Ice Nucleation and Propagation in Cranberry Uprights and Fruit Using Infrared Video Thermography

Infrared video thermography was used to study formation of ice in leaves, stems, and fruit of cranberry (Vaccinium macrocarpon Ait. ‘Stevens’). Ice formed on the plant surface at –1 or –2 °C by freezing of a droplet of water containing ice nucleation-active bacteria (Pseudomonas syringae van Hall). Samples were then cooled to a minimum of –8 °C. Observations on the initiation and propagation of ice were recorded. Leaves froze only when ice was present on the abaxial surface. Once initiated, ice propagated to the stem and then readily to other leaves. In both unripe and ripe fruit, ice propagation from the stem to the fruit via the pedicel was not observed. Fruit remained supercooled for up to 1 hour after ice was present in the stem. Fruit could only be nucleated when ice was present at the calyx (distal) end. Red (ripe) berries supercooled to colder temperatures and for longer durations than blush (unripe) berries before an apparent intrinsic nucleation event occurred. These observations provide evidence that leaves are nucleated by ice penetration via stomata. The ability of fruit to supercool appears to be related to the presence of barriers to extrinsic ice propagation at both the pedicel and fruit surface. Stomata at the calyx end of the fruit in the remnant nectary area may provide avenues for extrinsic ice nucleation. 1995; Fuller and Wisniewski, 1998; Le Grice et al., 1993; Wisniewski and Fuller, 1999; Wisniewski et al., 1997). With this method, freezing events are imaged as water freezes and the heat of fusion is released. Recent improvements in temperature sensitivity, accuracy, and spatial resolution have increased the potential for using IR thermography to study the location of ice nucleation, as well as the direction and rate of ice propagation (Wisniewski et al., 1997). Freezing events in intact plant tissue can also be observed in real time with IR video thermography. By observing the occurrence of freezing events as well as the extent of subsequent ice propagation, low temperature survival mechanisms (tolerance or avoidance of ice formation) of various plant parts can be determined. Ice formation in plants can be induced by either intrinsic (Andrews et al., 1986; Gross et al., 1988) or extrinsic (Lindow, 1995; Lindow et al., 1982) sources. Once ice has been initiated, barriers and avenues to ice propagation, both within the plant and at the plant surfaces, determine the subsequent pattern of ice formation and are a factor in the survival mechanism (tolerance of extracellular ice or avoidance of ice by supercooling) employed by a given plant tissue or organ. Barriers to ice propagation within a plant can be permanent or temporary (i.e., propagation is impeded until growth of ice crystals can continue). Some barriers may also only be present at particular times of the year, or upon completion of particular stages of development. Supercooling of the buds of some temperate woody species appear to be, at least in part, related to cell shape and structure in the bud axis area (Ashworth, 1982; Quamme et al., 1995) and a lack of vascular differentiation (Ashworth, 1982, 1984; Ashworth et al., 1992) during the dormant period. In wheat (Triticum aestivum L.), the propagation of ice was impeded for several hours at stem and rachis nodes (Single, 1964). Avenues to ice propagation require areas (Pearce and Ashworth, 1992) and pores (Ashworth and Abeles, 1984) large enough for ice crystals to grow. Wounds, Received for publication 26 Mar., 1999. Accepted for publication 8 July 1999. This work was supported by the College of Agriculture and Life Sciences, University of Wisconsin (UW), Madison, and by the Wisconsin State Cranberry Growers Association. We thank the staff of the UW, Madison Biotron and Heidi Barnhill of the Russell Laboratories Scanning Electron Microscopy Facility for their technical assistance, as well as Mustafa Özgen for assistance with the SEM work. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked advertisement solely to indicate this fact. Graduate research assistant. Campbell-Bascom professor of horticulture, to whom reprint requests should be addressed; email: [email protected]. Scientist. Freezing injury can occur in plants only after the formation of ice within their tissues. Knowledge of the patterns of ice nucleation and propagation and the nature of potential ice nucleating agents is important to understanding freezing-stress-resistance mechanisms in plants and for the development of frost control strategies (Ashworth, 1992; Lindow et al., 1982). Ice formation in plants has been studied by thermal analyses (Ashworth and Davis, 1984; Ashworth et al., 1985; Cary and Mayland, 1970; Proebsting et al., 1982; Quamme et al., 1972; Yelenosky, 1991). These techniques of measuring the heat released by freezing water in plants, however, do not yield direct data about either the location of ice initiation or the temperature at that location. Microscopic techniques can reveal sites of ice formation and accumulation (Ashworth et al., 1992; Pearce and Ashworth, 1992), but the sites of ice initiation must also be inferred in these cases. Nuclear magnetic resonance microimaging (Price et al., 1997) shows patterns of freezing in complex organs, such as the flower buds of woody plants, but does not allow for the viewing of such events over larger pieces of plant material or whole plants, or for viewing the freezing events in real time. Infrared (IR) video thermography has recently been used to visualize ice nucleation and propagation in plants (Ceccardi et al.,