Systems and methods for utilization of ice proteins

Abstract

Systems, compositions and methods for use of ice-structuring proteins (including antifreeze proteins and antifreeze glycoproteins) to prevent or reduce frost damage in seeds, seedlings, plants and plant products are disclosed. Compositions comprise one or more antifreeze proteins—derived from fish, insects, fungi, algae or bacteria—or modified variants thereof, and may include adjuvants, sugars, salts, oils, nanoparticles or microbial carriers. Delivery formats include foliar sprays, seed soaks or coatings, soil amendments, irrigation admixtures, nanoparticle-controlled release, and probiotic or endophytic microbes that secrete ice proteins in situ. Methods of application can lower freezing onset temperature, inhibit ice recrystallization to reduce tissue damage, and / or enhance plant cold-tolerance, depending on the chosen formulation. Representative formulations and experimental results (seed germination, seedling survival, reduced leaf freezing temperature and cryoprotection) are provided.

Description

Systems and Methods for Utilization of Ice ProteinsCOPYRIGHT NOTICE

[0001] A portion of the disclosure of this patent document contains or may contain material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent files or records, but otherwise reserves all copyright rights whatsoever.TECHNICAL FIELD

[0002] This disclosure relates to generation and utilization of antifreeze proteins, particularly in the agricultural field.BACKGROUND

[0003] Ice proteins, also known as Ice -Structuring Proteins (ISPs), are naturally occurring proteins that bind to water in ways that affect its ability to freeze either positively or negatively. Ice proteins that prevent water from freezing are known as Antifreeze Proteins (AFPs). AFPs follow a mechanism different from classic chemical antifreeze, allowing for strong antifreeze properties at low concentrations. AFPs come from a wide range of organisms, including many common foods. Certain AFPs and related antifreeze glycoproteins (AFGPs) are approved for use as food additives under some regulatory regimes.

[0004] Cold weather and frost events cause significant damage to crop production and are estimated to cause over $20 billion of global agricultural losses per year. A number of interventions are currently widely used to mitigate the effect of frost. These include:• Physical interventions, including wind machines, in-situ heaters, or manual helicopter flights designed to warm the air.• Physical barriers, which use covers or blankets to keep crops warm overnight.• Overhead continuous irrigation, where large volumes of running water are used to keep plants from freezing.• Insulating sprays, which can help to lock in heat but may be incompatible with other crop sprays.• Traditional antifreeze mixtures, which may help prevent frost but may also be limited in their use due to potential toxicity.• Hydrophobic sprays, which coat crops in a water-resistant coating to avoid icy buildup.Dkt. No.: 025526. OOlWOl Page 1 of 64 2025-11-20 PCT Patent ApplicationCrop biostimulants, which alter the metabolism of a plant to make it more tolerant to frost.

[0005] Many frost interventions available on the market are expensive and have limited effectiveness. Technologies that physically warm fields have high energy costs and low effectiveness due to thermal loss into the air, while antifreezes and biostimulants are highly limited by the range of concentrations that generally do not damage crops. Some provide only limited frost protection, while others have not been backed by any effectiveness studies. Against severe frost events, these interventions can be ineffective, and the cost is often too high for some agriculturalists. In addition to traditional mechanical interventions, various crop sprays, such as biostimulants and hydrophobic coatings, have been marketed to combat frost, though some of these products have been discontinued, and only a few have provided data on their effectiveness.

[0006] Addressing agricultural damage is complicated by the fact that frosts can vary significantly in their nature. In radiation (or “inversion”) frosts, cold air pools near the ground, allowing air-mixing devices like wind machines to provide limited frost protection. In advective frosts, cold air blows in on the wind, allowing physical barriers to offer limited frost protection. Frost damage to plants can take different forms as well. Wet frost, also known as white frost, occurs in humid environments where ice builds up outside the plant. Wet frost can damage the surface of a plant or even enter through its stomata, leading to ice damage that occurs because frost is able to accumulate on the plant’s surface. Dry frost, also known as black frost, occurs in drier environments where the internal tissues of a plant die without any visible external damage, resulting in ice damage that can depend on a plant’s internal state and hardiness. Typically, dry frost forms in the apoplast, a fluid-filled space between plant cells with lower concentrations of osmolytes that allow for easier freezing.

[0007] Prior strategies to confer AFP-mediated frost protection have focused on genetic engineering of plants to express AFPs. Such approaches have faced obstacles including inadequate folding, expression levels, protein stability, and distribution, as well as economic and practical limitations for application across many crop species. With dozens of crops and crop varieties currently suffering from frost, most frost damage could not be alleviated through genetic engineering without a huge investment of time, money, and expertise.

[0008] The application of recombinant protein in crop treatment remains highly theoretical. Nearly all proteins, when sprayed onto the surface of a plant, remain there, unable to cross the cell wall, pellicle, barrier cells, or some other obstacle whose main purpose is to prevent foreign substances from entering into a plant. A very small number of proteins or similar molecules are known to accumulate inside a plant when applied to the surface, but not as an intended function of thoseDkt. No.: 025526. OOlWOl Page 2 of 64 2025-11-20 PCT Patent Applicationapplications. Furthermore, any protein that does end up inside the plant is likely to end up in the apoplast, a space between plant cells, with only limited effect on plant or cellular function.

[0009] Better systems are needed to treat frost in crops. There remains a need for practical systems and methods that enable application of AFPs and related ice proteins to protect seeds, seedlings, mature plants and plant products from frost and freezing damage. To make antifreeze-based frost prevention a reality, what is needed are systems and methods to facilitate uptake of frost prevention / mitigation treatments by plants to reduce the effects of frost damage in crops.GENERAL DESCRIPTION

[0010] The disclosure provides systems, compositions, and methods for utilization of ice proteins, including antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs), to reduce freezing and freeze-induced damage in seeds, seedlings, mature plants, plant tissues, foliage, fruits and soil. In some embodiments, the AFPs and / or other biomolecules are delivered externally and / or exogenously into and around plant tissues and onto one or more plant surfaces. Embodiments comprise AFP-containing formulations suitable for exogenous application (for example, sprays, seed treatments, soil amendments, and microbial carriers), methods of producing and formulating AFPs, and application protocols adapted to achieve one or more of: (a) lowering the freezing temperature of water in or on plant tissues to reduce the rate of or lower the threshold for freezing; (b) reducing the extent of damage caused by ice crystals via ice recrystallization inhibition or cryoprotection; and (c) increasing plant cold-hardiness by induced metabolic response in crops.

[0011] A subset of antifreeze proteins possess specific features that make them uniquely suitable for internalizing into a plant when applied or sprayed. These advantageous features comprise one or more of: 1) extremely small size, on the order of 10 kDa or less, and / or with a molecular width in the low nanometers; 2) a tightly ordered tertiary structure (for example, stabilized by disulfide bonds or repeated motifs), which may help to keep the protein compact and able to fit through a smaller space, or increase its diffusion; and / or 3) a neutral charge, which makes these proteins less likely to interact with or bind to the plant surface and more likely to simply pass through barriers to entry.

[0012] Production methods comprise recombinant expression in bacteria, yeast (including cold-tolerant yeast), or other cell-based systems; cell-free expression; production of microbes that secrete or release AFPs on plant surfaces or within plant tissues; and production of microbes that deliver nucleotide sequences encoding AFPs to plant cells (for example, via Agrobacterium-based delivery) as a form of plant- targeted vaccine or transient expression system.Dkt. No.: 025526. OOlWOl Page 3 of 64 2025-11-20 PCT Patent Application

[0013] Proteins absorbed into a plant are more likely to end up in the apoplast, where they can only affect a limited set of plant functions; however, in the case of at least some of the embodiments disclosed herein, this is ideal. The apoplast of most plants tends to have a lower concentration of biomolecules and osmolytes than the cytoplasm inside cells; these osmolytes normally lower the temperature of freezing. Thus, freezing is more likely to begin in the apoplast than the cytoplasm. Some of the embodiments disclosed herein may have the ability to target or accumulate in the apoplast while also benefiting from this accumulation by increasing frost protection.

[0014] The systems and methods disclosed herein have the ability to prevent freezing of multiple points at once, in some embodiments. Frost in plants can form at a variety of locations in or around a plant, including on the surface or in the apoplast. In some embodiments, both internal and external frost may be prevented or reduced concurrently. The actual amount remaining on the surface vs. inside the apoplast depends on a number of factors including crop type and spray formulation, and formulations can be chosen to target higher or lower surface retention or absorption. Between 1 and 99% of the active ingredient remains on the surface of plants and between 1 and 99% is internalized into the plant’s tissue, cells, and / or apoplast, in some embodiments.

[0015] The systems and methods disclosed herein are capable of combating more than one mechanism by which frost typically damages crops by reducing the impact of frost through one or more of the following: 1) directly lowering the temperature at which water inside or outside of plants freezes; 2) reducing the damage caused by ice crystal formation to plant tissues; and 3) increasing natural cold-hardiness. In some embodiments, one or more of a single AFP-based ingredient, a combination of AFP and other biostimulant or antifreeze molecules, and a combination of 2-10 different AFPs is utilized to perform one or more frost mitigation methods.

[0016] AFPs may be used alone or in combination with other molecules to enhance antifreeze function, stability and crop compatibility. Supportive molecules include sugars and polyols, carbohydrates, salts and ions (for example citrate, succinate, malate, acetate), nutrients, plant hormones, polyphenols, surfactants and oil adjuvants, nanoparticles or nanostructured carriers, insulating agents, anti-ice- nucleation microorganisms, and other non-colligative or colligative antifreeze substances, as described in the detailed disclosure.

[0017] The described subject matter can be implemented in various ways to realize one or more of the following advantages.

[0018] The general description is provided to give a general introduction to the described subject matter as well as a synopsis of some of the technological improvements and / or advantages it provides. The general description and backgroundDkt. No.: 025526. OOlWOl Page 4 of 64 2025-11-20 PCT Patent Applicationare not intended to identify essential aspects of the described subject matter, nor should they be used to constrict or limit the scope of the claims. For example, the scope of the claims should not be limited based on whether the recited subject matter includes any or all aspects noted in the general description and / or addresses any of the issues noted in the background.DESCRIPTION OF DRAWINGS

[0019] The preferred and other embodiments are described in association with the accompanying drawings in which:

[0020] Fig. 1 depicts the effects of frost on a treated leaf and an untreated leaf. In freezing conditions, the untreated leaf is coated in ice and the treated leaf is merely wet.

[0021] Fig. 2 depicts engineered microbes producing an antifreeze protein and releasing it upon the microenvironment on the surface of a leaf (or internally).

[0022] Fig. 3 is a graph depicting the effect of antifreeze and temperature on frost protection.

[0023] Fig. 4 is a graph depicting survival rate of treated and untreated seeds when exposed to frost.

[0024] Fig. 5 is a photograph depicting AFP-treated seedlings and those treated with a negative control.

[0025] Fig. 6 is a graph depicting experimental results between a control spray treatment and an AFP spray treatment.

[0026] Fig. 7 depicts the direct effect of frost prevention applications on young Nicotiana plants.

[0027] Fig. 8 shows one embodiment of an electronic computing device that can be used as part of the systems and methods disclosed herein.

[0028] Fig. 9 shows various embodiments of the devices that can be included as part of the electronic computing device in Fig. 8.

[0029] Fig. 10 shows various embodiments of the electronic computing device in Fig. 8 communicatively linked to one or more additional electronic computing devices by way of a network.DETAIEED DESCRIPTION OF EMBODIMENTS

[0030] Before any embodiments of the present disclosure are explained in detail, it is to be understood that the systems and methods disclosed herein are not limited in their application to the details of construction and the arrangement of components set forth in the following description or illustrated in the accompanying drawings. TheDkt. No.: 025526. OOlWOl Page 5 of 64 2025-11-20 PCT Patent Applicationsystems and methods disclosed herein are capable of other embodiments and of being practiced or of being carried out in various ways. It should be noted that there are many different and alternative configurations, devices, and technologies to which the disclosed embodiments may be applied. The full scope of the embodiments is not limited to the examples that are described below.

[0031] In the following examples of the illustrated embodiments, references are made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration various embodiments in which the systems, methods, processes, and / or apparatuses disclosed herein may be practiced. It is to be understood that other embodiments may be utilized, and that structural and functional changes may be made without departing from the scope of the present disclosure.I. ICE PROTEIN APPLICATIONS

[0032] Disclosed herein are systems, compositions, and methods for the utilization of ice proteins comprising antifreeze proteins (AFPs) and related ice-structuring proteins to prevent or reduce negative effects of frost in seeds, seedlings, mature plants, and plant products. The disclosure provides compositional embodiments, methods of production and delivery, modes of application, potential formulae including some that may be able to enhance the activity or function of individual AFPs, or combine two or more AFPs for beneficial or synergistic effects, and experimental evidence of effectiveness in a range of crops and conditions.A. Agricultural Applications for Antifreeze Proteins

[0033] AFPs contribute to the frost tolerance of plants by one or more of the following three mechanisms:1. Reducing the freezing temperature of water. By reducing the freezing temperature of water, either in any of the various tissues inside of the plant or of water that collects on the surface of a plant, application of AFPs makes it more difficult for damaging ice to form, reducing the rate, frequency, or impact of frost damage to treated crops.2. Reducing the damage caused by water freezing. By affecting the structure of ice crystals as they form, AFPs reduce the damage to plant cells by forming ice or by the freezing process itself. Thus, AFPs are able to reduce the amount of direct or indirect damage suffered by crops that have partially or fully frozen.3. Increasing cold-hardiness. Some AFPs from fish, insects, plants, or microorganisms have the ability to change the expression of certain genes within plants or their mitochondria, increasing the strength of, switching on, or altering the natural pathways that exist in plants to resist damage from freezing. In this way, AFPs increase the cold hardiness of plant tissues, cells, and seeds regardless of whether orDkt. No.: 025526. OOlWOl Page 6 of 64 2025-11-20 PCT Patent Applicationnot the crop freezes, including reducing the damage suffered by plants from cold temperatures above the freezing point of 0° C, or in other cases where the plant is exposed to potentially damaging frost but does not freeze.

[0034] AFP formulations may comprise one or more different AFP components, each specifically selected to contribute to frost tolerance using one or more of these mechanisms. In some embodiments, an AFP formulation comprises two or more components wherein each component contributes to frost tolerance using at least one of the above mechanisms. In some embodiments, an AFP formulation comprises two or more components wherein each component contributes to frost tolerance using at least one of the above mechanisms, and wherein at least two of the frost tolerance mechanisms are utilized. Using more than one frost tolerance mechanism optimizes crop frost protection. Combined AFP formulations may be specifically designed for certain plant types, environments, and frost conditions providing the most optimal frost protection for specific applications.

[0035] Ice-structuring proteins used in the compositions and methods disclosed herein may be wild-type, naturally occurring proteins or modified derivatives thereof. Representative protein sources include, without limitation:• Type I, II, III, and IV antifreeze proteins, antifreeze glycoproteins, insect antifreeze proteins, or antifreeze proteins from other families• Antifreeze proteins, in combination with peptides, glycoproteins, carbohydrates, lipids, or glycolipids, in some embodiments; either in the same form as they naturally occur or with any chemical, post-transcriptional, or gene modifications designed to increase thermal hysteresis, ice recrystallization inhibition, antifreeze strength, stability, protease resistance, thermostability, long-term stability, increased disulfide bonding, decreased disulfide bonding, bio availability, bioabsorption, biodegradability, taste, color, melting point, freezing point, pH, pKI, energetic, entropic, or redox effects, persistence, duration, or retention in, on, or around any parts of any specific or general crops; rationally designed as a combination, fusion, or completely new peptide, carbohydrate, lipid, or combination or modification thereof, or taken from a naturally occurring organism; with such potential biological sources including but not limited to any plant, animal, fungus, protist, bacteria, archaea, or other organism adapted to, evolved for, or otherwise in possession of biomolecules capable of increasing frost tolerance or reducing frost damage, comprising: o fish such as Dissostichus mawsoni, Zoarces americanus, Pseudopleuronectes americanus, Myoxocephalus scorpius, Myoxocephalus octodecemspinosus, Osmerus mordax, ClupeaDkt. No.: 025526. OOlWOl Page 7 of 64 2025-11-20 PCT Patent Applicationharengus, Boreogadus saida, Arctogadus glacialis, Lycodapus fierasfer, Lycodichthys dearborni, Seleniolycus pectoralis, Seleniolycus robertsi, Anarhichas lupus, Mallotus villosus, Liposetta pinnifasciata, Ctenopharyngodon idella; o insects or other arthropods such as Tenebrio molitor, Belgica antarctica, Choristoneura fumiferana, Upis ceramboides, Dendroides canadensis, Hypogastrura harveyi, Ixodes scapularis, Tetranychus urticae, Nemoura arctica, Rhagium mordax, Rhagium imperator, Stephos longipes, Eurygaster maura; o plants such as Secale cereale, Triticum aestivum, Deschampsia antarctica, Solanum dulcamara, Daucus carota, Brachypodium distachyon, Avena sativa, Lolium perenne; algae such as Chlamydomonas raudensis, Chloromonas brevispina, Navicula glaciei, Chaetoceros neogracile; o fungi such as Typhula ishikariensis, Glaciozyma martinii, Glaciozyma antarctica, Glaciozyma spp., Leucosporidium creatinivorum, Candida parapsilosis, Leucosporidium antarcticum; o bacteria such as Flavobacterium frigoris, Marinomonas spp., Colwellia spp., Pseudomonas fluorescens, Pseudomonas syringae, Pseudomonas spp., Moraxella spp.Modifications and constructs

