The severity and duration of low temperatures that cause frost or freeze damage in plant tissues and organs are important abiotic factors limiting plant distribution and horticultural economics. Commercial growers often experience periodic frost/freeze damage to their crops even in areas where those species are normally well adapted. Many plant enthusiasts grow species at or even beyond their nominal U.S. Department of Agriculture (USDA) Plant Hardiness Zone ratings. Thus, there is great interest in developing methods to improve the cold tolerance characteristics of existing plant cultivars.
The variable ability of plant species to withstand frost or freeze conditions is genetically determined, and cold-tolerant plants, like their animal counterparts, exhibit two interrelated cold tolerance strategies: freeze tolerance and freeze avoidance (Lee, 1991). Freeze avoidance mechanisms use a variety of cryoprotectant molecules and other strategies that lower the intracellular freezing temperature (supercooling). For example, Francko and Wilson (2004) demonstrated that although cold-hardy palms (Palmae) exhibit a significant constitutive foliar cold-resistance capability, enhanced cold tolerance (an additional 5 to 10 °F) can rapidly be induced by exposure to above-freezing chilling temperatures. Freeze-point depression and thermocouple studies conducted on chinese windmill palm [Trachycarpus fortunei (Larcher et al., 1991)] and needle palm [Rhapidophyllum hystrix (Lokuge, 2006)] point to supercooling enhancement as a likely freeze avoidance mechanism involved in cold acclimation. However, supercooling alone is insufficient to protect plant tissues when temperatures drop low enough for ice crystals to form. Freeze tolerance mechanisms involve structural, anatomical, and biochemical adaptations to prevent or minimize damage to cells and tissues caused by ice formation. For example, addition of soluble silicates to soil media may enhance cold tolerance in crops via silica deposition-induced strengthening of cell walls (PQ Corporation, 2003). Larcher et al. (1991) suggested that low supercooling points noted in chinese windmill palm may have been due at least in part to cell walls strengthened with silica.
Thus, a topical spray designed to improve plant cold tolerance would ideally enhance both freeze avoidance and freeze tolerance mechanisms. To date, limited success has been reported in the development of foliar sprays designed to enhance supercooling in or on plant leaves and thus reduce frost, freeze damage, or both. These methods induce freezing point depression, inhibit ice-nucleating bacteria via inorganic salts, or use organic polymers to create a vapor barrier at the leaf surface to provide modest frost and freeze protection (from ≈31 to 28 °F) but have not been shown to enhance cold hardiness in frost-tolerant plants. Many existing technologies require the application of high concentrations of cryoprotectants dissolved in water [up to 10% by weight (Savignano and Hanafin, 1997)]. In the most successful patent in this genre that we are familiar with, Sekutowski et al. (2001) used a solution of hydrophobic materials to prevent ice crystal formation on leaf surfaces at temperatures at or below ≈21 °F and leaves were undamaged. In untreated leaves, water droplets froze and leaves were damaged at 29.3 °F. However, it is not clear whether this invention was persistent on leaf surfaces after precipitation events nor was an extensive range of plant species tested. Antidessicant spray manufacturers [e.g., Anti-Stress 2000 (Terra Tech, Eugene, OR) and Wilt-Pruf (Wilt-Pruf Products, Essex, CT)] claim that these agents may ameliorate freeze damage to plant foliage, but the mechanism appears to involve reduction of transpirational water loss rather than freeze damage per se (Francko, 2003).
The limited ability of existing spray products to protect frost-sensitive plants at temperatures much below freezing or to offer any significant freeze tolerance protection to cold-tolerant, USDA Plant Hardiness Zone 7 to 9a plants exposed to temperatures well below their typical tolerance limits (0 and 20 °F, respectively) may be due in part to the mechanism through which the above cryoprotectants function—increasing the solute content of extracellular water, intracellular cytoplasm, or both, and thus lowering the freezing point of these compartments. The very high solute concentrations required to significantly improve supercooling capacity can be toxic to cells and induce irreversible plasmolytic water loss from cell interiors, causing a detachment of the cell membrane from the cell wall and cell death.
We developed a novel foliar/floral/fruit topical spray containing two multiple-benefit, mechanistically distinct cryoprotectants, a surfactant, a cell wall reinforcing compound, and an antidessicant, which was designed to reduce both the environmental temperature at which foliage, flowers, and fruits first become damaged by sub-freezing cold and the environmental temperature at which plant structures are completely killed by cold. The formulation was designed to augment rather than replace a plant's native cold acclimation potential and to be effective over a broad range of species, both taxonomically and in terms of USDA Plant Hardiness Zone ratings.
Each of the five ingredients in our spray was chosen for its specific mode of action in hypothetically improving resistance to cold in plant tissues. In animals and insects, polyols like polyethylene glycol (PEG) appear to enhance cold tolerance through both freeze avoidance and freeze tolerance mechanisms. As solutes, they act colligatively via freezing point depression and non-colligatively by stabilizing cell membranes against freeze damage caused by ice crystals (Lee, 1991). Although no direct evidence for PEG-induced cold tolerance enhancement in plants has been reported, a plant tissue culture medium concentration of ≈2.5 mm (4% w/v) of high molecular weight PEG (PEG 8000, molecular weight 8 kDa; FisherBiotech, Fair Lawn, NJ) induces non-destructive cytorrhysis in arabidopsis (Arabidopsis thaliana), stabilizes cell membranes, and enhances production of the plant cryoprotectant proline (van der Weele et al., 2000; Verslues and Bray, 2004).
As a cell wall strengthening agent, AgSil 25, a proprietary solution of soluble potassium silicate (PQ Corporation, Valley Forge, PA) reduces crop stress due to drought, insect damage, and cold when applied as a foliar spray at a concentration of ≈10 mm soluble silicate (PQ Corporation, 2003). Glycerol at cellular concentrations of ≈5 mm is an important inducible low molecular weight cryoprotectant in animal systems but not in plants (Lee, 1991). As a non-plant-produced cryoprotectant, glycerol would not be expected to feedback-inhibit the production of natural plant cryoprotectants such as proline induced by cold acclimation. Most plants have waxy cuticles and a surfactant is required to aid assimilation of sprayed materials from the leaf surface into plant tissues. We employed a plant-specific silicone polyether copolymer-based surfactant (Silwet L-77; Setre Chemical Co., Memphis, TN) used with high efficacy (0.1% to 1% v/v) as an adjuvant to control plant diseases in many food crops (Tipping et al., 2003). As noted earlier, bicyclic oxazolidine-based antidessicant sprays are used in aqueous concentrations from 5% to 10% (v/v) to reduce transpirational water loss from leaves during cold weather, transplantation stress for several weeks after application, or both. We used Wilt-Pruf as an antidessicant in our spray to reduce both winter water loss and wash-off of the cryoprotective compounds introduced into leaf tissues by our spray methodologies.
Each ingredient mentioned earlier is biodegradable and non-toxic to plants, humans, and other animals. PEG and glycerol are used extensively as components of human food and consumer products, and AgSil 25, Silwet L-77, and Wilt-Pruf are routinely used in row crop and fruit horticulture to improve crop yield and vigor.
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