Reprinted from AGRONOMY JOURNAL Vol. 62, Nov.-Dec. 1970, p. 715-719 Factors Influencing Freezing of Supercooled Water in Tender Plants' J. W. Cary and H. F. Mayland2 ABSTRACT may increase the tolerance of tender plants to frost Seedlings of beans (Phaseolus vulgaris), corn (Zea mays), (9). There is essentially no information on how pro- and tomatoes (Lycopersicon esculentum) were grown in tective mechanisms interact with the natural physical the greenhouse and then exposed to controlled freezing environment around plants, particularly with respect conditions in a growth chamber. Variables were adjusted to the stability of supercooled water in the plant. to determine the influence of plant water potential, freez- ing time, and external dew formation on the seedlings' There is an immediate need for a basic understanding susceptibility to frost injury. Freezing, detected visually of what does occur during periods of light frost. and by release of latent heat, progressed rapidly through- out plants with high water potential and was always lethal. Spreading of the ice phase was impeded in plants with METHODS low water potential. In this case, the freezing injury ap• peared as spots on the leaves which gradually enlarged 'Pinto' beans (Phaseolus vulgaris) 'Green Giant code 20' sweet to encompass the entire leaf as the exposure continued. corn (Zea mays), and 'Sioux' tomatoes (Lycopersicon esculentum) In general, the plant water supercooled before freezing. were grown' in the greenhouse (9- to 12-hour day length) in 4- Supercooled water within the plant appeared to be in- liter pots of silt loam soil. Eight to 12 corn or bean plants and ternally nucleated if the leaf temperature remained above about 20 tomato plants were grown per pot. The corn and bean th atmospheric dewpoint temperature. Under these condi- plants were allowed to reach a height of 15 to 20 cm, and to- tions root temperature, plant water energy, and duration matoes a height of 8 cm before they were exposed to various of the freezing period all influenced the stability of the cold stress conditions in the growth chamber. supercooled water. On the other hand, external inocula- Prior to the freezing experiments, plants were preconditioned tion prevailed when the freezing temperatures were ac- in the growth chamber for a minimum of 40 min in the dark companied by condensation of water from the air and at 20C to induce closing of stomata. The air temperature was subsequent formation of ice crystals on the leaves. One then lowered at a rate of 0.5C/min to various specified points important exception was noted when ice on corn seedling from —2 to —5C. Air temperatures in the growth chamber leaves failed to nucleate the supercooled internal plant and /eat surface temperatures were measured with 127 am water with a potential of —18 bars. copper-constantan thermocouples connected to a multipoint strip chart recorder. Thermocouples were kept in contact with Additional Key Words: Microclimate, Plant survival, the leaves by inserting the tip of the thermocouple into the Freezing temperatures, Plant water potential. leaf tissue. Air temperatures in the darkened growth chamber cycled ± 0.7G and plant leaf temperatures closely followed this cycling. Humidity in the chamber was not controlled, though HILE freezing damage to plants has been studied it was normally about 80% relative himidity when temperatures W extensively during the past 50 years, most of the were lowered to the freezing range. The low water-vapor pres- sure was a result of the condensation and freezing of vapor on emphasis has been directed toward plants that have the cooling fins as the air was recirculated in the chamber. the ability to cold-harden, particularly biennials and A frost layer less than 1 mm thick formed on the cooling fins perennials which survive low winter temperatures in within a few minutes after the temperature reached zero and a dormant stage (12). An equally important problem thereafter no change was observed, indicating there was not a gradual buildup of ice crystals which could break loose and be is frost damage to growing, nonhardened plants. Frost circulated in the air flow. damage to seedlings or immature crops results in seri- Following exposure to freezing temperatures, plants were al- ous economic losses. This phase of the frost problem lowed to warm to 20C and were returned to the greenhouse. The has received relatively little research aside from air percentage of fatally frozen plants in each pot was recorded the following day. Each treatment was replicated in at least five temperature control which may be attempted for or- pots with each pot treated as one random replicate and the re- chards and other specialty crops during periods of un- sults were statistically analyzed with the Duncan multiple range seasonably cold weather (10). test. Differences between treatment means of 20 to 25% and 25 to 30% were generally required for significance at the 5% and Growing