[0036] Ice proteins may be utilized in native / wild-type form or as modified variants. In some embodiments, ice proteins provide frost protection to crops either with wildtype proteins or with proteins modified for increased effectiveness, such as in any of the following ways:• alterations to glycosylation or other post-translational states;• fusion to production or purification tags (for example, His-tags, GFP, or other fusion domains) to improve yield, purification or detection;• concatenation (concatemer constructs) in which two to ten individual small AFP units are linked via peptide linkers either for use as a single multi-site ice-binding molecule or as a production strategy to increase yield and subsequently cleave into monomeric units;• truncation to retain an active core sequence while removing non-essential residues;• anti-degradation modifications, for example by covalently linking an antidegradation tag, by fusion to a thermostabilizing partner, or other stabilizingDkt. No.: 025526. OOlWOl Page 8 of 64 2025-11-20 PCT Patent Applicationchanges to protect the protein from heat, UV, or proteolysis; such modifications can provide stability for at least 12 hours at about 5°C and, depending on modification, potentially for longer durations and at higher temperatures;• addition of internalization sequences that increase uptake into plant tissues to lower the effective dose required for protection;• Addition of or fusion with a hydrophobic sequence or attachment tag, that helps them cling to the plant’s waxy pellicle for longer duration on the plant surface, but still allows the AFP to function as an ice nucleation inhibitor or ice-binding protein; and• addition of hydrophobic sequences or attachment tags that increase retention on plant waxy surfaces.Functional variants

[0037] The systems, compositions, and methods disclosed herein support use of functional variants of AFPs that retain one or more of ice-binding, thermal hysteresis, ice recrystallization inhibition, cryoprotective, or cold-hardiness-inducing activities relative to a corresponding wild-type protein. Functional variants may be obtained by point mutation, truncation, concatenation, fusion, increasing or decreasing the number of water-binding repeats, comprehensive mutational screens, Al-guided protein optimization from high-throughput screening, or other molecular engineering techniques that preserve antifreeze-related activity.Combination and adjuvant components

[0038] AFPs may be combined with molecules that enhance efficacy, stability, or crop compatibility. Suitable combination components include, without limitation:• simple sugars and polyols (e.g., sucrose, glucose, fructose, trehalose, maltose, lactose, mannitol, sorbitol, myoinositol, galactinol);• carbohydrates and oligosaccharides (e.g., raffinose, stachyose, starch, fructans);• plant byproducts such as wheat or other grain bran, husks or husk-derived fiber, cellulose, derived cellulose products;• salts and ions such as potassium, magnesium, sodium, calcium, ammonium, zinc, iron, selenium, or manganese, as chlorides, sulfates, nitrates, iodides, or citrates, succinates, malates, malonates, or acetates• nutrients and osmoprotectants (e.g., glycine betaine, glutamine, proline, choline, polyamines);Dkt. No.: 025526. OOlWOl Page 9 of 64 2025-11-20 PCT Patent Application• fatty acids such as oleic acid, linoleic acid, linolenic acids; phospholipids or lysophospholipids, including lysophosphatidylcholine and lysophosphatidylethanolamine; sphingolipids such as ceramide and glycosylceramide; glycolipids, galactolipids, phosphatidic acid, oxylipins;• plant hormones and signaling molecules (e.g., abscisic acid, jasmonic acid, brassinosteroids);• polyphenols including flavonoids, antioxidants, and membrane protectants;• other molecules such as trehalose-6-phosphate, broad or narrow spectrum amino acids, phenols, terpenes, reactive oxygen species scavengers such as ascorbate or glutathione;• water channel proteins such as aquaporins;• various other natural or synthetic small molecule ice nucleation inhibitors;• bactericidal or suppression agents or probiotic or biocontrol microorganisms that limit ice-nucleating bacterial pathogens (for example, INP-negative Pseudomonas strains that out-compete ice-nucleating Pseudomonas syringae).Production and delivery modalities

[0039] Ice proteins may be produced and delivered by various methods, comprising:• recombinant expression in bacteria, yeast, or other selected hosts (optionally using cold-tolerant strains or production conditions to preserve protein integrity);• cell-free production, either with or without added enzymes to ensure specific glycosylation or post-translational states of the proteins in question;• cell-free expression systems, optionally supplemented with enzymes or components to enable desired post-translational modifications;• use of live microbial carriers (for example, probiotic bacteria or fungi) engineered or selected to produce and secrete AFPs on plant surfaces or within tissues (e.g. symbiotic endophytes) for sustained release or in situ production;• production of microbes that deliver nucleic acid constructs (for example, via Agrobacterium) encoding AFPs to plant cells for transient expression;• product as a dehydrated powder, wettable granules, or wettable powders that can be stored and then rehydrated at the time of application;• product as a concentrated or frozen solution, to be mixed with additional water before use;Dkt. No.: 025526. OOlWOl Page 10 of 64 2025-11-20 PCT Patent Application• product as a spray to be applied to crops via spraying with traditional agricultural sprayer equipment. Figure 1 depicts an embodiment in which antifreeze biomolecules are applied to a plant through standard spraying processes. Applied biomolecules either remain on the surface or find their way into the apoplast of plant tissue, with the apoplast and surface making up the two main points of ice formation during a frost. With the antifreeze biomolecules present, the treated leaf does not freeze when subjected to freezing temperatures, while the untreated crop does freeze;• applied through spray, drip, or overhead irrigation, or any other irrigation system, via mixing into the water, either to act on its own or in combination with any ability of the given irrigation system to diminish the effects of frost;• applied through hand sprayer, or any other manual spraying method that targets individual portions of a plant such as buds, flowers, fruit, leaves, or other plant tissue;• applied via drones, nanotractors, quadcopters, or any other automated / robotic system capable of applying chemical treatments to a site or crop; and• applied by any direct contact method including but not limited to paintbrush, eye-dropper, dip stick, or other methods for applying a chemical to a plant.Application formats and timing

[0040] A formulation may be designed to aid spreading, sticking, spraying, absorption, penetration, retention, protease inhibition, tissue targeting, or any combination of the above, including but not limited to adjuvants including nonionic surfactants, anionic surfactants, cationic surfactants, organosilicone surfactants, polysorbates, crop oils, crop oil concentrates, seed oils, methylated seed oils, high- surfactant oil concentrates, urea ammonium nitrate, buffering or conditioner agents, compatibility agents, thickeners, drift control agents, suspension agents, foam markers, tank cleaners, colorants, multifunctional adjuvants, penetration and retention agents, nanoparticles, nanostructured materials, or any other molecules that aid in or form microscopic structures that aid in penetration, retention, stability, delivery, or activity of the active ingredients.Representative general formulations

[0041] Example formulations comprise:1-3 high-activity fish or insect AFPs to maximize frost activity alone or in combination, by binding to different planes of ice, each at a concentration between 0.01 and 50 mg / mL;Dkt. No.: 025526. OOlWOl Page 11 of 64 2025-11-20 PCT Patent Application• 1-2 fish, insect, yeast, or bacterial AFPs to minimize damage to frozen cells, by presenting a high ice recrystallization inhibition effect, each at a concentration between 0.01 and 50 mg / mL;• 1-2 fish AFPs to boost the cold hardiness of affected plants, each at a concentration between 0.01 and 50 mg / mL;• With any of these antifreeze functions alone, in combination with each other, or in an embodiment whose composition comprises: o 1-2 nonionic surfactants such as polysorbate or organosilicones, each at a concentration between 0.01-1%, to increase sticking, spreading, spraying, application, penetration or absorption; o 1-2 oil-based adjuvants such as seed oil, methylated seed oil, or crop oil, each at a concentration between 0.01-1%, to increase sticking, spreading, spraying, application, penetration or absorption; o 1-3 small organic ions known or suspected to be cofactors for certain AFPs, such as citrate, succinate, malate, malonate, or acetate, at a concentration of 0.01-1 M, to boost the thermal hysteresis, ice recrystallization inhibition, stability, or other beneficial properties of the active ingredient(s), and possessing additional beneficial effect in their ability to boost crop hardiness or lower the freezing point of water; o salt or buffer to stabilize the formula pH in a range of 4-10 with the effect of increasing AFP activity, with buffers comprising phosphate, ammonium, carbonate, or tris buffers at an ionic concentration of 0.01-2 M, to boost the thermal hysteresis, ice recrystallization inhibition, stability, or other beneficial properties of the active ingredient(s), and possessing additional beneficial effect in their ability to boost crop hardiness or lower the freezing point of water; o with potentially up to 5 additional molecules known to act as non- colligative antifreezes such as starch, cellulose, simple sugars, NaCl or other basic ionic salts, or polyethylene glycol, at concentrations each of 0.01-2 M, possessing additional beneficial effect in their ability to boost crop hardiness or lower the freezing point of water.

[0042] Additional embodiments comprise any combination of the above identified formulations.Dkt. No.: 025526. OOlWOl Page 12 of 64 2025-11-20 PCT Patent ApplicationApplications for different stages of plant development

[0043] AFP formulations may be applied at any stage of plant development, dependent upon species and composition of the AFP formulation. In some embodiments, an AFP formulation may be applied at one or more stages of plant development.

[0044] In some embodiments, an AFP formulation is applied as a seed treatment, via one or more of soaking seeds, spraying seeds, infiltrating seeds, or applied as a coating to seeds, either with a liquid or dry formulation, either intended to be absorbed before planting, gradually after planting, or any method designed to be absorbed after germination, either slowly or quickly, or any combination thereof. This seed treatment reduces the damage of frost before germination, during, or after germination, including persistent effects from AFP that may last after sprouting.

[0045] Frost protection in seeds may last at least 1 hour and up to 1 month after planting of treated seeds, depending on crop and condition, and some additional frost protection may persist in treated seeds for at least 1 week and up to 1 month after planting. In some embodiments, the seeds are treated using a spray, soaking, or dry powdered formulation applied by dipping, brushing, or mixing. In some embodiments, these seed treatment embodiments have similar ingredients and composition to spray - based embodiments, with the specific AFPs, concentrations, or supporting molecules potentially changing within the same ranges. Treatments may differ in composition based on the specific characteristics of the crop(s) to be treated in order to optimize effectiveness.

[0046] In some embodiments, an AFP formulation is applied to seedlings, young plants, mature plants, or any part thereof such as buds, flowers, leaves, fruits, or any other parts or tissues, with the goal of at least one of:• reducing the temperature at which frost forms at a specific or general location inside or on the surface of crops;• reducing the rate at which frost forms, increasing the time until frost appears, or reducing the percentage of the crop affected by frost;• decreasing the amount of damage experienced by crops or specific parts or tissues thereof that experience partial, total, or external-only freezing;• increasing the natural ability of the given crop, tissue, or local part or area of the crop to withstand frost.

[0047] Frost protection from foliar applications may last at least 1 hour and up to 1 month after application, depending on crop and condition. Modifications to the protein or formulation, as described above, may provide considerable boosts to the duration of frost protection. In some embodiments, a spray formulation such as the formulaDkt. No.: 025526. OOlWOl Page 13 of 64 2025-11-20 PCT Patent Applicationdescribed above, will function if sprayed as little as 2 hours before damaging frost conditions, assuming that this gives adequate time for spray evaporation, with the maximum amount of spraying time to achieve high protection ranging from 4 hours to 2 weeks, depending on the specific AFPs and modifications or selected formulation. In some embodiments, whole plants / general foliage are sprayed, or specific targeted spraying may be focused on buds, seeds, or fruit / fruiting vegetables of a given crop.

[0048] Potential examples of use cases for embodiments include:• A spray formulation with nonionic surfactants designed to increase spreading, and optimized for quick uptake, which can be sprayed on crops as little as 2 hours before a frost, and last for at least 1 and up to 4 nights’ worth of frost, allowing growers to respond quickly to a last-minute frost and gain protection without preparation or warning• A spray formulation optimized to persist longer inside of plants when sprayed, and designed to be sprayed at least 1 full day and up to 14 days before an expected frost, with some amount (at least 5% of thermal hysteresis and / or ice recrystallization inhibition activity) of frost protection designed to last as long, allowing growers flexibility in their schedule of when to apply it before an expected frost.

[0049] In some embodiments, an AFP formulation is applied at any stage of growth, to either whole plants or targeting specific parts or tissues of the plant, including seedlings, young plants, mature plants, individual fruits, buds, or flowers, or at a specific vertical height or spray pattern intended to maximize contact with intended plant part. In some embodiments, the AFP product may be applied with automated Al or machine learning-based detection and spraying system that uses drone or automated photography, lidar, thermal imaging, or other detection methods to identify specific spots or areas on crops for a controlled sprayer to dispense the chemical.

[0050] In some embodiments, the product can be applied as an in-furrow application, to be added to or mixed into the irrigation water to protect against frost in important root crops such as potatoes

[0051] In some embodiments, the product can be applied alone or in combination with other agricultural products, including herbicide, fertilizer, pesticides, biostimulants, growth promoters, etc.

[0052] In some embodiments, the product can be applied as a soil amendment or soil spray containing AFP or other antifreeze biomolecules that can reduce the impact of frost on roots or tubers through any of the methods described in this patent, or intended to be absorbed through roots or tubers or uptaken, distributed, or transmitted via plant vasculature or other internal system, or any combination thereofDkt. No.: 025526. OOlWOl Page 14 of 64 2025-11-20 PCT Patent Application

[0053] In some embodiments, an AFP formulation is applied as a probiotic, endophyte, or other microorganism, including symbiotic or mutualistic bacteria, fungi, algae or protist or other microbe capable of living for at least 1 hour on plant surfaces, inside plants, or in some other way on or around crops; transgenically engineered or naturally able to produce AFP or other antifreeze biomolecules, including antifreeze proteins, glycoproteins, polypeptides, lipids, or other naturally occurring or artificial molecules capable of reducing frost effects or damage; which molecules are either secreted by these microbes, or remain in the cytoplasm of the microbes with anywhere between 1 and 100 percent released after natural or induced microbe death; as a way to produce an antifreeze biomolecule in situ on, around, or inside of plants to be absorbed gradually or continuously for an amount of time between 1 hour and 10 years after treatment with the microbe; applied via spraying, contact application, seed treatment, syringe infiltration, direct injection, or other method of application or infiltration. Ideal microbes for this purpose may include Pseudomonas fluorescens and Pseudomonas aeruginosa, which may have some synergistic ability to prevent frost due to potential probiotic ability to fight bacteria that otherwise increase frost sensitivity.• Figure 2 depicts an embodiment wherein an antifreeze biomolecule is delivered by probiotic microbes or other carriers. The exposure of crops to an active antifreeze molecule can be prolonged by use of a carrier. This can be a probiotic microbe, e.g. a bacterium, fungus or other microbe capable of surviving on the surface of a crop with no negative risks, which is engineered to release antifreeze molecules over time, a probiotic microbe that produces antifreeze molecules and releases them when it dies, or a nanoparticle or other carrier capable of gradually releasing stored antifreeze molecules over time.

[0054] In some embodiments, an AFP formulation is applied through a nanoparticle or nanostructured formulation, which either remains on the surface, is absorbed into some part of the plant, or a combination thereof, capable of slowly releasing antifreeze biomolecules at a steady or variable rate, for somewhere between 1 day and 1 year after application.

[0055] In some embodiments, an AFP formulation is applied via a plant vaccine, or mRNA vaccine, or infiltrated, applied, or sprayed mRNA, or microorganism designed to release AFP mRNA into or on plants or otherwise induce AFP production, or virus designed to either induce AFP production or directly infect and / or transform crops with an AFP production gene.Representative general formulations

[0056] Example formulations comprise:Dkt. No.: 025526. OOlWOl Page 15 of 64 2025-11-20 PCT Patent Application• Formula 1: a solution of Zoarces americanus AFP, modified with an N- terminal His-tag for purification purposes that may or may not be cleaved. AFP at a concentration between 0.1 and 1 mg / mL, depending on desired strength and manufacturing cost, mixed with water or mixed with a salt buffer of pH between 6 and 8 and concentration up to 0.5 mg / mL and containing electrolytes commonly used in foliar nutrition. This formulation may be used as a crop spray or as a liquid seed treatment in which seeds are soaked before planting to improve their frost resistance and germination rate. This formulation is capable of lowering the freezing temperature of crops by between 0.1°C and 3° C, able to reduce damage from freezing, and may be able to increase a plant’s natural cold hardiness.• Formula 2: a solution of Dendroides canadensis AFP at a concentration between 0.1 and 1 mg / mL, in a citrate buffer of strength 0.1-1 M and pH between 6 and 8, and used as a crop spray with the goal of lowering the freezing temperature of crops by 0.1°C to 6° C.• Formula 3: a solution of up to 3 high-activity AFPs from Tenebrio molitor, produced transgenically or extracted directly, potentially chosen for their ability to maximize thermal hysteresis in combination. Each AFP at a concentration between 0.05 and 1 mg / mL, in a buffer of electrolyte salts or foliar nutrient salts at a concentration of up to 1 mg / mL. In some embodiments, formulation 3 may be applied as a crop spray with the goal of lowering the freezing temperature of crops by between 0.1°C and 8° C.• Formula 4: high-activity AFPs from Tenebrio molitor, produced transgenically, with 1-3 extra coils added to the protein structure + sequence to increase ice-binding ability. AFP at a concentration between 0.1 and 1 mg / mL, in a buffer of electrolyte salts or foliar nutrient salts at a concentration of up to 1 mg / mL. In some embodiments, Formula 4 may be applied as a crop spray with the goal of lowering the freezing temperature of crops by somewhere between 0.1°C and 8°C.• Formula 5: a solution of Rhagium imperator or Rhagium mordax AFP, produced transgenically, at a concentration between 0.1 and 1 mg / mL. In some embodiments, Formula 5 may be applied as a crop spray with the goal of lowering the freezing temperature of crops by between 0.1°C and 6° C.• Formula 6: a solution of Liposetta pinnifasciata AFP, chosen for its small size, which may be able to cross cell barriers and enter plant tissues more easily, with unnecessary residues potentially removed through a trial-and-error process to achieve an even smaller active molecule. AFP at a concentration of 0.5-10 mg / mL, in water.Dkt. No.: 025526. OOlWOl Page 16 of 64 2025-11-20 PCT Patent Application• Formula 7: a solution of Hemitripterus americanus AFP or other type II fish AFP, chosen for its ice recrystallization ability, which may be able to reduce the damage received by plants when freezing. AFP at a concentration between 0.1 and 1 mg / mL when applied as a crop spray, or used as an iceresistance supplement and combined with one of the above formulations for increased frost tolerance.• Formula 8: a solution of Ctenopharyngodon idella AFP, chosen for its small size, produced transgenically and used at a concentration between 0.1 and 1 mg / mL. This formulation is capable of lowering the freezing temperature of crops by between 0.1 and 6° C, and may be able to increase a plant’s natural cold hardiness, or used as a hardiness-boosting supplement and combined with one of the above formulations for increased cold hardiness.• Formula 9: a crop spray comprising a mixture of Zoarces americanus and Rhagium spp. AFPs each at a concentration between 0.1 and 1 mg / mL, depending on desired strength and manufacturing cost, mixed with either water or a salt buffer of pH between 6 and 8 and concentration up to 0.5 mg / mL and containing electrolytes commonly used in foliar nutrition. This combination of AFPs provides boosted thermal hysteresis via combination effects or exhibit the strongest thermal hysteresis out of the two AFPs used. It provides resistance to freezing, frost tolerance, and improved plant hardiness.• Formula 10: a dry seed treatment comprising a mixture of AFPs, such as Z. americanus and Rhagium spp. Instead of being mixed with water, AFP is provided as a lyophilized powder, with the two or more AFPs in the mixture combined in the same concentration as they would be in solution for a spray. This powdered AFP is mixed with several stabilizing agents chosen to help it coat seeds before planting. Any of the above formulations may be combined to form other AFP products.B. Agricultural Combination Antifreeze

[0057] AFPs are non-colligative antifreeze proteins that operate differently and at lower concentrations than traditional antifreeze. Because AFPs act through non- colligative mechanisms and reach high relative activity at low concentrations, combinations of AFPs with colligative antifreezes (for example, sugars, salts, polysaccharides) or with insulating agents may provide synergistic frost protection beyond that achieved by either approach alone. Combination products may be formulated to leverage AFP-specific ice-binding and ice-recrystallization inhibition together with bulk freezing-point depression of colligative agents. While traditional antifreeze alters the freezing point of water and poses a risk to plants, the systems,Dkt. No.: 025526. OOlWOl Page 17 of 64 2025-11-20 PCT Patent Applicationcompositions, and methods disclosed herein may create a synergistic effect when paired with a method that physically warms the plant or boosts its cold tolerance.

[0058] Figure 3 is a graph depicting the effect of antifreeze and temperature on frost protection. Antifreeze proteins can be combined with other anti-frost interventions. As non-colligative antifreezes, antifreeze proteins use a unique mechanism of action that does not interfere with other methods for protecting crops. Traditional colligative antifreeze follows traditional dynamics, where a higher total concentration of antifreeze molecules lowers the freezing temperature, and antifreeze effect scales with concentration. In contrast, AFPs reach close-to-maximum effectiveness at a very low concentration. As a result of this, mixing a high concentration of colligative antifreeze with a much lower concentration of AFP can depress the freezing point by significantly more than would be possible with only one of these two types of antifreeze.Microbial and probiotic delivery

[0059] Microorganisms may be used as carriers for AFPs, either as live probiotic strains engineered or selected to produce and secrete AFPs, as microbial ghosts or extracts, or as colonizing endophytes that release AFPs into the apoplast. Such microbes may be selected for survival on plant surfaces and for lack of pathogenicity. Example carrier taxa include Pseudomonas fluorescens and other commensal or symbiotic strains. In some embodiments, INP-negative strains may be used to out- compete ice-nucleating pathogens such as Pseudomonas syringae.Nanoparticles and controlled release

[0060] AFPs may be loaded into or onto nanoparticles, nanostructured carriers, or encapsulated matrices that allow for controlled release over timescales ranging from hours to months. Such delivery systems can extend the functional window of protection and can be tailored for seed coatings, foliar adhesion, or soil persistence.II. EXPERIMENTAL RESULTSRepresentative AFP activity metrics

[0061] AFP activity is reported as thermal hysteresis or freezing-point depression under stated conditions. In experimental contexts: CB-1 (a type III Zoarces americanus AFP) exhibited freezing-point lowering on the order of about 0.5-2°C depending on buffer / salt conditions when measured in vitro, and reduced leaf freezing temperature by about 1.2°C when infiltrated into spinach leaves. Other AFPs (for example, insect-derived AFPs) are reported as having higher maximum described activities (for instance, CB-3, CB-4, CB-5 reported with maximum described activities of approximately 7°C, 3.5°C, and 6°C respectively CB-3, CB-4, CB-5 reported withDkt. No.: 025526. OOlWOl Page 18 of 64 2025-11-20 PCT Patent Applicationmaximum described activities of approximately 3.5°C, 7°C, and 6°C respectively, in the disclosure).A. Frost Protection in Commercial Crops

[0062] The experiments described herein show effectiveness in various crop-frost scenarios and represent novel applications that extend beyond previous literature findings on AFP’s ability to protect crops against frost.

[0063] Effectiveness has been observed in preventing simulated frost in several tested scenarios. These include tests of treated seeds, sprayed seedlings, and whole plants, as well as direct infiltration into leaves.TreatmentsSeed Treatment

[0064] To evaluate AFPs' ability to protect seeds from freezing, various crop seeds were soaked in a mixture of AFP and water, then exposed to freezing temperatures before germination. Tested AFPs included type I and type III fish AFPs from Zoarces americanus and Liposetta pinnifasciata, insect AFPs from Tenebrio molitor, Dendroides canadensis, Eurygaster maura, and Rhagium mordax, and mixtures of antifreeze glycoproteins (AFGPs) from a number of cold-water fish, in formulations comprised of some combination of water, nonionic surfactants at concentrations up to 0.2%, basic salts at concentrations of up to 1 M, simple sugars at concentrations of up to 1 M, organic ionic buffer, or nonionic organic buffer at concentrations of up to 1 M. Seeds were soaked in specified formulations for between 6 hours and 1 week. These tests were conducted on tomatoes, wheat, soybeans, and corn, with many tests showing significantly improved germination rates when treated with AFP. The most detailed experiments involved tomato and wheat seeds, as shown in Figure 4, with AFP-treated seeds sprouting at more than twice the rate of seeds mock-treated with a control solution consisting of the same formulation / ingredients without AFP. In each set of experiments, all formulations and conditions containing an AFP were compared to a mock solution consisting of all ingredients and conditions except the AFP itself. This figure shows seeds treated with CB-1, a formulation consisting of Zoarces americanus AFP at a concentration between 0.05 and 1 mg / mL, with between 0 and 1 M sodium citrate buffer at a pH between 6 and 8.

[0065] In each of these tested crops, at least one CB-1 solution meeting the above specifications proved effective for at least one period or condition of frost exposure. Figure 4 demonstrates a concentration of 0.3 mg / mL and a buffer concentration below 0.1 M. The results demonstrate that at least one type of basic seed treatment can improve the rate of survival for seeds or seedlings in at least some commodity crops. AFP demonstrates a strong ability to protect seeds from freezing and maintain their viability under tested freezing conditions.Dkt. No.: 025526. OOlWOl Page 19 of 64 2025-11-20 PCT Patent ApplicationSprout Treatment

[0066] Some commodity crops are highly vulnerable to frost at the seedling stage. While the seed treatment tests demonstrated continued protection against frost after germination, the effectiveness of an AFP spray on young seedlings was also evaluated.

[0067] To test the AFP on young sprouts, untreated seeds were germinated for a few days in vitro, then sprayed with either AFP formulation or a mock formulation and exposed to overnight freezing temperature (-3°C) before planting. Tested AFPs included type I and type III fish AFPs from Zoarces americanus and Liposetta pinnifasciata, insect AFPs from Tenebrio molitor, Dendroides canadensis, Eurygaster maura, and Rhagium mordax, and mixtures of AFGPs from a number of cold-water fish, in formulations comprised of some combination of water, nonionic surfactants at concentrations up to 0.2%, basic salts at concentrations of up to 1 M, simple sugars at concentrations of up to 1 M, organic ionic buffer, or nonionic organic buffer at concentrations of up to 1 M. Mock formulations consisted of the same conditions and ingredients as AFP formulations but without AFP as an active ingredient. Figure 5 shows the results of one such experiment, where all control-treated (sprayed with water) sprouts exposed to -3°C frost died before or soon after planting, while 50% of AFP-treated sprouts treated with a CB-1 formulation as described above (with an AFP concentration of 0.3 mg / mL and a buffer concentration below 0.1 M) survived. This experiment was independently repeated over 15 times in corn, soybeans, and wheat, with at least one set of conditions in each crop consistently yielding positive results, as AFP protected a higher percentage of sprouts from otherwise lethal frost conditions. It was observed that the seedlings with the highest survival rate were those first treated as seeds and then again with spray treatment. This figure shows sprouts treated with CB-1, a formulation consisting of Zoarces americanus AFP at a concentration between 0.05 and 1 mg / mL, with between 0 and 1 M sodium citrate buffer at a pH between 6 and 8.Plant Treatment

[0068] In a third set of experiments, crop plants of various ages were sprayed with a simple AFP mixture, then exposed to freezing temperatures. Plants sprayed with the AFP mixture showed significant benefits, with AFP reducing the rate at which whole plants or individual leaves froze.

[0069] Figure 6 displays a summary of several results, which are averaged over multiple trials. The plants were maintained at -3°C after being treated with either an AFP spray or a mock-control spray, and were regularly monitored for the rate of freezing, measured as the percentage of leaves that had frozen. Figure 6 shows plants treated with CB-1, a formulation consisting of Zoarces americanus AFP at aDkt. No.: 025526. OOlWOl Page 20 of 64 2025-11-20 PCT Patent Applicationconcentration between 0.05 and 1 mg / mL, with between 0 and 1 M sodium citrate buffer at a pH between 6 and 8.

[0070] Other AFPs tested in similar conditions, on live plants, included type I and type III fish AFPs from Zoarces americanus and Liposetta pinni fas data, insect AFPs from Tenebrio molitor, Dendroides canadensis, Eurygaster maura, and Rhagium mordax, and mixtures of AFGPs from a number of cold-water fish, in formulations comprised of some combination of water, nonionic surfactants at concentrations up to 0.2%, basic salts at concentrations of up to 1 M, simple sugars at concentrations of up to 1 M, organic ionic buffer, or nonionic organic buffer at concentrations of up to 1 M. Mock formulations consisted of the same conditions and ingredients as AFP formulations but without AFP as an active ingredient. Rate of freezing was determined by tactile interaction and manually and quickly counting the number of leaves frozen on each plant at relevant time points. The freezing parameter used was the point at which all plants began to experience frost. At this point, it was observed that a significantly smaller percentage of the plants treated with AFP was frozen compared to the control samples.

[0071] In other experiments, plants treated with a CB-1 spray, with an AFP concentration of 0.5 mg / mL and a citrate buffer concentration of 0.5 to 1 M, had individual leaves removed and attached to thermal probes before placement inside a slowly-cooling freezing chamber, allowing the temperature of freezing for each leaf to be measured. The experiment was conducted in spinach and N. benthamiana, and consistently showed lower freezing temperature for treated plants vs. those sprayed with a control solution consisting of the same formulation without the AFP.

[0072] Taken together, AFP demonstrates direct frost protection in plants in several different conditions, for different crops and application strategies. An optimized formulation for delivery will provide similar protection in a variety of crops. The optimized formulation lowers the freezing temperature of leaves, buds, tissues, or fruits within individual crops by up to 8° C, given the maximum thermal hysteresis of AFP and can increase the average time to freeze under typical frost conditions by 10% to 300%, depending on the crop, formulation, and conditions. In some embodiments, the average time to freeze exceeds 300%.

[0073] Figure 7 depicts the direct effect of frost prevention applications. Two young Nicotiana plants exposed to two hours of freezing at -3° C. The left plant was mock- treated with a control formulation and shows significant damage to all leaves as a result of freezing. The right plant was treated with AFP formulation and shows no damage, as none of the leaves have frozen.Dkt. No.: 025526. OOlWOl Page 21 of 64 2025-11-20 PCT Patent ApplicationQuantitative Tests of Capability

[0074] Research has primarily focused on CB-1, a formulation consisting of Zoarces americanus AFP at a concentration between 0.05 and 1 mg / mL, with between 0 and 1 M sodium citrate buffer at a pH between 6 and 8. The active ingredient is a type III antifreeze protein from Z. americanus known for its moderate antifreeze activity, which can reduce the freezing point of water by 0.5 to 2°C, depending on buffer and salt conditions. Additionally, Z. americanus AFP possesses significant cryoprotective properties, which help minimize damage during freezing.

[0075] The product may comprise a combination of two or more AFPs: one that effectively lowers the freezing point and another that provides strong cryoprotection. An example of this combination may comprise one or more of:• 1-3 high-activity fish or insect AFPs chosen to maximize frost activity alone or in combination, potentially by binding to different planes of ice, each at a concentration between 0.01 and 50 mg / mL;• 1-2 fish, insect, yeast, or bacterial AFPs chosen to minimize damage to frozen cells, potentially by presenting a high ice recrystallization inhibition effect, each at a concentration between 0.01 and 50 mg / mL; and• 1-2 fish AFPs selected to boost the cold hardiness of affected plants, each at a concentration between 0.01 and 50 mg / mL.

[0076] For instance, Tenebrio molitor produces three high-activity AFPs that each bind to different planes of ice, providing a higher thermal hysteresis (freezing-point- depression) effect when all three are present at concentrations in the range of 0.01 to 50 mg / mL. This may be further supplemented with a Zoarces americanus AFP at a concentration in the range of 0.01 to 50 mg / mL, which provides more ice recrystallization activity than would be possible with a high-thermal hysteresis AFP alone (or a combination thereof) at the same concentration, allowing the solution to provide additional protection from damage in the event of freezing.Freezing Point Reduction

[0077] AFP’s primary mechanism of action is to lower the freezing point of water, causing solutions to stay liquid at temperatures where they would otherwise freeze. This effect also applies to the freezing temperature of plants, as individual leaves treated with AFP freeze at a lower temperature compared to mock-treated leaves.

[0078] To quantify the exact effect of the first active ingredient on the freezing temperature of leaves, a quantification study was conducted on spinach, a frostsensitive crop whose leaves emit an easily detectable heat pulse when they freeze. Thermal probes attached to cut spinach leaves monitor the temperature of each leaf as it is slowly cooled, recording the exact temperature at which each leaf freezes.Dkt. No.: 025526. OOlWOl Page 22 of 64 2025-11-20 PCT Patent Application

[0079] Spinach leaves were infiltrated with the AFP mixture, CB-1, or a mock solution, using syringe infiltration — a procedure in which pressure is applied to force a solution into leaves through surface-applied pressure with a needle-less syringe. After infiltration, treated leaves were gradually cooled from room temperature to - 12 °C in several replicates, with probes recording the temperature at which each leaf froze. Leaves treated with CB-1 froze, on average, at temperatures 1.2° C lower than the freezing point of mock-treated leaves. Under optimal and natural conditions in fish, CB-1 can theoretically reduce the freezing temperature of a solution by up to 1.6 °C, meaning that it maintains around -75% of its effectiveness when infiltrated into leaves.Cryoprotection

[0080] A second effect of AFP is its cryoprotective ability, as the protein binds to ice and reduces the size and growth rate of ice crystals, thereby minimizing freezing damage to cells. While AFP has been used for this purpose in medical cryopreservation and ice cream manufacturing, this effect also functions in plants.

[0081] To test the cryoprotective effect of AFP in plants, tomato sprouts were sprayed with either the CB-1 solution or a mock control, then the sprouts were subjected to a prolonged hold at -3 °C until 100% of the samples froze, before being planted in soil. In these experiments, 0% of the water-treated sprouts recovered after planting, whereas approximately 40% of the AFP-treated sprouts survived and continued growing.Cold Tolerance

[0082] The third and least well-understood effect of AFP is that certain antifreeze proteins, including CB-1, can upregulate a plant’s natural defenses against cold damage. Some crops damaged by frost are also affected by temperatures above freezing. Of all affected commercial crops, coffee is the most severely impacted, where even a 4°C cold spell can cause lasting damage to plants. Tests on coffee showed that, when incubated with CB-1 ingredient, coffee leaves show significantly less damage after incubation at 4°C. AFP also confers additional cold tolerance to treated seeds, with the seed treatment increasing growth rate and recovery speed for seeds germinated after incubation at 4°C. Both cold tolerance effects were demonstrated in multiple trials.

[0083] AFP has demonstrated effectiveness on improving the plant’s ability to fight frost.

[0084] Experimental results yield the following AFP embodiments: CB-1, a type 3 fish AFP; CB-4, a type 1 fish AFP; and CB-3 and CB-5, strong insect AFPs. The other three AFPs are significantly stronger than CB-1 (with maximum described activity of 1.6°C, 7°C, 3.5°C, and 6°C for CB-1, CB-3, CB-4, and CB-5, respectively), with CB-5Dkt. No.: 025526. OOlWOl Page 23 of 64 2025-11-20 PCT Patent Applicationadditionally expressing extremely high yield, at approximately a 10-fold increase over the others.

[0085] Combinations of any one or more AFP, application method, and product formulation, as described herein, are possible according to the requirements and characteristics of the crops to be treated and the planting environment.Illustrative Embodiments

[0086] The following is a description of various embodiments of the disclosed subject matter. Each embodiment can include one or more of the various features, characteristics, or advantages of the disclosed subject matter. The embodiments are intended to illustrate a few aspects of the disclosed subject matter and should not be considered a comprehensive or exhaustive description of all possible embodiments.

[0087] Pl. Systems and methods for utilization of ice proteins in a plurality of applications.

[0088] P2. The systems and methods of Pl, wherein one of the plurality of applications comprises frost management in agriculture.

[0089] P3. The systems and methods of P2, comprising one or more of: Type I, II, III, and IV fish antifreeze proteins, or antifreeze glycoproteins; Tenebrio molitor, spruce budworm, or other insect-derived antifreeze proteins; fungal antifreeze proteins derived from cold-tolerant yeast; bacterial antifreeze proteins; plant-derived antifreeze proteins; and diatom-derived antifreeze proteins.

[0090] P4. The systems and methods of P3, wherein cold-tolerant yeast may comprise one or more of G. antarctica and T. ishikariensis.

[0091] P5. The systems and methods of P3, wherein the one or more proteins are modified by one or more of: addition or removal of glycosylation states or any other post-translational states, a fusion domain that allows proteins to be produced at higher yield in bacteria or cold-tolerant yeast, or any other suitable protein producing host, concatemer constructs, truncation, anti-degradation modification, addition of or fusion with an internalization sequence, addition of or fusion with a hydrophobic sequence or attachment tag, and increasing anti-freeze effect.

[0092] P6. The systems and methods of P5, wherein concatemer constructs comprise low- weight proteins expressed as a chain of two to ten units linked by a peptide linker to be one of: used as a single multi-potent antifreeze molecule, and cleaved post-production to produce a higher yield of protein units.

[0093] P7. The systems and methods of P5, wherein truncation comprises preserving only an active core sequence.Dkt. No.: 025526. OOlWOl Page 24 of 64 2025-11-20 PCT Patent Application

[0094] P8. The systems and methods of P5, wherein anti-degradation modification comprises one or more of protein modification, protein fusion, or the covalent linkage of an anti-degradation tag.

[0095] P9. The systems and methods of P8, wherein anti-degradation modification results in a modified protein that is stable when applied at temperatures of at least 5°C and up to 50°C, and for at least 12 hours and up to 30 days.

[0096] P10. The systems and methods of P5, wherein increasing the antifreeze effect comprises one or more of: lowering critical concentration, raising total degrees of frost protection, and increasing thermal hysteresis.

[0097] Pll. The system and methods of P10, wherein increasing the anti-freeze effect results in between 1°C and 20°C decrease of the freezing temperature of water when applied at saturation.

[0098] P12. The systems and methods of P5, wherein production and delivery methods comprise one or more of: recombinant production in production yeast or bacteria, cell-free production, and production of bacteria or fungi.

[0099] P13. The systems and methods of P12, wherein recombinant production in production yeast or bacteria occurs between 10°C and 40°C.

[0100] P14. The systems and methods of P12, wherein bacteria or fungi one of: internally express or secrete naturally or are artificially engineered to produce single or multiple antifreeze proteins and / or other antifreeze molecules.

[0101] P15. The systems and methods of P12, wherein bacteria may be used to otherwise deliver frost tolerance to a plant by inducing production of natural antifreeze or delivering a nucleotide construct coding for an antifreeze gene.

[0102] P16. The systems and methods of P12, wherein proteins are produced in one or more formats comprising: dehydrated powder, wettable granules, or wettable powders, concentrated or frozen solution, spray, seed treatment, soil additive, and application in conjunction with herbicide, fertilizer, pesticides, biostimulants, or growth promoters.

[0103] P17. The systems and methods of P2, a product comprising two or more of: antifreeze protein activity, food-safe conventional antifreezes, biostimulant molecules, growth promoters, seaweed extracts, or pesticides, nutrient molecules, insulating molecule, and anti-Pseudomonas bacterium or INP-negative P. syringae that can out- compete P. syringae.

[0104] P18. The systems and methods of P17, wherein insulating molecule comprises an insulating foam or nanocrystalline cellulose.Dkt. No.: 025526. OOlWOl Page 25 of 64 2025-11-20 PCT Patent Application

[0105] P19. A composition for agricultural frost protection comprising: one or more ice-structuring proteins selected from the group consisting of antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs); and a carrier suitable for application to plant surfaces or plant tissues, characterized in that the one or more ice-structuring proteins are present at a concentration between 0.01 mg / mL and 50 mg / mL.

[0106] P20. The composition according to P19, wherein the one or more icestructuring proteins are derived from sources selected from the group consisting of: fish, insects, fungi, bacteria, plants, algae, and archaea.

[0107] P21. The composition according to any one of P19 to P20, wherein the one or more ice-structuring proteins comprise Type I, Type II, Type III, or Type IV antifreeze proteins.

[0108] P22. The composition according to any one of P19 to P21, wherein the one or more ice-structuring proteins are sourced from organisms selected from the group consisting of: Zoarces americanus, Liposetta pinnifasciata, Tenebrio molitor, Dendroides canadensis, Rhagium mordax, Rhagium imperator, Eurygaster maura, Glaciozyma antarctica, and Typhula ishikariensis.

[0109] P23. The composition according to any one of P19 to P22, wherein the one or more ice-structuring proteins exhibit one or more of the following characteristics: a molecular weight of 10 kDa or less; a molecular width in the low nanometer range; a tightly ordered tertiary structure stabilized by disulfide bonds or repeated motifs; and a neutral charge.

[0110] P24. The composition according to any one of P19 to P23, further comprising one or more adjuvant components selected from the group consisting of: nonionic surfactants at concentrations between 0.01% and 1%; oil-based adjuvants at concentrations between 0.01% and 1%; organic ionic cofactors selected from citrate, succinate, malate, malonate, and acetate at concentrations between 0.01 M and 1 M; buffer agents at concentrations between 0.01 M and 2 M providing pH in a range of 4 to 10; simple sugars or polyols selected from sucrose, glucose, fructose, trehalose, maltose, lactose, mannitol, sorbitol, myoinositol, and galactinol; and salts selected from chlorides, sulfates, nitrates, citrates, succinates, malates, malonates, and acetates of potassium, magnesium, sodium, calcium, ammonium, zinc, iron, or selenium.

[0111] P25. The composition according to any one of P19 to P24, wherein the composition comprises a combination of two or more different antifreeze proteins, wherein at least one antifreeze protein provides thermal hysteresis activity and at least one antifreeze protein provides ice recrystallization inhibition activity.

[0112] P26. The composition according to P25, wherein the combination comprises: one to three high-activity fish or insect AFPs at concentrations between 0.01 mg / mLDkt. No.: 025526. OOlWOl Page 26 of 64 2025-11-20 PCT Patent Applicationand 50 mg / mL for lowering freezing temperature; and one to two fish, insect, yeast, or bacterial AFPs at concentrations between 0.01 mg / mL and 50 mg / mL for minimizing damage to frozen cells through ice recrystallization inhibition.

[0113] P27. The composition according to any one of P19 to P26, wherein the carrier is formulated as one or more of: an aqueous solution, a spray formulation, a seed coating, a wettable powder, wettable granules, a concentrated solution, or a frozen solution.

[0114] P28. The composition according to any one of P19 to P27, wherein the one or more ice-structuring proteins are modified by one or more modifications selected from the group consisting of: alteration of glycosylation states; fusion to a purification tag; concatenation of two to ten AFP units linked by peptide linkers; truncation to retain an active core sequence; addition of anti-degradation modifications providing stability for at least 12 hours at approximately 5°C; addition of internalization sequences that increase uptake into plant tissues; addition of hydrophobic sequences that increase retention on plant surfaces; and addition of hydrophobic sequences that increase attachment to waxy plant pellicles.

[0115] P29. A method of protecting plants from frost damage comprising the step of: applying to one or more plant surfaces or plant tissues a composition according to any one of P19 to P28, characterized in that the applying occurs at a time between 2 hours and 14 days before an expected frost event.

[0116] P30. The method according to P29, wherein the applying comprises one or more application methods selected from the group consisting of: spraying, drip irrigation, overhead irrigation, hand spraying, drone application, robotic application, syringe infiltration, direct injection, painting, and dip application.

[0117] P31. The method according to any one of P29 to P30, wherein the one or more plant surfaces or plant tissues comprise one or more selected from the group consisting of: leaves, stems, buds, flowers, fruits, seeds, seedlings, and whole plants.

[0118] P32. The method according to any one of P29 to P31, wherein the method achieves one or more of the following effects: lowering the freezing temperature of water in or on plant tissues by between 0.1°C and 8°C; reducing ice crystal damage to plant cells through ice recrystallization inhibition; increasing plant cold-hardiness through induced metabolic response; and increasing time to freeze under frost conditions by 10% to 300% or more compared to untreated plants.

[0119] P33. The method according to any one of P29 to P32, wherein between 1% and 99% of the applied composition remains on the surface of plants and between 1% and 99% is internalized into the plant's tissues, cells, or apoplast.Dkt. No.: 025526. OOlWOl Page 27 of 64 2025-11-20 PCT Patent Application

[0120] P34. The method according to any one of P29 to P33, wherein the composition is applied in combination with one or more additional agricultural treatments selected from the group consisting of: herbicides, fertilizers, pesticides, biostimulants, and growth promoters.

[0121] P35. The method according to any one of P29 to P34, wherein frost protection persists for a duration between 1 hour and 14 days after application.

[0122] P36. The method according to any one of P29 to P35, further comprising monitoring environmental conditions using one or more sensors selected from temperature sensors, humidity sensors, and light sensors, and triggering the applying step based on detected conditions indicative of potential frost.

[0123] P37. Use of a composition according to any one of P19 to P28 for protecting plants from frost damage.

[0124] P38. The use according to P37, wherein the composition is applied to seeds, seedlings, mature plants, or plant tissues.

[0125] P39. The use according to any one of P37 to P38, wherein the composition is applied between 2 hours and 14 days before an expected frost event.

[0126] P40. The use according to any one of P37 to P39, wherein the protection achieves one or more of: lowering freezing temperature by 0.1°C to 8°C, reducing ice crystal damage, or increasing plant cold-hardiness.

[0127] P41. A method of treating seeds to confer frost protection comprising the steps of: contacting seeds with a composition comprising one or more antifreeze proteins (AFPs) or antifreeze glycoproteins (AFGPs); and allowing the seeds to absorb at least a portion of the composition, characterized in that the one or more antifreeze proteins are present at a concentration between 0.01 mg / mL and 50 mg / mL and the contacting occurs for a duration between 6 hours and 1 week.

[0128] P42. The method according to P41, wherein the contacting comprises one or more of: soaking seeds in the composition, spraying seeds with the composition, infiltrating seeds with the composition, or applying the composition as a coating to seeds.

[0129] P43. The method according to any one of P41 to P42, wherein the composition further comprises one or more components selected from the group consisting of: nonionic surfactants at concentrations between 0.01% and 0.2%, basic salts at concentrations between 0.01 M and 1 M, simple sugars at concentrations between 0.01 M and 1 M, and organic ionic or nonionic buffers at concentrations between 0.01 M and 1 M.

[0130] P44. The method according to any one of P41 to P43, wherein frost protection persists for a duration between 1 hour and 1 month after planting of the treated seeds.Dkt. No.: 025526. OOlWOl Page 28 of 64 2025-11-20 PCT Patent Application

[0131] P45. The method according to any one of P41 to P44, wherein the treated seeds exhibit improved germination rates of at least 100% higher than untreated seeds when exposed to freezing temperatures.

[0132] P46. The method according to any one of P41 to P45, wherein the seeds are selected from crop species comprising: tomatoes, wheat, soybeans, corn, and coffee.

[0133] P47. Use of a composition comprising one or more antifreeze proteins (AFPs) or antifreeze glycoproteins (AFGPs) at a concentration between 0.01 mg / mL and 50 mg / mL for treating seeds to confer frost protection.

[0134] P48. The use according to P47, wherein the seeds are contacted with the composition for a duration between 6 hours and 1 week prior to planting.

[0135] P49. A modified antifreeze protein characterized in that it comprises: a wildtype antifreeze protein sequence that has been modified by one or more modifications selected from the group consisting of: concatenation to form a construct comprising two to ten individual AFP units linked by peptide linkers; truncation to retain only an active core sequence while removing non-essential residues; fusion to an antidegradation element providing stability for at least 12 hours at approximately 5°C; addition of an internalization sequence that increases uptake into plant tissues; and addition of a hydrophobic sequence or attachment tag that increases retention on plant waxy surfaces.

[0136] P50. The modified antifreeze protein according to P49, wherein the wild-type antifreeze protein is selected from proteins derived from Zoarces americanus, Tenebrio molitor, Dendroides canadensis, Rhagium mordax, Rhagium imperator, Liposetta pinni fas data, Hemitripterus americanus, Ctenopharyngodon idella, and Eurygaster maura.

[0137] P51. The modified antifreeze protein according to any one of P49 to P50, wherein the concatenation construct comprises individual AFP units that are cleavable post-production to yield monomeric AFP units.

[0138] P52. The modified antifreeze protein according to any one of P49 to P51, wherein the modification results in one or more enhanced properties selected from the group consisting of: increased thermal hysteresis activity, increased ice recrystallization inhibition, increased stability, increased protease resistance, increased bioavailability, and increased duration of frost protection.

[0139] P53. The modified antifreeze protein according to any one of P49 to P52, wherein the protein exhibits a freezing point depression of between 1°C and 20°C when applied at saturation.

[0140] P54. Use of a modified antifreeze protein according to any one of P49 to P53 for protecting plants from frost damage.Dkt. No.: 025526. OOlWOl Page 29 of 64 2025-11-20 PCT Patent Application

[0141] P55. A microbial carrier system for delivering antifreeze activity to plants comprising: a live microorganism selected from the group consisting of bacteria, fungi, and algae, characterized in that the microorganism is engineered or naturally capable of producing one or more antifreeze proteins (AFPs) or antifreeze glycoproteins (AFGPs), and the microorganism is capable of surviving on plant surfaces or within plant tissues for at least 1 hour.

[0142] P56. The microbial carrier system according to P55, wherein the microorganism is selected from the group consisting of: Pseudomonas fluorescens, Pseudomonas aeruginosa, symbiotic bacteria, endophytic bacteria, probiotic fungi, and cold-tolerant yeast.

[0143] P57. The microbial carrier system according to any one of P55 to P56, wherein the microorganism secretes the one or more antifreeze proteins onto plant surfaces or into plant tissues.

[0144] P58. The microbial carrier system according to any one of P55 to P57, wherein the antifreeze proteins remain in the cytoplasm of the microorganism and are released upon natural or induced microbe death, with between 1% and 100% of the antifreeze proteins being released.

[0145] P59. The microbial carrier system according to any one of P55 to P58, wherein the microorganism produces antifreeze proteins in situ on, around, or inside plants for a duration between 1 hour and 10 years after application.

[0146] P60. The microbial carrier system according to any one of P55 to P59, wherein the microorganism comprises an INP-negative Pseudomonas strain capable of out-competing ice-nucleating Pseudomonas syringae.

[0147] P61. The microbial carrier system according to any one of P55 to P60, wherein the microorganism delivers nucleotide sequences encoding AFPs to plant cells via Agrobacterium-based delivery for transient expression.

[0148] P62. Use of a microbial carrier system according to any one of P55 to P61 for protecting plants from frost damage.

[0149] P63. A system for automated agricultural frost protection comprising: one or more sensors configured to detect environmental conditions indicative of potential frost, wherein the sensors are selected from the group consisting of temperature sensors, humidity sensors, light sensors, and imaging devices; an electronic computing device comprising a processor and memory, wherein the electronic computing device is configured to receive data from the one or more sensors and to process the data to determine frost risk; and an automated spraying system communicatively linked to the electronic computing device, characterized in that theDkt. No.: 025526. OOlWOl Page 30 of 64 2025-11-20 PCT Patent Applicationautomated spraying system is configured to apply a composition according to any one of P19 to P28 to crops based on instructions from the electronic computing device.

[0150] P64. The system according to P63, wherein the temperature sensors are positioned to measure temperature at one or more locations selected from: ambient air temperature, crop surface temperature, and soil temperature.

[0151] P65. The system according to any one of P63 to P64, wherein the electronic computing device is configured to execute instructions to compare detected temperature to a threshold temperature and to trigger the automated spraying system when the detected temperature satisfies one or more frost risk conditions.

[0152] P66. The system according to any one of P63 to P65, wherein the automated spraying system comprises one or more devices selected from the group consisting of: automated sprayers, drones, quadcopters, and robotic applicators.

[0153] P67. The system according to any one of P63 to P66, further comprising one or more display devices for presenting information about environmental conditions and frost protection status to a user.

[0154] P68. The system according to any one of P63 to P67, wherein the electronic computing device is configured to determine timing of application based on one or more factors selected from the group consisting of: time until expected frost, crop type, formulation type, and desired duration of protection.

[0155] P69. A controlled-release frost protection system comprising: one or more antifreeze proteins (AFPs) or antifreeze glycoproteins (AFGPs); and a carrier selected from the group consisting of nanoparticles, nanostructured carriers, and encapsulated matrices, characterized in that the carrier is configured to release the antifreeze proteins over a time period ranging from 1 hour to 12 months.

[0156] P70. The controlled-release frost protection system according to P69, wherein the carrier is configured for one or more of: seed coating application, foliar adhesion, and soil persistence.

[0157] P71. The controlled-release frost protection system according to any one ofP69 to P70, wherein the release profile is tailored to provide frost protection for a duration of between 1 day and 1 year after application.

[0158] P72. The controlled-release frost protection system according to any one ofP69 to P71, wherein the nanoparticles or nanostructured carriers facilitate one or more of: penetration into plant tissues, retention on plant surfaces, and stability of the antifreeze proteins.

[0159] P73. Use of a controlled-release system according to any one of P69 to P72 for protecting plants from frost damage.Dkt. No.: 025526. OOlWOl Page 31 of 64 2025-11-20 PCT Patent Application

[0160] P74. A method of producing recombinant antifreeze proteins for agricultural applications comprising the steps of: expressing one or more antifreeze proteinencoding nucleotide sequences in a host cell selected from the group consisting of bacteria, yeast, and cold-tolerant yeast; recovering the expressed antifreeze proteins from the host cells or culture medium; and formulating the recovered antifreeze proteins into a composition suitable for agricultural application, characterized in that expression occurs at a temperature between 10°C and 40°C.

[0161] P75. The method according to P74, wherein the host cell is selected from cold-tolerant strains that preserve protein integrity during production.

[0162] P76. The method according to any one of P74 to P75, wherein the antifreeze protein-encoding nucleotide sequences encode modified antifreeze proteins comprising one or more modifications selected from concatenation, truncation, fusion to purification tags, addition of anti-degradation elements, addition of internalization sequences, and addition of hydrophobic sequences.

[0163] P77. The method according to any one of P74 to P76, wherein the formulating step produces one or more product formats selected from the group consisting of: dehydrated powder, wettable granules, wettable powders, concentrated solution, frozen solution, and ready-to-use spray formulation.

[0164] P78. The method according to any one of P74 to P77, further comprising a cell-free expression step, optionally supplemented with enzymes or components to enable desired post-translational modifications including glycosylation.

[0165] P79. A combination composition for synergistic frost protection comprising: one or more non-colligative antifreeze proteins (AFPs) at a concentration between 0.01 mg / mL and 50 mg / mL; and one or more colligative antifreeze agents selected from the group consisting of sugars, salts, polysaccharides, starches, cellulose, polyethylene glycol, and sodium chloride, characterized in that the colligative antifreeze agents are present at concentrations between 0.01 M and 2 M, and the combination provides greater freezing point depression than either component alone.

[0166] P80. The combination composition according to P79, wherein the colligative antifreeze agents are present at concentrations that do not cause significant damage to treated crops.

[0167] P81. The combination composition according to any one of P79 to P80, wherein the composition achieves a freezing point depression of at least 0.5°C greater than the sum of freezing point depressions achievable by each component independently.Dkt. No.: 025526. OOlWOl Page 32 of 64 2025-11-20 PCT Patent Application

[0168] P82. The combination composition according to any one of P79 to P81, further comprising one or more insulating agents selected from insulating foam and nanocrystalline cellulose.

[0169] P83. Use of a combination composition according to any one of P79 to P82 for protecting plants from frost damage.Electronic Computing Device

[0170] Fig. 8 shows one embodiment of an electronic computing device 101 (alternatively referred to as an electronic controller, programmable logic controller, electronic control system, or electronic computing system) that can be part of the process of frost protection, either by sensing weather conditions indicative of a potential frost and alerting growers to apply the innovation, or by directly tying in to and activating automated spraying systems of some kind. Fig. 9 shows exemplary subcomponents that can be included as part of the electronic computing device 101.

[0171] The electronic computing device 101 includes one or more processors 103 (alternatively referred to as a digital processing unit or microprocessor) and memory 105 communicatively linked to each other by way of a system bus 107. In some embodiments, the electronic computing device 101 can also include one or more other interfaces and / or devices communicatively linked to the system bus 107.

[0172] For example, one or more storage devices 109 can be communicatively linked to the system bus 107 byway of one or more storage interfaces 111. One or more display devices 113 can be communicatively linked to the system bus 107 by way of one or more graphics interfaces 115. One or more input devices 117 can be communicatively linked to the system bus 107 by way of one or more input interfaces 119. One or more output devices 121 can be communicatively linked to the system bus 107 byway of one or more output interfaces 123. One or more communication devices 125 can be communicatively linked to the system bus 107 by way of one or more communication interfaces 127.

[0173] It should be appreciated that the electronic computing device 101 can have a variety of configurations. For example, in some embodiments, the various components of the electronic computing device 101 can be positioned near each other in one or more housings and on a single circuit board or multiple circuit boards communicatively linked together, or the like. In other embodiments, the various components of the electronic computing device 101 can be located remotely. For example, the one or more input devices 117 and / or the one or more output devices 121 can be located remotely or at a distance from the one or more processors 103 and / or the memory 105.Dkt. No.: 025526. OOlWOl Page 33 of 64 2025-11-20 PCT Patent ApplicationProcessor

[0174] Each of the one or more processors 103 is an electric circuit such as an integrated circuit that executes program instructions. The processor 103 can perform operations such as arithmetic operations, logic operations, controlling operations, and input / output (I / O) operations specified by the program instructions. In some embodiments, the processor 103 includes a control unit (CU), an arithmetic logic unit (ALU), and / or a memory unit (alternatively referred to as cache memory).

[0175] The control unit can direct the operation of the processor 103 and / or instruct the memory 105, arithmetic logic unit, and output devices 121 how to respond to instructions in the program. It can also direct the flow of data or information between the processor 103 and other components of the electronic computing device 101. It can also control the operation of other components by providing timing and control signals.

[0176] The arithmetic logic unit is an electric circuit in the processor 103 that performs integer arithmetic and bitwise logic operations. The arithmetic logic unit receives input in the form of data or information to be operated on and code describing the operation to be performed. The arithmetic logic unit provides the result of the performed operation as output. In some configurations, the arithmetic logic unit can also include status inputs and / or outputs that convey information about a previous operation or the current operation between the arithmetic logic unit and external status registers.

[0177] It should be appreciated that the processor 103 can have any suitable configuration. For example, the processor 103 can range from a simple processor specially built or configured to execute one or more programs for a specific application or device to a complex central processing unit configured to be used in a wide variety of ways and an equally wide variety of applications.

[0178] Examples of processors 103 can range from simple controllers to complex computing units. For instance, the processor 103 can be a microcontroller (MCU), a general-purpose central processing unit (CPU) implementing an instruction-set architecture such as ARM, RISC-V, or x86, a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA), or a more complex system-on-a-chip (SoC) that integrates multiple subsystems (e.g., memory, peripherals, and I / O) onto a single die or chiplet fabric. The processor 103 can be single-core or multi-core and can include specialized co-processors such as a graphics processing unit (GPU), a neural / Al processing unit (NPU), a cryptographic or security engine, a video-encode / decode accelerator, or any combination thereof.Dkt. No.: 025526. OOlWOl Page 34 of 64 2025-11-20 PCT Patent ApplicationMemory

[0179] The memory 105 (alternatively referred to as primary memory, main memory, or a computer-readable medium) is a semiconductor device or system used to store information for immediate use by the processor 103. The memory 105 is generally directly accessible to the processor 103. The processor 103 can read and execute program instructions stored in the memory 105 as well as store data and / or other information in the memory 105 that is actively being operated on. The memory 105 is generally more expensive and operates at higher speeds compared to the storage device 109. The memory 105 can be volatile such as random-access memory (RAM) or non-volatile such as read-only memory (ROM).

[0180] Exemplary memory technologies include static RAM (SRAM), dynamic RAM such as DDR4 or DDR5, low-power DRAM such as LPDDR4 / LPDDR5, and emerging non-volatile memories such as magnetoresistive RAM (MRAM), resistive RAM (ReRAM), or phase-change RAM (PCRAM). In some embodiments, particularly in microcontrollers or SoCs, the memory 105 can be integrated directly with the processor 103 on the same semiconductor die, including types such as SRAM or embedded flash memory.System Bus

[0181] The system bus 107 broadly refers to the communication system through which information is transferred between the processor 103, the memory 105, and / or other components such as peripherals that can be considered part of the electronic computing device 101. The system bus 107 can include a physical system of connectors, conductive pathways, optical pathways, wires, or the like through which information travels.

[0182] The system bus 107 can have a variety of physical configurations. In some embodiments, the system bus can be configured as a backbone connecting the processor 103, the memory 105, and / or the various devices and / or interfaces as shown in the figure. In other embodiments, the system bus 107 can be configured as separate buses that communicatively link one or more components together. For example, the system bus 107 can include a bus communicatively linking the processor 103, the memory 105, and / or a circuit board (the bus can alternatively be referred to as the front-side bus, memory bus, local bus, or host bus). The system bus 107 can include multiple additional I / O buses communicatively linking the various other devices and / or interfaces to the processor 103.

[0183] It should be appreciated that information shared between the components of the electronic computing device 101 can include program instructions, data, signals such as control signals, commands, bits, symbols, or the like. The information can be represented using a variety of different technologies and techniques. For example, inDkt. No.: 025526. OOlWOl Page 35 of 64 2025-11-20 PCT Patent Applicationsome embodiments, the information can be represented by voltages, currents, electromagnetic waves, magnetic fields or particles, optical fields, or the like.

[0184] The system bus 107 can also be used for other purposes besides sharing information. For example, the system bus 107 can be used to supply power from the power source 129 to the various devices and / or interfaces connected to the system bus 107. Likewise, the system bus 107 can include address lines which match those of the processor 103. This allows information to be sent to or from specific memory locations in the memory 105. The system bus 107 can also provide a system clock signal to synchronize the various devices and / or interfaces with the rest of the system.

[0185] The system bus 107 can use a variety of architectures and protocols depending on the application. For example, in a high-performance computing system, the system bus 107 can implement high-speed peripheral interconnects such as PCI Express (PCIe), Universal Serial Bus (USB), Compute Express Link (CXL), or Thunderbolt. In simpler electronic controllers or within a system-on-a-chip (SoC), the bus architecture can include a network-on-chip (NoC) implementing on-chip communication standards like the advanced microcontroller bus architecture (AMBA, e.g., AXI or CHI) interconnects such as the inter-integrated circuit (I2C) bus, serial peripheral interface (SPI), universal asynchronous receiver-transmitter (UART), or proprietary chiplet interconnects (e.g., Infinity Fabric). For connecting storage devices, architectures can include serial ATA (SATA), universal flash storage (UFS), or NVM express (NVMe) over a PCIe or CXL bus. Interfaces such as HBM, GDDR, or LPDDR5 can be used for high-bandwidth memory subsystems. Protocols such as CAN bus, Modbus, DNP, BACnet, ControlNet, or EtherNet / IP can be used for industrial applications. In some embodiments, the system bus 107 can incorporate wireless or optical interconnects for distributed systems.Program Instructions

[0186] The instructions stored in the electronic computing device 101 can include software algorithms and / or application programs. It should be appreciated that the software algorithms can be expressed in the form of methods or processes performed in part or entirely by the electronic computing device 101 or as instructions stored in a computer-readable medium such as the memory 105 and / or the storage device 109. Likewise, the software algorithms are shown in the flowcharts and described in the methods and / or processes.

[0187] It should be appreciated that instructions can take the form of entirely software (including firmware, resident software, microcode, or the like), entirely hardware, or a combination of software and hardware. If implemented in software executed by the processor 103, the information can be stored on or transmitted over a computer-readable medium such as the memory 105 and / or the storage device 109. InDkt. No.: 025526. OOlWOl Page 36 of 64 2025-11-20 PCT Patent Applicationsome embodiments, the instructions can be contained in any tangible medium of expression having program code embodied in the medium. In some embodiments, the instructions can be written in any combination of one or more programming languages, which can be text-based or graphical languages.

[0188] It should also be appreciated that the flowcharts, block diagrams, methods, and / or processes describe algorithms and / or symbolic representations of information operations. The algorithmic descriptions and representations are the means used by those skilled in the data processing arts to convey the substance of their work most effectively to others skilled in the art. These operations, while described functionally or logically, are understood to be implemented by software and / or hardware that can be readily and easily created from the functional or logical descriptions of the algorithms.

[0189] For example, the instructions can include an algorithm for making a decision — e.g., determining whether a parameter satisfies one or more conditions and performing various operations based upon the parameter satisfying the one or more conditions. This can be represented in the instructions with a conditional statement or conditional expression written in a programming language. An example of such a conditional statement or expression is shown below. It should be appreciated that the syntax for the conditional statement or expression will vary depending on the chosen programming language. if conditionlsatisfied then perform operation 1 elseif condition2satisfied then perform operation 2 elseif condition3satisfied then perform operation 3 else perform operation 4; end if;

[0190] The instructions can be used to perform a variety of operations. For example, the instructions can be used to control the receipt and processing of data from the input devices 117. The instructions can also be used to control hardware such as any of the output devices 121.

[0191] In some embodiments, the instructions can include firmware 131, which can be a basic input / output system (BIOS), a more modern unified extensible firmware interface (UEFI), or a bootloader. The instructions can further include a hypervisor or virtual-machine monitor, an operating system 133, one or more application programs 135, program data 137, and the like. In simpler embodiments, such as an embeddedDkt. No.: 025526. OOlWOl Page 37 of 64 2025-11-20 PCT Patent Applicationcontroller, the instructions can include solely firmware that directly controls the hardware without a separate operating system. In some embodiments, the firmware supports secure boot and over-the-air (OTA) updates to maintain system integrity.

[0192] In some embodiments, the instructions and / or program data 137 can include or be generated from a trained machine learning model, such as a neural network, decision tree, or support vector machine. Such models can be configured to process input data and generate outputs for controlling the device or making decisions.

[0193] In some embodiments, the instructions are used to control the rate or timing of spraying, or the portion of the plant or specific plants to which the product may be applied by some form of automated or software-controlled sprayer or drone.Storage Device

[0194] Each of the one or more storage devices 109 (alternatively referred to as secondary memory, or a non-transitory computer-readable medium) is a device or system used to store information that is not needed for immediate use by the processor 103. The storage device 109 can be communicatively linked to the system bus 107 by way of a storage interface 111. The storage device 109 is generally not directly accessible to the processor 103. The storage device 109 is generally less expensive and operates at lower speeds compared to the memory 105. The storage device 109 is also generally non-volatile and used to permanently store the information.

[0195] The storage device 109 can take a variety of physical forms and use a variety of storage technologies. For example, in some embodiments, the storage device 109 can be in the form of a hard disk storage device, solid-state storage device, optical storage device, or the like. The storage device 109 can use a variety of storage technologies. For example, storage can be based on semiconductor memory such as flash memory, which is used in solid-state drives (SSDs), embedded MultiMediaCards (eMMC), secure digital (SD) cards and variants (microSD, UHS-II), or USB flash drives. Other technologies include magnetic disks (e.g., hard disk drives), optical discs (e.g., CD, DVD, or Blu-ray), or, for archival purposes, magnetic tape. In some embodiments, the storage device 109 can include or interface with remote or cloud-based storage accessible via a network.Display Device

[0196] Each of the one or more display devices 113 (alternatively referred to as a human-machine interface (HMI) or screen) is a device that visually conveys text, graphics, video, and / or other information. In some embodiments, the information shown on the display device 113 exists electronically and is displayed for a temporary period of time. It should be appreciated that the display device 113 can operate as an output device and / or input device (e.g., touchscreen display or the like).Dkt. No.: 025526. OOlWOl Page 38 of 64 2025-11-20 PCT Patent Application

[0197] The display device 113 can be communicatively linked to the system bus 107 by way of one or more graphics interfaces 115. In some embodiments, the graphics interface 115 can be used to generate a feed of output images to the display device 113. In some embodiments, the graphics interface 115 can be a separate component such as a dedicated graphics card or chip or can be an integrated component that is part of or a subset of the processor 103.

[0198] It should be appreciated that the display device 113 can include a variety of physical structures and / or display technologies. For example, in some embodiments, the display device 113 can be a screen integrated into a specific application or technology, a separate screen such as a monitor, or the like. The display device 113 can also be a liquid crystal display, a light emitting diode display, a plasma display, a quantum dot display, or the like.Input Devices

[0199] Each of the one or more input devices 117 is a physical component that provides information to the processor 103 and / or the memory 105. The input device 117 can be communicatively linked to the system bus 107 by way of one or more input interfaces 119. The input device 117 can be any suitable type and can provide any of a variety of information. For example, the input device 117 can be a digital and / or analog device and can provide information in a digital or analog format. Also, the input device 117 can be used to provide user input for controlling the electronic computing device 101 or operational input for controlling aspects of a specific application.

[0200] The input device 117 can include one or more sensors 139 and / or one or more other miscellaneous input devices 141. It should be appreciated that the input device 117 is not limited to only providing information. In some embodiments, the input device 117 can also receive information. Such devices can be considered both an input device 117 and an output device 121.

[0201] The miscellaneous input device 141 can include a variety of devices or components. In some embodiments, the miscellaneous input devices 141 can include switches such as limit switches, level switches, vacuum switches, pressure switches, or the like, as well as buttons including pushbuttons or the like. In some embodiments, the miscellaneous input devices 141 include user interface components such as a pointing device, for example a mouse, text input devices, for example a keyboard, a touch screen, or the like.Sensors

[0202] Each of the one or more sensors 139 can be used to provide information about a wide variety of measured parameters. In general terms, the sensor 139 is used to measure or detect information about its environment and send the information to the processor 103 and / or the memory 105. In some embodiments, the sensor 139 canDkt. No.: 025526. OOlWOl Page 39 of 64 2025-11-20 PCT Patent Applicationoperate as a transducer and generate an electrical signal as a function of the measured parameter. The electrical signal is communicated to the processor 103 and / or the memory 105 where it can be used for a variety of purposes.

[0203] The sensor 139 can be a digital sensor and / or an analog sensor. For example, in some embodiments, the sensor 139 provides digital information to the processor 103 and / or the memory 105. In other embodiments, the sensor 139 provides analog information to the processor 103 and / or the memory 105. Also, in some embodiments, the information can be converted from one type to the other — e.g., from digital to analog or from analog to digital.

[0204] The sensor 139 can measure the parameter directly (i.e., direct measurement) or indirectly (i.e., indirect measurement). A direct measurement sensor directly measures the parameter itself. An indirect measurement sensor measures a secondary parameter that can be translated into the parameter of interest.

[0205] The sensor 139 can communicate information to the processor 103 and / or the memory 105 in a variety of ways and / or using a variety of protocols. In some embodiments, the sensor 139 can be a protocol-based sensor that uses a protocol to communicate with the processor 103 and / or the memory 105, or it can be a nonprotocol-based sensor that does not use a protocol to communicate with the processor 103 and / or the memory 105. A protocol-based sensor communicates with the processor 103 by sending a data stream by way of a communication protocol. In some embodiments, the protocol-based sensor includes a separate processor that is part of the sensor and used to communicate using the protocol.

[0206] It should be appreciated that the information provided by the sensor 139 can be used in a variety of ways by the processor 103. For example, in some embodiments, the processor 103 can compare the information to a setpoint. In some embodiments, analog information is amplified before being compared to the setpoint.

[0207] In some embodiments, the sensor 139 can be used to measure one or more parameters. Some examples of what the sensors 139 can be used to measure are described herein. In some embodiments, the sensors 139 can include micro-electro-mechanical systems (MEMS) based sensors. For example, the sensors 139 can include an inertial measurement unit (IMU), which can comprise an accelerometer for detecting linear acceleration and a gyroscope for detecting angular velocity. Other examples include magnetometers for detecting magnetic fields, Global Navigation Satellite System (GNSS) receivers for determining geographic location, and biometric sensors such as fingerprint readers or heart rate monitors. The sensors 139 can also include environmental sensors (e.g., humidity, barometric pressure, gas, smoke, or air-quality), proximity or time-of-flight (ToF) sensors, capacitive touch orDkt. No.: 025526. OOlWOl Page 40 of 64 2025-11-20 PCT Patent Applicationforce sensors, lidar or radar units for three-dimensional mapping, and biosensors such as ECG, SpO2, or EEG electrodes.Temperature Sensors

[0208] In some embodiments, the sensor 139 is a temperature sensor used to measure the temperature of any relevant location on or around a farm, or on the surface of, base of, or inside any relevant part of any crop grown on said farm that might be vulnerable to frost. Temperature is the physical quantity expressing the thermal energy present in matter. In some embodiments, the temperature sensor acts as a transducer and generates an electrical signal as a function of the measured temperature.

[0209] The temperature sensor can be a contact type temperature sensor or a non-contact type temperature sensor. Contact type temperature sensors are positioned in physical contact with the material and rely primarily on conduction to detect changes in its temperature. Non-contact type temperature sensors are not positioned in physical contact with the material and rely primarily on convection and / or radiation to detect changes in its temperature.

[0210] The temperature sensor can be any of a variety of types of temperature sensors. For example, suitable temperature sensors include thermocouples (type K, J, T, E, N, S, R, or the like), resistance temperature detectors (RTDs), thermistors, bimetallic strips, semiconductor temperature sensors, thermometers, vibrating wire temperature sensors, infrared temperature sensors, or the like.Light Sensors

[0211] In some embodiments, the sensor 139 is a light sensor. The light sensor can be used to determine the presence and / or intensity of light by measuring the radiant energy that exists in a certain range of frequencies, which typically include the infrared, visible, and / or ultraviolet light spectrum. In some embodiments, the light sensor acts as a transducer and generates an electrical signal as a function of the measured radiant energy.

[0212] The light sensor can include a variety of different light sensing technologies. In some embodiments, the light sensor generates electricity when illuminated. Examples of such light sensors include photovoltaic light sensors and photo -emissive light sensors. In some embodiments, the light sensor changes its electrical properties when illuminated. Examples of such light sensors include photoresistor light sensors and photoconductor light sensors.Image Sensors

[0213] In some embodiments, the sensor 139 is an image sensor. In general, an image sensor is a device that detects and conveys information used to make an image.Dkt. No.: 025526. OOlWOl Page 41 of 64 2025-11-20 PCT Patent ApplicationThe image sensor converts the variable attenuation of radiation waves (infrared, visible, and / or ultraviolet spectrum radiation as well as other frequencies) into signals that convey the information.

[0214] The image sensor can be any of a variety of types of image sensors. For example, suitable image sensors include electronic image sensors such as a charge-coupled device (CCD), active-pixel sensor (CMOS sensor), or the like. The image sensor can be part of a camera or other imaging device.Output Devices

[0215] Each of the one or more output devices 121 is a physical component that receives information from the processor 103 and / or the memory 105. The output device 121 can be communicatively linked to the system bus 107 byway of one or more output interfaces 123. The output device 121 can be any suitable type and can receive any of a variety of information. For example, the output device 121 can be a digital and / or analog device and can receive information in a digital and / or analog format. Also, the output device 121 can be used to provide information to the user or perform various operations related to the specific application.

[0216] The output device 121 can include one or more actuators 143 and / or one or more other miscellaneous output devices 145. It should be appreciated that the output device 121 is not limited to only receiving information. In some embodiments, the output device 121 can also send information. Such devices can be considered both an output device 121 and an input device 117.

[0217] The miscellaneous output devices 145 can include a variety of devices or components. In some embodiments, the miscellaneous output devices 145 can include audio output devices such as speakers as well as other output devices.Actuators

[0218] Each of the one or more actuators 143 can be used to activate movement or an operation. In general terms, the actuator 143 is used to activate something in response to an instruction or control signal sent from the processor 103. In some embodiments, the actuator 143 can act as a transducer by receiving an electrical signal and transforming it into the desired movement or operation.

[0219] The information received by the actuator 143 can take a variety of forms and use a number of technologies. For example, the information can be in the form of an electric voltage or current, pneumatic or hydraulic fluid pressure, binary data, or the like. The information can be provided as digital and / or analog format. For example, in some embodiments, the actuator 143 receives digital information from the processor 103 or other component(s) in the electronic computing device 101. In other embodiments, the actuator 143 receives analog information from the processor 103 orDkt. No.: 025526. OOlWOl Page 42 of 64 2025-11-20 PCT Patent Applicationother component(s) in the electronic computing device 101. Also, in some embodiments, the information received by the actuator 143 can be converted from one type to the other — e.g., from digital to analog or from analog to digital.

[0220] The actuator 143 can use a variety of energy sources to operate. For example, the actuator 143 can operate using electrical energy, hydraulic energy, pneumatic energy, thermal energy, magnetic energy, or the like. Likewise, the actuator 143 can be an electric actuator, hydraulic actuator, pneumatic actuator, thermal actuator, magnetic actuator, or the like. The actuator 143 can also be used to produce a variety of movements. For example, the actuator 143 can be used to produce linear movement and / or rotary movement.Motors

[0221] In some embodiments, the actuator 143 can include an electric motor. In general, the electric motor is a device that converts electrical energy to mechanical energy. In some embodiments, the mechanical energy produced by the electric motor is in the form of the rotation of a shaft. The mechanical energy can be used directly or converted into other mechanical movement using levers, gears, ratchets, cams, or the like. The motor can be a DC motor or an AC motor.Relays

[0222] In some embodiments, the actuator 143 can include a relay. In general, a relay is an electrically operated switch. In some embodiments, the relay includes one or more input terminals to receive information or control signals and one or more operating contact terminals electrically linked to a separate electrical device.

[0223] In some embodiments, the relays can include electromechanical relays having contacts that mechanically open and close. For example, the relay can include an electromagnet that opens and closes the contacts. In other embodiments, the relays can include solid state relays that use semiconductor properties to control the on or off state of the relay without any moving parts. Solid state relays can include thyristors and transistors to switch currents up to a hundred amps or more.Communication Devices

[0224] Each of the communication devices 125 is a physical component that allows the electronic computing device 101 to communicate with other devices, components, and / or networks. The communication device can be communicatively linked to the system bus 107 by way of one or more communication interfaces 127. The communication device 125 can include one or more wired communication devices 147 and / or one or more wireless communication devices 149.

[0225] It should be appreciated that the communication device 125 can be any suitable physical device. For example, in some embodiments, the communicationDkt. No.: 025526. OOlWOl Page 43 of 64 2025-11-20 PCT Patent Applicationdevice 125 is a network interface controller used to connect the electronic computing device 101 to a larger network such as a local area network (LAN), wide area network (WAN), or the Internet.

[0226] It should also be appreciated that the communication device 125 can use a variety of communication protocols. For example, in some embodiments, the wired communication device 147 can use communication protocols such as Ethernet, RS-232, RS-485, USB, industrial real-time Ethernet variants such as EtherCAT or time-sensitive networking (TSN) or the like. Also, in some embodiments, the wireless communication devices 149 can use communication protocols such as Wi-Fi (e.g., 802.11ax, Wi-Fi 6, 802.11be Wi-Fi 7, or Wi-Fi HaLow 802.11ah), Bluetooth (including Bluetooth Low Energy), Zigbee or 802.15.4, Thread and Matter (loT frameworks), Near Field Communication (NFC), LoRa or Sigfox for long-range low-power links, or cellular standards like LTE, NB-IoT, 5G, or C-V2X.

[0227] In some embodiments, the communication devices 125 allow distributed computing, allowing the electronic computing device 101 to offload processing to remote servers or edge devices via cloud services. This can include protocols for Internet of Things (loT) integration, such as MQTT or CoAP, facilitating real-time data exchange in networked applications.Power Source

[0228] The power source 129 can be used to supply electric power to the electronic computing device 101. The power source 129 can provide any suitable type of power including AC power, DC power, or the like. The power source 129 can also comprise power-over-ethernet (PoE) injectors, wireless charging coils (e.g., Qi or magnetic -resonant), or supercapacitor banks that supply short-term peak current. The power source 129 can obtain power from any suitable source including an AC power source (standard wall outlet), DC power source (a transformer plugged into a wall outlet), battery, generator, solar panels, or energy-harvesting mechanisms such as kinetic or thermal harvesters.

[0229] In some embodiments, the power source 129 includes a power supply that converts electric current from a source to a desired voltage, current, and / or frequency to power the electronic computing device 101. In some embodiments, the power supply can convert AC power ranging from 110-240 VAC to DC power ranging from 6- 60 VDC and can support low-power modes for energy-efficient operation in embedded systems.

[0230] In some embodiments, the electronic computing device 101 includes a dedicated power management unit (PMU), which can be a separate chip or integrated into the processor 103. The PMU can manage power rails, battery charging, and transitions between various power states, such as active, idle, sleep, or deep-sleepDkt. No.: 025526. OOlWOl Page 44 of 64 2025-11-20 PCT Patent Applicationmodes, to optimize power consumption. This allows for energy-efficient operation, particularly in battery-powered or energy-harvesting applications.Circuit Board

[0231] The electronic computing device 101 can include one or more circuit boards (alternatively referred to as logic boards) to which one or more of the components can be coupled. For example, the processor 103, the memory 105, the storage device 109, the display device 113, the input device 117, the output device 121, the communication device 125, and / or the power source 129 can be coupled to one or more circuit boards. In some embodiments, the processor 103, the memory 105, and / or the storage device 109 can be coupled to one circuit board.

[0232] In some embodiments, the circuit board can contain a series of conductive tracks, pads, and / or other features etched from one or more sheet layers of copper laminate laminated onto and / or between sheet layers of nonconductive substrate. The conductive features can be part of the system bus 107 communicatively linking the various components of the electronic computing device 101. In some embodiments, the circuit board can be a printed circuit board. In some embodiments, the circuit board can be a motherboard.Multiple Electronic Computing Devices Communicatively Linked

[0233] Referring to Fig. 10, the electronic computing device 101 can be communicatively linked to and / or controlled by one or more additional electronic computing devices 151. For example, the additional electronic computing device(s) 151 can be used to send data to or receive data from the electronic computing device 101. The additional electronic computing device(s) 151 can also be used to control or operate the electronic computing device 101. For example, the additional electronic computing device(s) 151 can be used to control the electronic computing device 101 to perform any of the methods, processes, or other operations described above.

[0234] The additional electronic computing device(s) 151 can be the same as or similar to the electronic computing device 101. The additional electronic computing device(s) 151 can also be a different device than the electronic computing device 101 even though it can have any of the components and / or features described in connection with the electronic computing device 101. The additional electronic computing device 151 can be a mobile electronic computing device, a personal electronic computing device, a wearable electronic computing device, a general-purpose electronic computing device, a special-purpose electronic computing device (e.g., designed for a specific purpose, application, or field of applications), an industrial electronic computing device, or the like.

[0235] By way of example, the additional electronic computing device 151 can be a mobile electronic computing device such as a mobile phone, smartphone, tabletDkt. No.: 025526. OOlWOl Page 45 of 64 2025-11-20 PCT Patent Applicationcomputer, handheld personal computer, or the like. The additional electronic computing device 151 can also be a personal electronic computing device such as a laptop computer, desktop computer, or workstation. The additional electronic computing device 151 can also be a wearable electronic computing device such as a smartwatch, smartband, smartglasses, or the like. The additional electronic computing device 151 can also be an industrial electronic computing device such as a programmable logic controller, system on a module, or the like.

[0236] The electronic computing device 101 can be communicatively linked with the additional electronic computing device(s) 151 using any suitable wired or wireless communication protocol. For example, the electronic computing devices 101, 151 can communicate using one or more of the following wired communication protocols: ethernet, HDMI, SATA, CAN, RS-232, RS-485, UART, USART, USB, or the like. The electronic computing devices 101, 151 can communicate using one or more of the following wireless communication protocols: Wi-Fi, Bluetooth, Bluetooth Low Energy, Zigbee, Z-wave, GSM / GPRS, CDMA, NFC, RFID, 6L0WPAN, or the like.

[0237] The additional electronic computing device(s) 151 can be connected directly to the electronic computing device 101 without connecting to any intermediate electronic computing devices, or the additional electronic computing device(s) 151 can be connected to the electronic computing device 101 by way of one or more intermediate electronic computing devices — e.g., a network 153. Likewise, the additional electronic computing device(s) 151 can be positioned adjacent to or nearby the electronic computing device 101 (e.g., same room, line of sight, etc.), or it can be positioned remotely relative to the electronic computing device 101 (e.g., different rooms, out of sight, different continents, etc.).

[0238] In one example, the additional electronic computing device 151 can be a mobile electronic computing device capable of running applications obtained from an app source (e.g., an app store) including an application designed to communicate with and / or control the electronic computing device 101. In another example, the additional electronic computing device 151 can be a personal electronic computing device such as a laptop computer capable of running software designed to communicate with and / or control the electronic computing device 101. It should be appreciated that there are numerous other ways the additional electronic computing device 151 can connect to, communicate with, and / or control the electronic computing device 101.Network Computing

[0239] One or more of the electronic computing devices 101, 151 can be part of or communicatively linked to a network 153 of computing devices having a variety of topologies. The network 153 can include a local area network (LAN), a wide area network (WAN), a personal area network (PAN), or a combination thereof, includingDkt. No.: 025526. OOlWOl Page 46 of 64 2025-11-20 PCT Patent Applicationthe Internet. The network 153 can support various topologies. For example, the devices can be arranged in a star topology, where multiple electronic computing devices 151 connect to a central device 101 (or a server). Alternatively, they can be arranged in a mesh topology, where devices can communicate directly with multiple other devices, providing redundant communication paths. Other topologies, such as bus, ring, or hybrid configurations can also be used. The program instructions can be implemented by a single electronic computing device or by multiple electronic computing devices in a distributed fashion over the network 153.

[0240] Communication between devices can leverage various architectures and protocols. The protocols can be conceptualized as a stack of layers, such as in the open systems interconnection (OSI) model or the TCP / IP model, where each layer provides services to the layer above it. These protocols establish rules for data formatting, addressing, transmission, routing, and reception.

[0241] For example, communication can utilize protocols from various layers, including but not limited to: (a) application layer protocols for device-specific services, such as hypertext transfer protocol (HTTP / HTTPS), file transfer protocol (FTP / SFTP), simple mail transfer protocol (SMTP), domain name system (DNS), and protocols for the Internet of things (loT) like MQTT and CoAP; (b) transport layer protocols for end-to-end data transmission, such as transmission control protocol (TCP) for reliable, connection-oriented communication and user datagram protocol (UDP) for low-latency, connectionless communication; (c) network layer protocols for addressing and routing packets across the network, most commonly the Internet protocol (IP); and (d) link and physical layer protocols for communication on the local network segment, such as Ethernet, Wi-Fi, industrial Ethernet variants (e.g., EtherCAT, PROFINET, TSN), controller area network (CAN bus), Wi-Fi (e.g., 802.11ax / be), Bluetooth (including BLE), cellular standards (e.g., 4G LTE, 5G), and low-power wide-area network (LPWAN) protocols like Zigbee, Thread, LoRa, and cellular loT standards (e.g., NB-IoT, LTE-M).

[0242] Network management protocols, such as Simple Network Management Protocol (SNMP) and Internet Control Message Protocol (ICMP), can be used to monitor network health and diagnose problems. Network security can be provided by protocols such as Transport Layer Security (TLS / SSL) and Secure Shell (SSH), which provide for data encryption and secure authentication.

[0243] This layered approach allows electronic computing devices 101, 151 with different hardware and software to communicate effectively and reliably over the network 153.Dkt. No.: 025526. OOlWOl Page 47 of 64 2025-11-20 PCT Patent ApplicationGeneral Terminology and Interpretative Conventions

[0244] The articles “the,” “a,” and “an” shall be interpreted as referring to both singular and plural forms. Additionally, unless preceded by the word “either” or similar language indicating exclusivity, the term “or” should be interpreted inclusively (for example, “x or y” refers to one or both x and y).

[0245] The term “and / or” shall be interpreted inclusively; for example, “x and / or y” refers to either x, y, or both. When “and / or” or “or” is used to connect three or more items, the phrase shall be interpreted to encompass any individual item, all items collectively, or any combination of the items.

[0246] The phrase “based on” shall be interpreted to mean an open set of conditions unless it is explicitly limited (for example, based on only a given condition). For instance, if a step is described as being based on a particular condition, it may depend on both the stated condition and additional unstated conditions.

[0247] The term “can,” when used as an auxiliary verb, indicates that the described subject matter optionally has the feature, aspect, ability, capacity, or the like, but it is not required in any given instance.

[0248] The terms "have," "having," "contain," "containing," "include," "including," and "characterized by" shall be construed as synonymous with "comprise" and "comprising" — that is, these terms are inclusive or open-ended and do not preclude additional, unrecited subject matter. The use of these terms shall also be interpreted as providing disclosure and support for narrower alternative embodiments in which these terms are substituted with "consisting of," "consisting of the recited subject matter plus impurities and / or trace amounts of other materials," or "consisting essentially of."

[0249] It shall be understood that features described in separate embodiments can be combined as a single embodiment. Similarly, features described together in a single embodiment can be implemented separately or in various subcombinations across multiple embodiments. Additionally, although certain features may be initially presented or claimed as part of specific combinations, it is possible to remove one or more features from a claimed combination so that the claim pertains to a subcombination or a variation thereof.

[0250] Numerous aspects or features are described as optional, often indicated by terms such as "can" or similar expressions. This document does not detail every possible combination or permutation arising from selecting among these optional elements. Nevertheless, all such combinations and permutations are considered expressly disclosed herein. For instance, an item described with three optional aspects may be embodied in seven distinct configurations: any single aspect, any pair of aspects, or all three aspects incorporated together.Dkt. No.: 025526. OOlWOl Page 48 of 64 2025-11-20 PCT Patent Application

[0251] The methods described in this document shall not be interpreted to require steps to be performed in a particular order unless explicitly indicated or if it is impossible to do otherwise. The methods should be understood to provide support or basis for the steps to be carried out in any sequence.

[0252] The configurations presented in this document are provided as examples and do not encompass all possible implementations within the scope of the claims. The term “example” is used to indicate an instance or illustration, without implying that it is preferred or superior to other possibilities.

[0253] Unless otherwise indicated, all numerical values or expressions regarding dimensions, physical characteristics, or similar parameters in the specification (excluding the claims) shall be interpreted as qualified by the term “approximately.” Additionally, if there is an absence of functional, qualitative, or other interpretative guidelines, each numerical value identified as "approximately" within the specification and claims shall be interpreted based on the stated number of significant digits and / or standard rounding conventions, without limiting the application of the doctrine of equivalents to the claims.

[0254] All disclosed ranges shall be interpreted as encompassing, and providing support for, claims that specify any subranges or individual values contained within each stated range. For instance, if a range is described as 1 to 10, it is intended to include and support claims directed to all subranges and individual values falling between, and including, the minimum value of 1 and the maximum value of 10. This includes all subranges commencing at a value of 1 or higher and concluding at a value of 10 or lower (e.g., 5.5 to 10, 2.34 to 3.56), as well as any individual values from 1 to 10 (e.g., 3, 5.8, 9.9994). The values in the range may be recited independently, as a minimum value (e.g., at least 5.8), or as a maximum value (e.g., no more than 9.9994).

[0255] All disclosed numerical values shall be interpreted as variable within a range of 0-100% in either direction. This interpretation provides support for claims referencing these values, whether stated individually or as a minimum or maximum (e.g., at least <value> or no more than <value>), as well as any ranges or subranges that can be derived from such values. For instance, a specified value of 8 should be considered as extending from 0-16 (reflecting 100% variability in both directions) and supports claims referring to the entire range (e.g., 0-16), any subrange within this interval (e.g., 2-12.5), or any individual value within the range (e.g., 15.2), including its use as a minimum value (e.g., at least 4.3) or a maximum value (e.g., no more than 12.4).

[0256] The terms in the claims shall be interpreted according to their ordinary and customary meanings, as established by relevant entries in widely recognized general or technical dictionaries and by commonly accepted meanings within the pertinentDkt. No.: 025526. OOlWOl Page 49 of 64 2025-11-20 PCT Patent Applicationfield. The interpretation should reflect the broadest meaning derived from any single source or from a combination of these sources (e.g., by synthesizing multiple dictionary definitions to arrive at the most comprehensive understanding), except under the following circumstances: (a) if a term is employed in a manner that extends beyond its ordinary usage, it should be accorded its customary meaning along with the broader meaning or (b) if a term is expressly defined within the document by language such as “as used in this document <term> shall mean,” “this term means,” “this term is defined as,” or “for the purposes of this disclosure this term shall mean.” References to specific examples, use of “i.e.,” or use of the word “invention” are not intended to invoke exception (b) or to otherwise narrow the scope of the claim terms. Except where exception (b) applies, nothing in this document should be construed as a disclaimer or limitation of claim scope.

[0257] The limitations in the claims shall not be interpreted as invoking 35 U.S.C. 112(f) unless the claim specifically includes the terms “means for” or “step for.”

[0258] Unless otherwise specified or clearly indicated by the context, terms such as “processing,” “computing,” “calculating,” “determining,” and “displaying” refer to actions and operations performed by an electronic computing device that includes a processor and memory.

[0259] The subject matter recited in the claims is not coextensive with and should not be interpreted as coextensive with any embodiment, feature, or combination of features described or illustrated in this document. This is the case even if only one version of a feature or combination is depicted and discussed.Joining or Fastening Terminology and Interpretative Conventions

[0260] The term “coupled” means the joining of two members directly or indirectly to one another. Such joining can be stationary in nature or movable in nature. Such joining can be achieved with the two members or the two members and any additional intermediate members being integrally formed as a single unitary body with one another or with the two members or the two members and any additional intermediate member being attached to one another. Such joining can be permanent in nature or alternatively can be removable or releasable in nature.

[0261] The term “coupled” includes joining that is permanent in nature or releasable and / or removable in nature. Permanent joining refers to joining the components together in a manner that is not capable of being reversed or returned to the original condition. Releasable joining refers to joining the components together in a manner that is capable of being reversed or returned to the original condition.

[0262] Releasable joining can be further categorized based on the difficulty of releasing the components and / or whether the components are released as part of their ordinary operation and / or use. Quickly releasable joining (i.e., quick-release) refers toDkt. No.: 025526. OOlWOl Page 50 of 64 2025-11-20 PCT Patent Applicationjoining that can be released without the use of tools. Readily or easily releasable joining refers to joining that can be readily, easily, and / or promptly released with little or no difficulty or effort. Some joining can qualify as both quickly releasable joining and readily or easily releasable joining. Other joining can qualify as one of these types of joining but not the other. For example, one type of joining can be readily or easily releasable but also require the use of a tool.

[0263] Non-quickly releasable joining (i.e., non-quick-release) refers to joining that can only be released with the use of tools. Difficult or hard to release joining refers to joining that is difficult, hard, or arduous to release and / or requires substantial effort to release. Some joining can qualify as both non-quickly releasable joining and difficult or hard to release joining. Other joining can qualify as one of these types of joining but not the other. For example, one type of joining can require the use of a tool but may not be difficult or hard to release.

[0264] The joining can be released or intended to be released as part of the ordinary operation and / or use of the components or only in extraordinary situations and / or circumstances. In the latter case, the joining can be intended to remain joined for a long, indefinite period until the extraordinary circumstances arise.

[0265] It should be appreciated that the components can be joined together using any type of fastening method and / or fastener. The fastening method refers to the way the components are joined. A fastener is generally a separate component used in a mechanical fastening method to mechanically join the components together. A list of examples of fastening methods and / or fasteners is given below. The list is divided according to whether the fastening method and / or fastener is generally permanent, readily released, or difficult to release. A general reference to fastening or fasteners without specifying a particular fastening method(s) or fastener(s) should be interpreted as including any type of fastening method and / or fastener.

[0266] Examples of permanent fastening methods include welding, soldering, brazing, crimping, riveting, stapling, stitching, some types of nailing, some types of adhering, and some types of cementing. Examples of permanent fasteners include some types of nails, some types of dowel pins, most types of rivets, most types of staples, stitches, most types of structural ties, and toggle bolts.

[0267] Examples of readily releasable fastening methods include clamping, pinning, clipping, latching, clasping, buttoning, zipping, buckling, and tying. Examples of readily releasable fasteners include snap fasteners, retainer rings, circlips, split pins, linchpins, R-pins, clevis fasteners, cotter pins, latches, hook and loop fasteners (VELCRO), hook and eye fasteners, push pins, clips, clasps, clamps, zip ties, zippers, buttons, buckles, and / or confirmat fasteners.Dkt. No.: 025526. OOlWOl Page 51 of 64 2025-11-20 PCT Patent Application

[0268] Examples of difficult to release fastening methods include bolting, screwing, most types of threaded fastening, and some types of nailing. Examples of difficult to release fasteners include bolts, screws, most types of threaded fasteners, some types of nails, some types of dowel pins, a few types of rivets, a few types of structural ties.

[0269] It should be appreciated that the fastening methods and fasteners are categorized above based on their most common configurations and / or applications. The fastening methods and fasteners can fall into other categories or multiple categories depending on their specific configurations and / or applications. For example, rope, string, wire, cable, chain, or the like can be permanent, readily releasable, or difficult to release depending on the application.Drawing Related Terminology and Interpretative Conventions

[0270] Reference numbers in the drawings and corresponding description refer to identical or similar elements although such numbers can be referenced in the context of different embodiments.

[0271] The drawings are intended to illustrate embodiments that are both drawn to scale and / or not drawn to scale. This means the drawings can be interpreted, for example, as showing: (a) everything drawn to scale, (b) nothing drawn to scale, or (c) one or more features drawn to scale and one or more features not drawn to scale. Accordingly, the drawings can serve to provide support to recite the sizes, proportions, and / or other dimensions of any of the illustrated features either alone or relative to each other. Furthermore, all such sizes, proportions, and / or other dimensions are to be understood as being variable from 0-100% in either direction and thus provide support for claims that recite such values or any ranges or subranges that can be formed by such values.

[0272] Spatial or directional terms, such as “left,” “right,” “front,” “back,” or the like, relate to the subject matter as it is shown in the drawings and / or how it is commonly oriented during manufacture, use, or the like. However, it is to be understood that the described subject matter can assume various alternative orientations and, accordingly, such terms are not to be considered as limiting.Composition Related Terminology and Interpretative Conventions

[0273] Values expressed as a percentage, parts of, or a ratio are by weight unless expressly stated otherwise.

[0274] The description of a group or class of materials as suitable or preferred for a given purpose shall be understood as disclosing that a single member of the group or class or a mixture of any two or more members of the group or class is equally suitable or preferred.Dkt. No.: 025526. OOlWOl Page 52 of 64 2025-11-20 PCT Patent Application

[0275] The description of constituents in chemical terms refers to the constituents:(a) at the time of addition to any combination specified in the description (e.g., the formal or analytical concentration of a salt that dissociates in solution) and / or (b) generated in situ by chemical reactions with other constituents. The description of the constituents does not preclude other chemical interactions among the constituents of a mixture once mixed unless expressly stated otherwise.

[0276] The description of materials in ionic form additionally implies the presence of sufficient counter ions to produce electrical neutrality for the composition.Incorporation by Reference

[0277] The entire content of each document listed below is incorporated by reference into this document (the documents below are collectively referred to as the “incorporated documents”). If the same term is used in both this document and one or more of the incorporated documents, then it should be interpreted to have the broadest meaning imparted by any one or combination of these sources unless the term has been explicitly defined to have a different meaning in this document. If there is an inconsistency between any incorporated document and this document, then this document shall govern. The incorporated subject matter should not be used to limit or narrow the scope of the explicitly recited or depicted subject matter.Benefit / priority patent documents incorporated by reference:- U.S. Prov. App. No. 63 / 722,698, titled “Systems and Methods for Utilization of Ice Proteins,” filed on 20 Nov 2024.Dkt. No.: 025526. OOlWOl Page 53 of 64 2025-11-20 PCT Patent Application

Claims

CLAIMS:

1. A composition for agricultural frost protection comprising: one or more ice-structuring proteins selected from the group consisting of antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs); and a carrier suitable for application to plant surfaces or plant tissues, characterized in that the one or more ice-structuring proteins are present at a concentration between 0.01 mg / mL and 50 mg / mL.

2. The composition according to claim 1, wherein the one or more ice-structuring proteins are derived from sources selected from the group consisting of: fish, insects, fungi, bacteria, plants, algae, and archaea.

3. The composition according to claim 1 or 2, wherein the one or more icestructuring proteins comprise Type I, Type II, Type III, or Type IV antifreeze proteins.

4. The composition according to any one of claims 1 to 3, wherein the one or more ice-structuring proteins are sourced from organisms selected from the group consisting of: Zoarces americanus, Liposetta pinnifasciata, Tenebrio molitor, Dendroides canadensis, Rhagium mordax, Rhagium imperator, Eurygaster maura, Glaciozyma antarctica, and Typhula ishikariensis.

5. The composition according to any one of claims 1 to 4, wherein the one or more ice-structuring proteins exhibit one or more of the following characteristics: a molecular weight of 10 kDa or less; a molecular width in the low nanometer range; a tightly ordered tertiary structure stabilized by disulfide bonds or repeated motifs; and a neutral charge.

6. The composition according to any one of claims 1 to 5, further comprising one or more adjuvant components selected from the group consisting of: nonionic surfactants at concentrations between 0.01% and 1%; oil-based adjuvants at concentrations between 0.01% and 1%; organic ionic cofactors selected from citrate, succinate, malate, malonate, and acetate at concentrations between 0.01 M and 1 M; buffer agents at concentrations between 0.01 M and 2 M providing pH in a range of 4 to 10; simple sugars or polyols selected from sucrose, glucose, fructose, trehalose, maltose, lactose, mannitol, sorbitol, myoinositol, and galactinol; and salts selected from chlorides, sulfates, nitrates, citrates, succinates, malates, malonates, and acetates of potassium, magnesium, sodium, calcium, ammonium, zinc, iron, or selenium.Dkt. No.: 025526.001W01 Page 54 of 64 2025-11-20 PCT Patent Application7. The composition according to any one of claims 1 to 6, wherein the composition comprises a combination of two or more different antifreeze proteins, wherein at least one antifreeze protein provides thermal hysteresis activity and at least one antifreeze protein provides ice recrystallization inhibition activity.

8. The composition according to claim 7, wherein the combination comprises: one to three high-activity fish or insect AFPs at concentrations between 0.01 mg / mL and 50 mg / mL for lowering freezing temperature; and one to two fish, insect, yeast, or bacterial AFPs at concentrations between 0.01 mg / mL and 50 mg / mL for minimizing damage to frozen cells through ice recrystallization inhibition.

9. The composition according to any one of claims 1 to 8, wherein the carrier is formulated as one or more of: an aqueous solution, a spray formulation, a seed coating, a wettable powder, wettable granules, a concentrated solution, or a frozen solution.

10. The composition according to any one of claims 1 to 9, wherein the one or more ice-structuring proteins are modified by one or more modifications selected from the group consisting of: alteration of glycosylation states; fusion to a purification tag; concatenation of two to ten AFP units linked by peptide linkers; truncation to retain an active core sequence; addition of anti-degradation modifications providing stability for at least 12 hours at approximately 5°C; addition of internalization sequences that increase uptake into plant tissues; addition of hydrophobic sequences that increase retention on plant surfaces; and addition of hydrophobic sequences that increase attachment to waxy plant pellicles.

11. A method of protecting plants from frost damage comprising the step of: applying to one or more plant surfaces or plant tissues a composition according to any one of claims 1 to 10; characterized in that the applying occurs at a time between 2 hours and 14 days before an expected frost event.

12. The method according to claim 11, wherein the applying comprises one or more application methods selected from the group consisting of: spraying, drip irrigation, overhead irrigation, hand spraying, drone application, robotic application, syringe infiltration, direct injection, painting, and dip application.Dkt. No.: 025526. OOlWOl Page 55 of 64 2025-11-20 PCT Patent Application13. The method according to claim 11 or 12, wherein the one or more plant surfaces or plant tissues comprise one or more selected from the group consisting of: leaves, stems, buds, flowers, fruits, seeds, seedlings, and whole plants.

14. The method according to any one of claims 11 to 13, wherein the method achieves one or more of the following effects: lowering the freezing temperature of water in or on plant tissues by between 0.1°C and 8°C; reducing ice crystal damage to plant cells through ice recrystallization inhibition; increasing plant cold-hardiness through induced metabolic response; and increasing time to freeze under frost conditions by 10% to 300% or more compared to untreated plants.

15. The method according to any one of claims 11 to 14, wherein between 1% and 99% of the applied composition remains on the surface of plants and between 1% and 99% is internalized into the plant's tissues, cells, or apoplast.

16. The method according to any one of claims 11 to 15, wherein the composition is applied in combination with one or more additional agricultural treatments selected from the group consisting of: herbicides, fertilizers, pesticides, biostimulants, and growth promoters.

17. The method according to any one of claims 11 to 16, wherein frost protection persists for a duration between 1 hour and 14 days after application.

18. The method according to any one of claims 11 to 17, further comprising monitoring environmental conditions using one or more sensors selected from temperature sensors, humidity sensors, and light sensors, and triggering the applying step based on detected conditions indicative of potential frost.

19. Use of a composition according to any one of claims 11 to 18 for protecting plants from frost damage.

20. The use according to claim 19, wherein the composition is applied to seeds, seedlings, mature plants, or plant tissues.

21. The use according to claim 19 or 20, wherein the composition is applied between 2 hours and 14 days before an expected frost event.

22. The use according to any one of claims 19 to 21, wherein the protection achieves one or more of: lowering freezing temperature by 0.1°C to 8°C, reducing ice crystal damage, or increasing plant cold-hardiness.

23. A method of treating seeds to confer frost protection comprising the steps of: contacting seeds with a composition comprising one or more antifreeze proteins(AFPs) or antifreeze glycoproteins (AFGPs); and allowing the seeds to absorb at least a portion of the composition,Dkt. No.: 025526. OOlWOl Page 56 of 64 2025-11-20 PCT Patent Applicationcharacterized in that the one or more antifreeze proteins are present at a concentration between 0.01 mg / mL and 50 mg / mL and the contacting occurs for a duration between 6 hours and 1 week.

24. The method according to claim 23, wherein the contacting comprises one or more of: soaking seeds in the composition, spraying seeds with the composition, infiltrating seeds with the composition, or applying the composition as a coating to seeds.

25. The method according to claim 23 or 24, wherein the composition further comprises one or more components selected from the group consisting of: nonionic surfactants at concentrations between 0.01% and 0.2%, basic salts at concentrations between 0.01 M and 1 M, simple sugars at concentrations between 0.01 M and 1 M, and organic ionic or nonionic buffers at concentrations between 0.01 M and 1 M.

26. The method according to any one of claims 23 to 25, wherein frost protection persists for a duration between 1 hour and 1 month after planting of the treated seeds.

27. The method according to any one of claims 23 to 26, wherein the treated seeds exhibit improved germination rates of at least 100% higher than untreated seeds when exposed to freezing temperatures.

28. The method according to any one of claims 23 to 27, wherein the seeds are selected from crop species comprising: tomatoes, wheat, soybeans, corn, and coffee.

29. Use of a composition comprising one or more antifreeze proteins (AFPs) or antifreeze glycoproteins (AFGPs) at a concentration between 0.01 mg / mL and 50 mg / mL for treating seeds to confer frost protection according to any one of claims 23 to 28.

30. The use according to claim 29, wherein the seeds are contacted with the composition for a duration between 6 hours and 1 week prior to planting.

31. A modified antifreeze protein characterized in that it comprises: a wild-type antifreeze protein sequence that has been modified by one or more modifications selected from the group consisting of: concatenation to form a construct comprising two to ten individual AFP units linked by peptide linkers; truncation to retain only an active core sequence while removing non-essential residues; fusion to an anti-degradation element providing stability for at least 12 hours at approximately 5°C; addition of an internalization sequence that increases uptake into plant tissues; and addition of a hydrophobic sequence or attachment tag that increases retention on plant waxy surfaces.Dkt. No.: 025526. OOlWOl Page 57 of 64 2025-11-20 PCT Patent Application32. The modified antifreeze protein according to claim 31, wherein the wild-type antifreeze protein is selected from proteins derived from Zoarces americanus, Tenebrio molitor, Dendroides canadensis, Rhagium mordax, Rhagium imperator, Liposetta pinni fas data, Hemitripterus americanus, Ctenopharyngodon idella, and Eurygaster maura.

33. The modified antifreeze protein according to claim 31 or 32, wherein the concatenation construct comprises individual AFP units that are cleavable postproduction to yield monomeric AFP units.

34. The modified antifreeze protein according to any one of claims 31 to 33, wherein the modification results in one or more enhanced properties selected from the group consisting of: increased thermal hysteresis activity, increased ice recrystallization inhibition, increased stability, increased protease resistance, increased bioavailability, and increased duration of frost protection.

35. The modified antifreeze protein according to any one of claims 31 to 34, wherein the protein exhibits a freezing point depression of between 1°C and 20°C when applied at saturation.

36. Use of a modified antifreeze protein according to any one of claims 31 to 35 for protecting plants from frost damage.

37. A microbial carrier system for delivering antifreeze activity to plants comprising: a live microorganism selected from the group consisting of bacteria, fungi, and algae, characterized in that the microorganism is engineered or naturally capable of producing one or more antifreeze proteins (AFPs) or antifreeze glycoproteins (AFGPs), and the microorganism is capable of surviving on plant surfaces or within plant tissues for at least 1 hour.

38. The microbial carrier system according to claim 37, wherein the microorganism is selected from the group consisting of: Pseudomonas fluorescens, Pseudomonas aeruginosa, symbiotic bacteria, endophytic bacteria, probiotic fungi, and cold-tolerant yeast.

39. The microbial carrier system according to claim 37 or 38, wherein the microorganism secretes the one or more antifreeze proteins onto plant surfaces or into plant tissues.

40. The microbial carrier system according to any one of claims 37 to 39, wherein the antifreeze proteins remain in the cytoplasm of the microorganism and are releasedDkt. No.: 025526. OOlWOl Page 58 of 64 2025-11-20 PCT Patent Applicationupon natural or induced microbe death, with between 1% and 100% of the antifreeze proteins being released.

41. The microbial carrier system according to any one of claims 37 to 40, wherein the microorganism produces antifreeze proteins in situ on, around, or inside plants for a duration between 1 hour and 10 years after application.

42. The microbial carrier system according to any one of claims 37 to 41, wherein the microorganism comprises an INP-negative Pseudomonas strain capable of out- competing ice-nucleating Pseudomonas syringae.

43. The microbial carrier system according to any one of claims 37 to 42, wherein the microorganism delivers nucleotide sequences encoding AFPs to plant cells via Agrobacterium-based delivery for transient expression.

44. Use of a microbial carrier system according to any one of claims 37 to 43 for protecting plants from frost damage.

45. A system for automated agricultural frost protection comprising: one or more sensors configured to detect environmental conditions indicative of potential frost, wherein the sensors are selected from the group consisting of temperature sensors, humidity sensors, light sensors, and imaging devices; an electronic computing device comprising a processor and memory, wherein the electronic computing device is configured to receive data from the one or more sensors and to process the data to determine frost risk; and an automated spraying system communicatively linked to the electronic computing device, characterized in that the automated spraying system is configured to apply a composition according to any one of claims 1 to 44 to crops based on instructions from the electronic computing device.

46. The system according to claim 45, wherein the temperature sensors are positioned to measure temperature at one or more locations selected from: ambient air temperature, crop surface temperature, and soil temperature.

47. The system according to claim 45 or 46, wherein the electronic computing device is configured to execute instructions to compare detected temperature to a threshold temperature and to trigger the automated spraying system when the detected temperature satisfies one or more frost risk conditions.

48. The system according to any one of claims 45 to 47, wherein the automated spraying system comprises one or more devices selected from the group consisting of: automated sprayers, drones, quadcopters, and robotic applicators.Dkt. No.: 025526. OOlWOl Page 59 of 64 2025-11-20 PCT Patent Application49. The system according to any one of claims 45 to 48, further comprising one or more display devices for presenting information about environmental conditions and frost protection status to a user.

50. The system according to any one of claims 45 to 49, wherein the electronic computing device is configured to determine timing of application based on one or more factors selected from the group consisting of: time until expected frost, crop type, formulation type, and desired duration of protection.

51. A controlled-release frost protection system comprising: one or more antifreeze proteins (AFPs) or antifreeze glycoproteins (AFGPs); and a carrier selected from the group consisting of nanoparticles, nanostructured carriers, and encapsulated matrices, characterized in that the carrier is configured to release the antifreeze proteins over a time period ranging from 1 hour to 12 months.

52. The controlled-release frost protection system according to claim 51, wherein the carrier is configured for one or more of: seed coating application, foliar adhesion, and soil persistence.

53. The controlled-release frost protection system according to claim 51 or 52, wherein the release profile is tailored to provide frost protection for a duration of between 1 day and 1 year after application.

54. The controlled-release frost protection system according to any one of claims 51 to 53, wherein the nanoparticles or nanostructured carriers facilitate one or more of: penetration into plant tissues, retention on plant surfaces, and stability of the antifreeze proteins.

55. Use of a controlled-release system according to any one of claims 51 to 54 for protecting plants from frost damage.

56. A method of producing recombinant antifreeze proteins for agricultural applications comprising the steps of: expressing one or more antifreeze protein-encoding nucleotide sequences in a host cell selected from the group consisting of bacteria, yeast, and cold-tolerant yeast; recovering the expressed antifreeze proteins from the host cells or culture medium; and formulating the recovered antifreeze proteins into a composition suitable for agricultural application, characterized in that expression occurs at a temperature between 10°C and 40°C.

57. The method according to claim 56, wherein the host cell is selected from cold- tolerant strains that preserve protein integrity during production.Dkt. No.: 025526. OOlWOl Page 60 of 64 2025-11-20 PCT Patent Application58. The method according to claim 56 or 57, wherein the antifreeze proteinencoding nucleotide sequences encode modified antifreeze proteins comprising one or more modifications selected from concatenation, truncation, fusion to purification tags, addition of anti-degradation elements, addition of internalization sequences, and addition of hydrophobic sequences.

59. The method according to any one of claims 56 to 58, wherein the formulating step produces one or more product formats selected from the group consisting of: dehydrated powder, wettable granules, wettable powders, concentrated solution, frozen solution, and ready-to-use spray formulation.

60. The method according to any one of claims 56 to 59, further comprising a cell- free expression step, optionally supplemented with enzymes or components to enable desired post-translational modifications including glycosylation.

61. A combination composition for synergistic frost protection comprising: one or more non-colligative antifreeze proteins (AFPs) at a concentration between 0.01 mg / mL and 50 mg / mL; and one or more colligative antifreeze agents selected from the group consisting of sugars, salts, polysaccharides, starches, cellulose, polyethylene glycol, and sodium chloride, characterized in that the colligative antifreeze agents are present at concentrations between 0.01 M and 2 M, and the combination provides greater freezing point depression than either component alone.

62. The combination composition according to claim 61, wherein the colligative antifreeze agents are present at concentrations that do not cause significant damage to treated crops.

63. The combination composition according to claim 61 or 62, wherein the composition achieves a freezing point depression of at least 0.5°C greater than the sum of freezing point depressions achievable by each component independently.

64. The combination composition according to any one of claims 61 to 63, further comprising one or more insulating agents selected from insulating foam and nanocrystalline cellulose.

65. Use of a combination composition according to any one of claims 61 to 64 for protecting plants from frost damage.

66. Systems and methods for utilization of ice proteins in a plurality of applications.

67. The systems and methods of claim 66, wherein one of the plurality of applications comprises frost management in agriculture.

68. The systems and methods of claim 67, comprising one or more of:Dkt. No.: 025526. OOlWOl Page 61 of 64 2025-11-20 PCT Patent ApplicationType I, II, III, and IV fish antifreeze proteins, or antifreeze glycoproteins;Tenebrio molitor, spruce budworm, or other insect-derived antifreeze proteins; fungal antifreeze proteins derived from cold-tolerant yeast; bacterial antifreeze proteins; plant-derived antifreeze proteins; and diatom-derived antifreeze proteins.

69. The systems and methods of claim 68, wherein cold-tolerant yeast may comprise one or more of G. antarctica and T. ishikariensis.

70. The systems and methods of claim 68, wherein the one or more proteins are modified by one or more of: addition or removal of glycosylation states or any other post-translational states, a fusion domain that allows proteins to be produced at higher yield in bacteria or cold-tolerant yeast, or any other suitable protein producing host, concatemer constructs, truncation, anti-degradation modification, addition of or fusion with an internalization sequence, addition of or fusion with a hydrophobic sequence or attachment tag, and increasing anti-freeze effect.

71. The systems and methods of claim 70, wherein concatemer constructs comprise low- weight proteins expressed as a chain of two to ten units linked by a peptide linker to be one of: used as a single multi-potent antifreeze molecule, and cleaved post-production to produce a higher yield of protein units.

72. The systems and methods of claim 70, wherein truncation comprises preserving only an active core sequence.

73. The systems and methods of claim 70, wherein anti-degradation modification comprises one or more of protein modification, protein fusion, or the covalent linkage of an anti-degradation tag.

74. The systems and methods of claim 73, wherein anti-degradation modification results in a modified protein that is stable when applied at temperatures of at least 5°C and up to 50°C, and for at least 12 hours and up to 30 days.

75. The systems and methods of claim 70, wherein increasing the antifreeze effect comprises one or more of: lowering critical concentration, raising total degrees of frost protection, and increasing thermal hysteresis.Dkt. No.: 025526. OOlWOl Page 62 of 64 2025-11-20 PCT Patent Application76. The system and methods of claim 75, wherein increasing the anti-freeze effect results in between 1°C and 20°C decrease of the freezing temperature of water when applied at saturation.

77. The systems and methods of claim 70, wherein production and delivery methods comprise one or more of: recombinant production in production yeast or bacteria, cell-free production, and production of bacteria or fungi.

78. The systems and methods of claim 77, wherein recombinant production in production yeast or bacteria occurs between 10°C and 40°C.

79. The systems and methods of claim 77, wherein bacteria or fungi one of: internally express or secrete naturally or are artificially engineered to produce single or multiple antifreeze proteins and / or other antifreeze molecules.

80. The systems and methods of claim 77, wherein bacteria may be used to otherwise deliver frost tolerance to a plant by inducing production of natural antifreeze or delivering a nucleotide construct coding for an antifreeze gene.

81. The systems and methods of claim 77, wherein proteins are produced in one or more formats comprising: dehydrated powder, wettable granules, or wettable powders, concentrated or frozen solution, spray, seed treatment, soil additive, and application in conjunction with herbicide, fertilizer, pesticides, biostimulants, or growth promoters.

82. The systems and methods of claim 67, a product comprising two or more of: antifreeze protein activity, food-safe conventional antifreezes, biostimulant molecules, growth promoters, seaweed extracts, or pesticides, nutrient molecules, insulating molecule, and anti-Pseudomonas bacterium or INP-negative P. syringae that can out-compete P. syringae.

83. The systems and methods of claim 82, wherein insulating molecule comprises an insulating foam or nanocrystalline cellulose.Dkt. No.: 025526. OOlWOl Page 63 of 64 2025-11-20 PCT Patent Application

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