Extent of the problem.

The success of the agricultural production is heavily dependent on the weather. For example, in April 2014, the American Southeastern states suffered one of the worst frosts in recent years. Blasts of arctic air brought prolonged record-breaking low temperatures. In Florida, citrus, strawberries, tomatoes, beans and other crops were damaged. In anticipation of compromised supplies, prices for many affected fruits and vegetables shot up. Frost damage to crops is not unusual; it causes American farmers to lose billions of dollars annually.

Frost forms when an outside surface cools past the dew point. The dew point is the point where the air gets so cold, the water vapor in the atmosphere turns into liquid. If it gets cold enough, little bits of ice, or frost, form. The ice is arranged in the form of ice crystals. In general, -2 to -3° C frost over a period of at least an hour can be expected to cause damage to crops, -1° C for an extended period such as 3 to 4 hours can also cause similar damage.

Current solutions. Currently farmers’ attempts to prevent frost damage are decidedly low-tech. Methods include burning smudge pots to produce warm smoke; running wind machines to move the frigid air; and spraying water on the plants to form an insulating coat of ice.

It is a surprising experimental observation that frost protection can be achieved by preventing ice crystal formation on the plant surface. Indeed, this approach may be more important than inhibiting ice formation in the interior of the plant cell. Evidence suggests that ice crystals formed on the surface of plants must physically grow into the interior of the plant in order to initiate freezing in the plant. This can occur through stomates (very small openings in the epidermis of a leaf or stem through which gases and water vapor pass — singular = “stoma”) or cracks in the cuticle. Based on this data showing how freezing occurs, one method of frost protection might be by prevention of the ice nucleation of leaves by delaying the penetration of ice from a frozen droplet on the leaf surface. The efficiency of this protection was such that ice penetration was delayed on average by up to 2 h and in a short freezing test this was sufficient to enable a higher proportion of plants to supercool and avoid freezing and thus remain undamaged. Several new approaches to frost protection are under investigation. Exogenously applied cryoprotectants may provide a barrier that can prevent external ice from inducing plants to freeze. Many compounds have been screened, such as sorbitol or polyethylene glycol. However, most appear to either inhibit plant growth at active concentrations or only marginally effective in controlling ice nucleation in vegetative field crops. Some acrylic compounds have been marketed on the basis of giving frost protection by covering the leaf surfaces with an inert layer. Hydrophilic formulations of kaolin dust have been reported to protect tomato plants from frost damage.

Biologic solution.

Animals and plants living in cold climates produce natural antifreeze proteins (AFP) that serve as a survival mechanism and prevent organic fluids from crystallizing and forming ice. The production of antifreeze proteins in living things is one of the major evolutionary routes taken by a variety of organisms. First described in fish, they have also been reported in insects. Antifreeze protein activity has also been identified in many plants, but with low activity.

A synthetic biology solution to the frost damage problem is a spray-on film of a nonpathogenic strain of E. coli that covers the crop and contains a secreted antifreeze protein (AFP). AFP's possess the ability to bind to the ice crystals surface, inhibiting the formation of ice. The growing ice surface becomes energetically unfavorable for further absorption of water molecules as the surface curvature increases, leading to the stopping of ice growth.

RiAFP refers to an antifreeze protein (AFP) produced by the Rhagium inquisitor longhorned beetle. It is a type V antifreeze protein with a molecular weight of 12.8 kDa; this type of AFP is noted for its hyperactivity. R. Inquisitor is a freeze-avoidant species, meaning that, due to its AFP, R. inquisitor prevents its body fluids from freezing altogether. This contrasts with freeze-tolerant species, who’s AFPs simply depress levels of ice crystal formation in low temperatures. The antifreeze protein from the Rhagium inquisitor beetle is the most active antifreeze protein discovered to date — hundreds of times more potent than salt in inhibiting the formation of ice.

Rhagium Inquisitor

The story of a biotechnology solution (Frostban™) to frost damage frozen out by federal regulators.

An ice-minus bacterium is a common name given to a mutant of the common bacterium Pseudomonas syringae (P. syringae). This strain of P. syringae lacks the ability to produce a certain surface protein, usually found on wild-type P. syringae. This lack of surface protein provides a less favorable environment for ice formation when the bacteria cover plant surfaces.

Both the wild-type and mutant strains of P. syringae occur naturally. However, the ice-minus bacteria to be used for spraying crops are made on a large scale using recombinant DNA technology. In the mid 1980’s, Advanced Genetic Sciences, a pioneering agricultural biotechnology company headquartered in Oakland, California, developed Frostban, a bacterial-based treatment capable of reducing frost damage to fruit and nut crops. As the first U.S. field trial, Frostban became a lightening rod for opponents of the emerging biotechnology industry. In 1987, the ice-minus strain of P. syringae became the first genetically modified organism (GMO) to be released into the environment when a strawberry field in California was sprayed with the ice-minus strain of bacteria. The results were promising, showing lowered frost damage to the treated plants. The testing was very controversial and drove the formation of US biotechnology policy. Frostban was never marketed.

Broader anti-icing applications.

Anti-icing’s primary function is to prevent the bond of snow and ice freezing to aircraft and pavement surfaces. The composition of anti-icing fluids varies considerably depending upon the specific application and may include propylene glycol or ethylene glycol, sugar beet by-product added to salt brine, and potassium acetate. However, a common concern is the toxicity of these different formulations including corrosion to vehicles, impact on infrastructure, and, of course, damage to environment. A synthetic biology solution in the mold of PlantiFreeze may be an approach to these issues.