plants may survive freezing temperatures 1% levels, respectively. in two ways. The water in the plant may supercool When needed, two identical growth chambers were used simul- and not form ice crystals, or the plant may tolerate taneously and temperatures were monitored continuously with some ice crystals without having a significant number the same multipoint recorder. In some experiments, the pots were insulated by wrapping the outside in aluminum foil and of cell membranes ruptured (6). Single (15) reported by covering the soil surface with approximately 2 cm of dry that wheat plants could, under certain conditions, be vermiculite. Plant water potential was measured (2) just prior kept at temperatures of —3 to —5C "almost indefinite- to initiating the freezing period. The soil moisture treatments ly" without having ice form in the tissue, provided were grouped into two general categories, wet and dry. Dry indicated that the surface appeared dry and pots had not been there were no ice crystals in the external environment. watered for at least 2 days. Wet indicated a moist soil surface When ice crystals do form in the tender, growing tissue which had been irrigated within the previous 12- to 24-hour of many plants, the cells rupture and die. However, period. Olien (13) has postulated that some plants contain The preceding description of methods applies to all of the experiments in which the survival of corn, beans, and tomatoes polysaccharides which impede the growth of ice crys- are reported. The individual treatments in each experiment tals so that the plant cells are not all killed. It has are pointed out as the results are discussed. Observations of ice been reported that the application of some chemicals formation in sugarbeets (Beta vulgaris), peas (Pisum sativurn), alfalfa (Medicago saliva), and lettuce (Lactuca saliva) were made in the growth chamber on individual potted plants rather than 'Contribution from the Northwest Branch, Soil and Water on plants in complete replicated designs. However, the observa- Conservation Research Division, Agricultural Research Service, tions were repeated from three to five times to give confidence USDA; Idaho Agricultural Experiment Station cooperating. Re- in their validity. ceived Dec. 22, 1969. Separate experiments were also conducted to measure super- 'Research Soil Scientists, Snake River Conservation Research cooling and the spontaneous freezing point of the intact plant Center, Kimberly, Idaho 83341. leaves. Thermocouples were inserted in the leaves and the 715 716 AGRONOMY JOURNAL, VOL. 62, NOVEMBER-DECEMBER 1970 chamber temperature lowered from 20C at a rate of about 0.25 affected differently by the pretreatments; nor did degree per minute until freezing was detected by latent heat either pretreatment cause a different response to freeze release in the leaves. damage at the 1% significance level. RESULTS AND DISCUSSION To test the hypothesis that nucleation might occur randomly from external contact with airborne parti- Supercooling and Internal Nucleation of cles, experiments were conducted simultaneously with Plant Water two growth chambers. In one chamber, plants were Preliminary observations suggested that beans with subjected to a continuous freeze lasting 10.5 hours. leaf temperatures of —2 to —3C could be kept in the Plants in the second chamber were also subjected to chamber for at least 2 hours before any ice formed. a total freezing time of 10.5 hours, except that every For periods longer than 3 hours, the number of plants 1.5 hours the temperature was raised to + 2C under having the ice phase slowly increased. After 24 hours, full light for a 5- to 7-min period. Short breaks in most plants were frozen. The difference in color in- the freezing cycle generally favored survival of the tensity between frozen and unfrozen leaves made it plants (Table 1). This was also true when the treat- possible to detect the initiation and progress of freez- ments were reversed in the two growth chambers. ing. This method of detection was verified by measure- Since the frost accumulation on the heat exchanger was ments of latent heat release and by the immediate constant after the first few minutes of cooling, the death of frozen tissue upon thawing. density of airborne ice particles, if present, should It was noted on recently irrigated bean plants that have been similar in the two chambers. Thus, if ice once freezing began somewhere in the plant, it pro- nucleation was from an external source, the survival gressed through the entire plant top within a few sec- in the two chambers should have been the same. It onds. On the other hand, if the soil was dry and the appears that, under these conditions where the rela- leaves were visually suffering from water stress, small tive humidity was always less than 100% so that no frozen spots appeared on the leaves and the spread of water condensed on the leaves, nucleation was oc- ice through the plant was slowed.