by a grant from the Rockefeller Foundation. Solanum acaule Bitt. (PI 210029) and S. tuberosum L. subsp. tuberosum Hawkes (var. Red Pontiac) were used in these studies. S. acaule is one of the hardiest of the tuber-bearing Solanum species while Red Pontiac, like most cultivated varieties, has little frost resistance. Genotypically uniform plants were grown from rooted stem cuttings. After 4 weeks in the greenhouse they were transferred to a growth chamber and maintained for two weeks at a 12-hr photoperiod and a day/ night temperature regime of 12 C/2 C, prior to study. The fifth through the eight leaf from the apex were used in all studies to reduce any age-dependent variability. Temperatures were monitored with cooper-constantan thermocouples and a multipoint recording potentiometer. Analysis of variance and t tests were used for assessing validity of results. Freezing Curve Studies. Excised leaves with thermocouples attached were cooled in 15 cm long glass tubes placed in a cooling bath. The leaves were inoculated with ice crystals by touching them with a frost-covered pipe cleaner introduced into the tubes when the temperature reached -2 C. The temperature was subsequently lowered at the rate of 10 per hr, while the leaf temperatures were recorded. Leaves were removed from the bath at different test temperatures and transfered to a refrigerator maintained at 0 C for slow thawing. After 2 hr in the refrigerator they were transferred to a humid chamber at room temperature and injury was estimated visually the next day. Microscopic Observations. Leaf sections were frozen at different rates of cooling on a microscope stage equipped with a thermoelectric cooler. The 60to 100-x. cross sections cut with a Vibratome were suspended in FC-47 fluorocarbon fluid: