EA040708B1 - PREPARATIONS AND METHODS FOR PROCESSING PHOTOSYNTHESIS ORGANISMS AND INCREASING THEIR QUALITY AND YIELD USING GLYCAN COMPOSITE PREPARATIONS - Google Patents

Description

Cross-reference to related applications

This application claims priority to U.S. Provisional Application No. 62/329,226, filed April 29, 2016, the contents of which are incorporated herein by reference.

The field of technology to which the invention belongs

The embodiments disclosed in the invention relate to methods for treating photosynthetic organisms, for example, fields with photosynthetic organisms, including crops, and / or enhancing their growth using preparations that include compositions of glycan composites containing components of a branched deglycosylated glycan with components of a coordination connections. Moreover, in the case of liquid compositions, the above preparations may include one or more preservatives that ensure the stability of the preparations during their transportation and storage. Embodiments disclosed in the invention include systems for delivering glycan mixtures to photosynthetic organisms.

State of the art

The embodiments disclosed herein relate to the application to photosynthetic organisms of glycan composite preparations consisting of one or more branched deglycosylated glycans and ²⁺ transition metal coordination compounds.

The viability of photosynthetic organisms in crops depends on their biological production of primary photosynthetic products, particularly sugars, and the compositions of the present embodiments enhance the ability to use these primary photosynthetic products to improve crop viability and growth. Recent significant advances in improving crop yields relate to water-soluble glucosides described in the patent literature. These glucosides have proven effective at foliar application at application rates in the range of several kilograms per hectare, but there is a need for formulations that are more active and effective at application rates in the range of several grams per hectare and are disclosed in embodiments of the present invention. The branched deglycosylated glycans of the present embodiment have higher activity than conventional compositions, with the transition metal ²⁺ coordination compound components of the glycan composites further enhancing activity. In addition, by influencing the movement of nectar juice, glycan composites increase the flux efficiency of the resulting primary photosynthetic products in crops. Moreover, methods and compositions of glycan composites can be used to improve the quality and/or yield of crops while maintaining the activity of the glycan composite for a long time.

The essence of the invention

The purpose of the embodiments disclosed herein is to provide methods and formulations containing a glycan composite for improving crop viability and the growth of photosynthetic organisms, for treating photosynthetic organisms, and improving crop quality and yield. The formulations comprise a branched deglycosylated glycan with a glycopyranose or acylglycosamine terminal ligand in admixture with a Ca ²⁺ coordination compound and one or more D-transition metal ²⁺ coordination compounds.

Yet another object of the embodiments disclosed herein is to provide liquid formulations comprising one or more formulations of glycan composites with preservatives to maintain high potency.

Another object of the embodiments disclosed in the invention is to provide a method for improving crop viability and growth of photosynthetic organisms by enhancing the respiratory metabolism of primary photosynthetic products by treating photosynthetic organisms such as plants, and for enhancing the growth of the photosynthetic organism, for example, in plant crops, by application of the glycan composite preparation.

Yet another object of the embodiments disclosed herein is to provide glycan composite preparations wherein the one or more ²⁺ D-transition metal coordination compounds comprise both ²⁺ metal components and one or more anionic components selected from the group consisting of chelating polydentate alkylamides, aconitates, citrates, fumarates, glutarates, malates, oxaloacetates, succinates, their acids, salts and esters.

Yet another object of the embodiments disclosed herein is to provide glycan composite formulations comprising soluble D-transition metal ²⁺ coordination compounds.

Another object is to provide methods for treating photosynthetic organisms, in particular plants, and preparations for the growth of photosynthetic organisms, including glycan composite preparations, packaged in liquid and/or dry form, which are accessible and have penetrating power when using transcuticular, transepidermal and/ or

- 1 040708 transmembrane transport, when taken up by seeds, foliage and roots from the liquid phase, during germination, and/or which support growth under low light conditions.

Another goal is to provide preparations for the treatment and growth of photosynthetic organisms and their parts, and in particular in relation to agricultural crops.

Another goal is to develop preparations for the treatment of flowers and / or fruits, including a glycan composite, to control the quality of growth of photosynthetic organisms, in particular in relation to roots, foliage, flowers and / or fruits that are attached to the plant before harvest, to plucked flowers and/or fruits after harvest, and/or to improve palatability.

Another goal is to create new plant growth regulators based on glycan compositions to control the respiration of photosynthetic organisms.

Yet another goal is to provide a glycan composition-based environment for photosynthetic organisms in cultivation that promotes respiration at low light intensity under shade to dark conditions.

Another goal is to create an environment that provides acceleration of respiration for photosynthetic organisms during cultivation, by processing with glycan composites. In some embodiments, roots at any time of the day or night and/or shoots in the dark of night are exposed to respiration accelerators that increase oxygen (O2) levels in the environment either by treating the photosynthetic organism with >25% O2 gas or by applying O ₂ generators. In addition, it is another object to provide a respiration-promoting environment for photosynthetic organisms when cultured with glycan composites, in which photosynthetic organisms when cultured with glycan composites can be exposed to respiration accelerators.

Another goal is to create an environment conducive to the accumulation of primary photosynthetic products for photosynthetic organisms during cultivation by treatment with glycan composites. Photosynthetic organisms during cultivation by treatment with glycan composites can be exposed to respiration retarders, which contribute to the accumulation of primary products of photosynthesis.

Another goal is to create composites of glycans and/or their components for use as natural biostimulants to increase nutrient efficiency, increase tolerance to abiotic stress and/or to increase the yield and quality of crops.

In particular embodiments, such as for ease of use in the field, the formulation containing the glycan composite is easy to apply to photosynthetic organisms and is completely safe to photosynthetic organisms when formulated in liquid and/or dry form formulations and can be used as an additive in the processing of agricultural crops.

Another goal is to provide treatments and compositions for photosynthetic organisms, including compositions of exogenous glycan composites, to control endogenous deconjugation of primary photosynthetic products that increase the quality and quantity of harvested crops in agriculture.

Another goal is to provide a preparation for the treatment and growth of photosynthetic organisms, including compositions of exogenous glycan composites, for the treatment of endogenous primary photosynthetic products that enhance the taste characteristics of harvested crops in agriculture.

Yet another object is to provide a formulation for the treatment and growth of photosynthetic organisms comprising compositions of exogenous glycan composites that are protected from spoilage for storage by one or more plant-friendly preservatives.

Another goal is to provide a preparation for the treatment and growth of photosynthetic organisms, including systems of glycan composites, to improve the growth of crops when grown in hydroponics.

Another goal is to develop a preparation for the treatment and growth of photosynthetic organisms, including composites of glycans in the form of synergistic systems, to improve the aesthetic appearance of crops by reducing the incidence of sunburn.

Yet another object is to provide a method of treating photosynthetic organisms that enhances the nutritional value of field flower sap nectar, comprising applying glycan composites to food-grade flowering plants to greatly enhance their health benefits for pollinators and grazing livestock.

In specific embodiments, the glycan composite is prepared in the form of a complex consisting of one or more components of methyl-a-O-mannopyranosyl (Man)i_ ₃ , a citrate-Ca ²⁺ coordination compound, and with one or more D-transition metals ²⁺ , and/or additionally prepared with one or more preservatives.

- 2 040708

In specific embodiments, the implementation of the glycan composite is prepared in the form of a complex consisting of one or more components of methyl-α-D-Man ₁ . ₃ , a malate-Ca ²⁺ coordination compound, in admixture with one or more D-transition metals ²⁺ , and/or with one or more preservatives.

In specific embodiments, the implementation of the glycan composite is prepared in the form of a complex consisting of one or more components of methyl-α-d-Man ₁ . ₃ , a coordination compound of succinateCa ²⁺ and with one or more D-transition metals ²⁺ and/or additionally with one or more preservatives.

In specific embodiments, the glycan composite is prepared in the form of a complex consisting of one or more Manl.3N-linked-glycan components, a citrate-Ca ²⁺ coordination compound component, and with one or more ²⁺ d-transition metals, and/or additional with one or more optional preservatives.

In specific embodiments, the glycan composite is prepared in the form of a complex consisting of one or more Manl.3N-linked-glycan components, a Ca ²⁺ coordination compound component with one or more ²⁺ d-transition metals, and/or additionally with one or more optional preservatives.

In specific embodiments, the glycan composite is prepared in the form of a complex consisting of one or more methyl-α-D-Glc ₁ components, a citrate-Ca ²⁺ coordination compound component, with one or more ²⁺ d-transition metals, and/or additionally with one or more optional preservatives.

In specific embodiments, the glycan composite is prepared in the form of a complex consisting of one or more deglycosylated O-linked glycan components from vegetable resins, components of Ca ²⁺ coordination compounds with one or more d-transition metals ²⁺ , and/or additionally with one or more optional preservatives.

In specific embodiments, the glycan composite is prepared in the form of a complex consisting of one or more components of a deglycosylated N-linked glycan from invertase, components of Ca ²⁺ coordination compounds with one or more ²⁺ d-transition metals, and/or additionally with one or more optional preservatives.

Brief description of the drawings

In FIG. 1 schematically depicts, in turn, from top to bottom, the processes that underlie methods and compositions for treating photosynthetic organisms with a glycan composite, in accordance with specific embodiments. In the example shown in FIG. 1, the application of the glycan composite results in the effect of the solution on the plant cell, which improves the quality and yield of crops. The glycan composite is delivered to the cell, metabolized, resulting in energy transfer. This metabolic pathway, determined by the glycan composite, aimed at releasing the primary products of photosynthesis from the stored stock and triggering O ₂ ^ CO ₂ respiration, leads to an increase in plant productivity with an increase in their quality and yield.

In FIG. 2 is a graphical representation of examples of branched deglycosylated glycans that can be used to prepare glycan composites. The main structure at the top left of the page corresponds to trimannopyranosyl-N-glycan, while the other structures are examples of glycans with higher degrees of branching. The basic structures of the invertase shown are suitable for selecting one or more branched deglycosylated glycans with preferred terminal ligands of the glycan composite. Examples of N-glycans containing a high amount of mannopyranosyl, with terminal mannopyranosyl ligands, tend to have a higher degree of branching from top to bottom on the page. The structure of Man ₃ GlcNAc ₂ depicted in the upper left corner of the page corresponds to Ethan in examples 1, 6, 9, 16 and 17. Abbreviations used: Glc - glucopyranosyl, Man - mannopyranosyl, NAc - N-acetyl.

Detailed description of the invention

Unless otherwise indicated, all technical and scientific terms used in the invention have their traditional meaning for this field of technology. The terms used in the invention have the following meanings.

M denotes molar concentration, μM denotes micromolar concentration, and mM denotes millimolar concentration.

kDa stands for kilodalton

PGR stands for Plant Growth Regulator.

Percent or % is percent by weight unless otherwise noted.

Ppb stands for parts per billion by mass.

Ppm stands for parts per million by mass.

Ppt stands for parts per thousand by weight.

Nomenclature - botanical names are usually given in the form of ordinary names, which

- 3 040708 can be referred to the scientific nomenclature used in most scientific publications.

Statistics - Comparison of means using Student's t-test (two-tailed) shows significance at p<0.05.

Enhanced growth or growth enhancement means promoting, increasing or improving the growth rate of a photosynthetic organism, such as a plant, and/or increasing or promoting an increase in the size and/or yield and/or improving the quality of the photosynthetic organism or parts thereof, regulation of the flow of primary photosynthetic products , increasing the flow of primary photosynthetic products for respiration, improving organoleptic properties, increasing hydrostatic pressure, improving aroma, accumulation of primary photosynthetic products in the photosynthetic organism and / or improving the taste characteristics of the photosynthetic organism, in particular the Brix sugar content (Brix) in its seeds, fruits, flowers, nectar, roots, stem or parts thereof.

Photosynthetic organism means life forms that synthesize the primary products of photosynthesis, including plants with C ₃ -, C ₄ - and CAM photosynthesis, and photosynthetic eukaryotes, including, but not limited to, eukaryotes from the following preferred supergroups: Archaeplastida (Archaeplastid algae), such as Plantae, Chlorophyta (green algae) and Rhodophyta (red algae), and Chromoveolata (chromalveolates) such as Phaeophyta (brown algae). Photosynthetic organisms may also refer to plants, ground cover and ornamental plants, crops including food, fodder, fiber, food and agricultural crops and their crops, and plants, both higher and lower plants, and plant-like organisms. Systems, methods and preparations can be successfully applied to any kind of photosynthetic living organisms.

The compositions can be applied to virtually any kind of living photosynthetic organism. Photosynthetic organisms that may benefit include, but are not limited to, all Plantae species, in particular plant species in all crop groups recognized by the US Environmental Protection Agency (2012: 40 CFR 180.41), such as alfalfa, allspice, amaranth, angelica, anise, annatto, arugula, bach ciao, lemon balm, barley, basil, beans, beets, borage, breadfruit, broccoli, Brussels sprouts, burdock, burnet, cabbage, cantaloupe, capers, cumin, cardamom, cardon, carrot, cassava, castor bean, cauliflower, cavalio, broccoli, allspice celery, salad celery, asparagus lettuce, cereals, chard, chayote, chervil, chickpeas, chicory, chives, cilantro, cinnamon, cloves, clover, coffee tree, kale, coriander, corn, cotton plant, cranberry, watercress, cucumber, cumin, curry, daikon, daylily, dill, endive, spurge, eggplant, fennel, fenugreek, flax, food plant, hazel grouse, gherkin, pumpkin bottled, vee nograd, cereal, garlic, hywar, hay grass, hemp, shandra, hosta, hyssop, swordfish, jicama, jojoba, kale, kohlrabi, kudzu, leek, hyacinth bean, lavender, green leafy vegetables, leek , legume, lemongrass, lentil, lespedeza, lettuce, lupine, nutmeg, marjoram, melon, millet, mint, mizuna, momordica, musky melon, mustard, nasturtium, nutmeg, oats, onion, quinoa, parsley, parsnip , pasture plants, peas, peanuts, peppers, peppermint, perilla, popcorn, potatoes, poppy, pumpkin, purslane, radicchio, radish, green rapeseed, rhubarb, rice, rosemary, swede, rye, safflower, saffron, sage, sainfoin , goat's beard, sugar beetroot, sesame, shallot, sorghum, soybean, spinach, large-fruited table squash, stevia, strawberry, sunflower, noble laurel, sweet potato, sugar beet, sugar cane, leaf beet, xiphoid canal, taro, taro, wormwood, tea plant, teosinte, thyme, tobacco, tomato, clover, triticale, turmeric, turnip, vanilla, vernia, vetch, watermelon, wheat, water rice, wintergreen, woodruff, wormwood, yam, marrow and other similar plants; fruit-bearing plants such as almond, apple tree, apricot tree, avocado, azarol hawthorn, banana tree, beech hazel, blackberry, blueberry, brazil nut, breadfruit, gray walnut, cashew nut, cherry, chestnut, dwarf chestnut, citrus plant, tree cocoa, cocoon, coffee tree, currant, pitahaya, elderberry, fig, hazel, lure, gooseberry, grapefruit, guava, hickory, blueberry, kiwi, kumquat, lemon, lime, loganberry, medlar, macadamia nut, mango, mangosteen , martinia, summer hawthorn, naranjilla, nectarine, nopales, walnut, okra, olive, orange tree, papaya, passion fruit, peach tree, pear tree, pecan, pepper, pistachio tree, plum, plumcot, house plum, pomello, quince , raspberry, rosella, tangelo, mandarin, tangor, Mexican hawthorn, Mexican tomato, agli, walnut, spices and other similar plants; floral and ornamental plants such as yarrow, adenium, agave, ageratum, aloe, alyssum, anemone, aquilegia, aster, azalea, begonia, strelitzia, dicentra splendid, borage, bromeliad family, bougainvillea, buddleia, cactus, calendula, camellia, bluebell , sedge, garden carnation, celosia, chrysanthemum, clematis, cleome, coleus, cosmos, saffron, croton, cyclamen, dahlia, yellow daffodil, daisy, dandelion, krasnodnev, delphinium, carnation, dietes, digitalis, mandevilla, sorrel, cornflower, euonymus , forget-me-not, fremontodendron, fuchsia, gardenia, gazania, geranium, gerbera, gloxinia, gladiolus, hibiscus, hydrangea, balsam, jasmine, lily, lilac, lisianthus, lobelia, marigolds, mesembryanthemum, sponge stick, forget-me-nots, narcissus, Hawker's balsam, water lily, primrose, oleander, orchid, ornamental plants, oxalis, pansies, penstemon, peony, petunia, plumeria, poinsettia, cyanosis, buckwheat, poppy, purslane, primrose, ranunculus, rhododendron, rose, sage, ragwort, dode katheon, antirrinum, nightshade, goldenrod, matthiola, ti, torenia, tulip, verbena, periwinkle, violet, violet, yucca, zinnia and other similar plants; indoor flowers and plants such as African violet, Aglaonema variably, succulent, dieffenbachia, dragonfly, ficus, hosta, spathiphyllum, philodendron, scindapsus, hevea, sansevieria, chlorophytum and other similar plants; trees such as fir, aspen, birch, cedar, cinnamon, dogwood, cycads, cypress, metasequoia, elm, fig tree, spruce, ginkgo, gymnosperms, deciduous trees, rosewood, jacaranda, juniper, laurel, legume, liriodendron , Magnolia, Mahogany, Maple, Oak, Palm Tree, Spruce, Resinseed, Plantain, Poplar, Evergreen Sequoia, Rosewood, Giant Saber, Willow, Sycamore, Yew, Teak, Willow, Yew, Christmas Tree, Production Trees lumber, trees for paper production and other similar plants; herbs such as lawn grass, turf, bluegrass, bent grass, bermuda grass, bonfire, reed grass, sedge, marsh bent grass, couch grass, fescue, oat grass, emperorata, miscanthus, molina, millet, paspalum, pennisetum, canary grass, roa, meadow seeds herbs and other similar plants; dwarf plants; grafted plants; cuttings; hybrids and other similar plants. In addition to the above crops, the preparations can also be applied to secondary metabolites of sources of photosynthetic organisms, such as switchgrass, jatropha, spurge, tobacco, lichen, brown algae, diatoms, cyanobacteria, bacteria, dunalyella, nannochloropsis, chlorella, hematococcus, eucheuma; mosses such as lichen and fern, and other similar plants. This list should be considered as an example only and is not intended to be exhaustive. Those skilled in the art can easily identify other photosynthetic organisms that may benefit from the compositions and methods of the present embodiments. The methods and preparations disclosed in the invention can be used to enhance the growth of juvenile and mature photosynthetic organisms, as well as cuttings, tissues, seeds, meristems, callus, cells, and vegetative propagation. Glycan composites can be used in soaking and coating seeds prior to sowing at a rate in the range of 10-1000 µg of glycan composites per seed, preferably in the range of 20-300 µg/seed.

Alternatively, seeds, tubers, bulbs, underground shoots and cuttings can be worked in the furrow at the same time as sowing. Generally, the anatomical site to which the composition is applied by the method of the invention should have a large enough surface area for the photosynthetic organism to absorb the composition. For example, it is desirable to cover a sprouted cotyledon (ie, cotyledon), underground potato shoot, bulb, tuber, or other large surfaces that facilitate the absorption process, such as prominent leaves and roots. Bearing plants can be treated before and after the onset of bud, fruit and seed formation. For plants such as annuals, perennials, trees, orchids, the Gesneriaceae and cacti, in which stems, roots and/or stems may be treated, application methods include spraying shoots and/or simultaneously spraying and irrigating shoots and roots. or application by dipping or by separate application to roots and shoots. Commercially grown aquatic and marine crops such as spirulina, aonori, laver, seaweed, macrocystis, nori, and wakame, glycan composites can be applied by spraying, spraying, brushing, or dipping in sterile solutions in fresh or sea water. with a content of 10 ppb - 3% glycan composites for a time of 15-90 minutes, sufficient for the absorption process.

Glycan Composite

Methods and formulations in accordance with specific embodiments disclosed herein are for example for treating any of the photosynthetic organisms mentioned above, such as plants, and for improving the quality, enhancing growth and/or improving the quality and yield of crops grown. This can be achieved by applying preparations of a glycan composite consisting of the following components: one or more branched deglycosylated glycans with specific ²⁺ transition metal coordination compounds. The formulations can be applied in dry or liquid form directly to photosynthetic organisms. In specific embodiments, the implementation of liquid preparations may additionally include a preservative to prevent deterioration during transportation and storage. The methods disclosed herein make the glycan composites readily available for uptake by photosynthetic organisms.

Branched deglycosylated glycan component

In specific embodiments disclosed herein, branched, deglycosylated glycans are provided that are components of a glycan composite. This branched deglycosylated glycan component will be referred to hereinafter as the glycan or deglycosylated glycan component of the glycan composite.

Chemically synthesized glycans are quite expensive and, with the exception of some of the embodiments of the invention, their use in agriculture cannot

- 5 040708 be economically justified. However, the embodiments disclosed herein provide a range of cost-effective products resulting from the deglycosylation of macromolecules of specific inexpensive substances, and these products can be used as glycan components in a glycan composite. Due to this, the use of specific branched deglycosylated glycans makes the treatment of crops economically justified.

Suitable glycans can be obtained by cleavage, ie deglycosylation of the glycan subunits in their parent molecule. Typically, macromolecules with a molecular weight of more than a few 1000 kDa are chemical structures that are too large to be processed and taken up by a photosynthetic organism, therefore, the preferred components of a branched deglycosylated glycan are deglycosylated glycans with a molecular weight of less than a few 10 kDa. Deglycosylated glycans are typically derived from macromolecules such as proteins, glycoproteins, N-linked-glycan macromolecules, and/or O-linked-glycan macromolecules. They may be products of hydrolysis or other known processes resulting from the action of acids, bases, enzymes and/or micro-organisms that break chemical bonds. The biosynthesis of branched glycan components with a plant or yeast can be cost effective compared to chemical synthesis and purification of products, which are too costly. For example, the cost of pure highly branched N-linked mannan glycans can be several $1000 per gram, while suitable highly branched N-linked mannan glycans obtained by deglycosylation from proteins in accordance with the embodiments disclosed in the invention can cost several cents per gram. .

Plant sources of suitable glycan subunits include the following: Cyanaposis tetragonalobus and Cyanaposis psoraloides, guar gums, GalMan2; Caesalpinia spinosa, tara gums, GalMan ₃ ; Ceratonia siliqua, locust bean gums, GalMan _1-8 ; Amorphophallus konjac, konjac gums, Glc2Man2; Canavalia ensiformis, xiphoid canal, N-linked glycans; phytelefas fruit, Mann; carob; coffee beans; Greek fenugreek; barley; palms, lilies, irises and leguminous plants, endosperm tissues, Mann; coniferous wood and bark of various trees; birch; gymnosperms; Norway spruce and green algae Chlorophytum such as Dasycladales, Characeae, Codium fragile, Caulerpa and Acetabularia acetabulum mannan containing algae. In addition, the structures of branched mannan derivatives such as those shown in FIG. 2 can be found in fungi such as Hansenula holstii, Rhodotorula acheniorum, in glycoproteins such as concanavalins and enzymes; and preferably in invertases. Other natural sources include microbes, bacteria, fungi, animals such as arthropods, crustaceans, molluscs, fish, krill and insects, and waste products such as guano, giblets, blood, bone marrow, liver, animal organs, bark, sawdust, wood, bones, exoskeleton, dough, by-catch and manure.

The gums, proteins and other macromolecules mentioned above can be deglycosylated using processes known in the industry. For example, some branched glycan macromolecules can be microbiologically processed using highly controlled fermentation, and other macromolecules can be subjected to various other known enzymatic processing processes, and as a result, branched glycan macromolecules can be partially hydrolyzed to cleave gums with a molecular weight of >100,000 kDa. to deglycosylated glycan with average molecular weights of 0.2-10 kDa. As a result, deglycosylated glycans with significantly lower molecular weights can be taken up by plants. When compared with various natural sources, branched deglycosylated glycans from invertases showed the best technical and economic performance, characterized by low cost and high activity, which makes their production on an industrial scale possible, see table. 1.

Table 1. Comparison of natural sources for obtaining branched deglycosylated glycans. Relative activities and costs were compared after obtaining final products from different sources. In most cases, higher activity resulted in lower cost. Invertase was the lowest cost and highest potency source of deglycosylated glycans available for production.

Source Activity Relative cost Invertase 1000-1000000 $ vegetable gums 10-100 $$$ shellfish 5-10 $$$$ sawdust 1 $$$$

The activity of glycans depended to a very large extent on their terminal ligands, which gave

- 6 040708 reason to consider the identification of this part of the glycan structure as a very important point. Suitable glycan terminal ligands have been identified in glycopyranoses such as galactopyranoses, glucopyranoses and preferably mannopyranoses, in their alkyl, acyl and aryl substituted derivatives, and in acylglycosamines. From this it followed that suitable glycans are cationic, anionic and neutral polymers, aldosyl and/or ketosyl, and branched glycans with any of the above terminal ligands. Molecular weights typically range from 0.1 to >500 kDa, preferably from 0.2 to 10 kDa, and most preferably in the range from 0.5 to 2 kDa.

The following abbreviations for glycans are used:

Gal is galactopyranosyl;

Glc is glucopyranosyl;

GlcNAc is N-acetylglucosaminosyl;

GalNAc is N-acetylgalactosaminosyl;

Gly is glycopyranosyl;

Lac is lactosyl;

Ara is arabinosyl;

Man stands for mannopyranosyl and

Mann stands for poly-map.

j, m, n subscripts indicate the respective lengths of the chains, where m=1-24 and n=1-24, unless otherwise indicated. For example, GalGlcMann is galactopyranosylglucopyranosylmannopyranosyl _n and GlcmMann is glucopyranosyl _m mannopyranosyl _n .

Numbers separated by hyphens indicate a range of values. For example Man ₈ . ₁₄ GlcNAc1. ₂ denotes branched mannopyranosyl ₈ - ₁₄ N-acetylglucosamine ₁ . ₂ , where Man ₈ _ ₁₄ denotes a range of 8 to 14 Man units in a branched chain.

Examples of preferred branched chains include MannGly with alkyl, acyl, aryl, polyacyl, polyalkyl, amine or unsubstituted; aldosils; ketosyls; GlcNAcn; alkyl Glc _n ; methyl Glc _n ; methyl GlcGly _n ; _alkylMann ; Gal _n Man ₂ ; pentoses; arabinose; riboses; xylose; hexoses; mannoses, mannosides, mannans; glucose, glucosides, glucans; galactoses, galactosides, galactans, raffinoses; Gly ₂ eg Glc ₂ sucrose, trehalose, maltose, gentiobiose, cellobiose, GalGlc lactose, xylobiose, laminaribiosa; GalmMann; Gal _m GlCjMan _n ; GlCjMann; XyloMann; AraMann; AraGaln; fructofuranosyl _m GlCi; Lac; maltopyranosyl; Man _1-3 such as Man1, methyl-α-D-Man, methyl-a-DMan1_ ₃ , methyl-α-D-Man ₃ Gal; triosyls such as Man ₃ ; and their derivatives and combinations.

Suitable glycans in a mixture of deglycosylated glycans of a glycan composite are, for example, Man1, Man ₃ , methyl-D-Mann and methyl-D-GlCn. Deglycosylated glycans can be selected from short chains such as Mann, where n is a number from 1 to 8, preferably Man1_ ₃ ; Linked-branched chains such as Man _m _ _n Gly, Man _m _ _n Glc, Man _m _ _n Gal and Man _m _ _n GalGlc, where m is a number from 1 to 8 and n is a number from 1 to 8. Even more suitable glycans are chains with a molecular weight >1 kDa, such as, for example, Gal _m Man _n , Gal _n Man _n , Glc _m Man _n , GaljGlc _m Man _n , j = 1-24, m=1 -24, n=1-24; preferred are deglycosylated glycans with molecular weight <1 kDa, where j = 1-8, m=1-8, n=1-8, such as, for example, Gal ₂ Man ₂ and in combinations and mixtures.

Preferred deglycosylated glycans are O-linked glycans and N-linked glycans with Gly _m . _n where m=1-8 and n=1-8. Additional examples include the following basic Mann structures such as α-mannobioses, mannotetroses, mannopentoses; amino-functionalized-mannoses, for example glycylMan _n , alanylMan _n and aminylMan _n , where n=1-24.

Preferred branched deglycosylated glycans may include one or more N-linked glycans, such as, for example, Man _n GlcNAc1_ ₃ and Man _m . _n GlyNAc1. ₃ , for example Man ₈ _ ₁₅ GlcNAc ₂ . Preferred N-linked-glycans are selected from low molecular weight MannN-glycans, therefore, Man ₃ GlcNAc1_ ₃ and trimannopyranosyl-N-glycans are most preferred. Suitable derivatives may have higher degrees of branching, such as Man _m _ _n GalNAc1_ ₃ , for example Man ₈ _ ₁₅ GlcNAc ₁ _ ₂ and Man ₉ . ₂₀ GlcNAc1. ₃ , and derivatives such as N-glycan, acyl-, alkyl- and aryl-substituted. In addition, suitable branched GalGlcManN glycans include Glc:Gal:Man in ratios of 1:2:16-9:2:20, such as Gal ₄ Man ₁₀ GlcNAc ₂ . In addition, N-acetylglycosaminyl-terminal ligands such as N-acetylgalactosamines, N-acetylglucosamines and N-acetylneuroamines can be selected from GalNAc1_ ₃ , GlcNAc1_ ₃ , GlcNAc ₂ , Man ₁ _ ₈ GlcNAc ₁ _ ₃ , derivatives and combinations thereof.

Components of a transition metal coordination compound ² *

In the embodiments disclosed herein, transition metal coordination compound ² *glycan composite compositions are provided. In particular embodiments, the ² * transition metal coordination compound consists of a ² * metal component and one or more anionic components. Certain metals ² * are introduced into the structure of the holoprotein for reliable binding of glycans. In the absence of certain metals ^2* , the structure of the protein is incomplete, not about

- 7 040708 matching configuration for conjugation. Therefore, these preferred ²⁺ metals include calcium (Ca ²⁺ ) and manganese (Mn ²⁺ ), and their combined use is preferred, since both Ca ²⁺ and Mn ²⁺ occur at natural holoprotein binding sites. However, in addition to Mn ²⁺ , other suitable ²⁺ transition metals, including one or more D-transition metals ²⁺ selected from cobalt (Co ²⁺ ), nickel (Ni ²⁺ ), and zinc, may be added, used in place of, or incorporated into the formulation. (Zn ²⁺ ) and combinations thereof, but always in the presence of Ca ²⁺ . In addition, the presence of iron (Fe ²⁺ ) and magnesium (Mg ²⁺ ) and/or one or more of the ²⁺ d-transition metals mentioned above may further support the structural conformation of the holoprotein when exposed to Ca ²⁺ and Mn ²⁺ . These ²⁺ metals and/or their water-soluble salts can be introduced into the glycan composite in the form of liquid or solid substances, for example, with a content in the ranges of 0.1-100 ppm Ca ²⁺ , 0.1-100 ppm Mg ²⁺ , 0, 1-10 ppm Fe ²⁺ , 0.110 ppm Mn ²⁺ , 0.1-10 ppm Zn ²⁺ , 0.001-1 ppb Co ²⁺ and 0.001-0.1 ppb Ni ²⁺ .

Preferred anionic components of the above transition metal coordination compounds ²⁺ glycan composites can be selected from sequestering anions, additionally acting as respiration accelerators, which are listed below: oxaloacetates, acetates, aconitates, citrates, isocitrates, fumarates, glutarates, ketoglutarates, malates and succinates. Suitable acids for their derivatives, mentioned in the invention, but not excluding others, were selected from aconitic, citric, fumaric, glutaric, malic, oxaloacetic, succinic and other similar acids for coordination compounds of transition metals ²⁺ , and, preferably, at molar ratios anion:cation 10:1 or more, ranging from 100 ppb to 30% by weight. Aconitic acids include aconitates, cis- and trans-aconitic acids, salts, and the like.

Citric acids include citrates, citric, isocitric and methylcitric acids, citric anhydrides, citrate phosphates, salts, and the like. Fumaric acids include fumarates, fumaric acids, alkyl fumarate salts, and the like. Glutaric acids include glutarates, ketoglutarates, glutaric acid, glutaric anhydride, alkyl glutarates, glutamates, salts, and the like. Malic acids include malates, malic acids, maleic acids, maleates, maleic anhydrides, alkylmaleic anhydrides, maleyl proteins, salts, and the like. Oxaloacetic acids include acetates, acetic acids such as glacial acetic acid and vinegar, acetyl-CoA; acetyl phosphate, acetic anhydrides; alkyl acetates, alkyl acetoacetates, oxaloacetates, salts and the like. Succinic acids include succinates, succinic acids, alkyl succinic acids, succinyl anhydride, salts, and the like. The salts mentioned above include one or more Ca, Mg, Na and transition metal ²⁺ coordination compounds with the acids mentioned above. The anionic components of the ²⁺ transition metal coordination compounds may preferably be selected from their phosphates, such as, for example, malate phosphates, citrate phosphates and the like. Typically, components of pure ²⁺ transition metal coordination compounds are commercially produced in large batches. The anionic components of the ²⁺ transition metal coordination compounds may be selected from suitable polydentate chelating agents such as alkylamide chelating agents as follows: ammonium, sodium and/or potassium salts of alkylamide chelating agents such as ethylenediaminetetraacetic acids (EDTA), N-hydroxyethylethylenediaminetetraacetic acids (HeEDTA), ethylenediamine-N,N'-bis-2hydroxyphenylacetic acids (EDDHA), di(orthohydroxybenzyl)ethylenediaminedioacetic acids (HBED), diethylenetriaminepentaacetic acids (DTPA); methylglycine N,N-diacetic acids (MGDA); glutamine diacetic acids (GLDA) and other similar compounds. Anionic components are typically added to liquid metal solutions at a minimum anion:cation mole ratio of 7:1 and preferably 10:1 or more.

Preferred ²⁺ transition metal coordination salts can be prepared by reacting the ²⁺ metal and anionic components. In addition, suitable commercially available salts include N, P, K, S, C, H, O, Cl derivatives, secondary derivatives and micronutrients thereof, and other agriculturally useful combinations of known compounds. For example, N in the form of amines, amides, nitrates, polyacylamines; C in the form of carbonates; Cl in the form of chlorides; P in the form of phosphates, phosphites; S in the form of sulfates; H in the form of acids; OH in the form of bases and in the form of other similar compounds.

Influence of the Components Taken as Examples on the Functional Characteristics of Glycan Composites

Glycan composites (GC) are composed of several compounds having different chemical characteristics, each of which contributes to the desired properties of the composites as a whole. This is best supported by the fact that when the components of the glycan composite are applied separately to crops in the field, their actions are not coordinated with each other. Direct application of each component to separate groups of plants is possible, but is not preferred due to the lack of a positive effect. Therefore, experiments were carried out to study the functional characteristics of glycan composites taken as examples, and, along with this, it was proved that each of the components separately did not have an action adequate to that of

- 8 040708 to the performance of the composite.

The growth of photosynthetic crops occurs as a result of the respiratory metabolism of the primary products of photosynthesis, which ensures the formation, existence and reproduction. However, the ratio of respiration to photosynthesis is less than one third. To increase this ratio, the regulation of crop growth by more efficient use of the primary products of photosynthesis for respiration than before was used, through the use of glycan composites in accordance with the embodiments disclosed in the invention. This was facilitated by efficient treatment with glycan composites in the dark (i.e., in an environment that is not exposed to sunlight), for example, either seeds and roots underground during the day, or roots and/or seedlings at night. Agriculture would greatly benefit from increased yields by optimizing the glycan treatment of seeds, fruits, flowers, juice nectar, primary photosynthetic products, roots, stems and/or trunks with composites, i.e. by applying to seedlings and/or roots of these new systems. Taken together, the use of glycan composites to achieve a beneficial effect is realized using the embodiments disclosed in the invention.

Unlike photosynthetic leaves, seeds are completely respiratory. As a result, the acceleration of seed germination was achieved by applying extremely low doses of GC to them compared to the regulation of nutrient production. Early growth responses using the separate components were compared with the use of complex GCs, and the results were analyzed for statistical significance of the compared means. It was clearly shown that the separate components did not work, but together they contributed to the overall efficiency. In addition, the ²⁺ transition metal coordination compounds of the embodiment, which included the full set of ² /d-transition metals, improved the performance of the composite preparations.

Respiration is dependent on available oxygen (O ₂ ), and O ₂ supply was enhanced by co-application or by treating crops with GC to increase O ₂ . Targeting O ₂ in specific locations, particularly roots or seeds, has been more effectively achieved in the field by the use of O ₂ -generating compounds such as peroxides. Suitable inexpensive peroxides include H ₂ O ₂ and urea peroxides, where well known O ₂ generating granular compounds such as CaO ₂ and/or MgO ₂ slowly release O ₂ when crops are treated with GC. CaO ₂ and MgO ₂ provided O2 enriched environments that supported respiration, particularly when applied to seeds or roots as separate oxygen sources in combination with the formulations of the embodiment. Peroxides are O ₂ generating components that can be included in dry preparations, but are preferably stored and applied to the plant before, during or after GC treatment. Peroxides tend to destabilize and degrade GC concentrates, reducing their shelf life as a result.

An exemplary joint application method for generating O ₂ was as follows. Prior to GC treatment, liquid H ₂ O ₂ , 10-100 g granular CaO ₂ and/or MgO ₂ were applied to the soil to a depth of 15-30 cm at a rate of 50 kg/ha during the growing season, and/or a solution of 10-20 g/l was introduced with stirring into potting media or planting holes in the soil before transplanting. GC was applied by row dressing or spray irrigation to a photosynthetic organism in the same crop area. Then, after the plant was treated with GC, the peroxide slowly released O ₂ , increasing respiration in conjunction with the actions of the GC. The introduction of gaseous O2 can be carried out, mainly by bubbling through liquid media until saturation. The combined use of O ₂ generators with glycan composites produced a synergistic effect, which led to an increase in yield.

Under conditions where the O2 content cannot be increased, alternative methods have been used to create a respirable environment in which photosynthetic organisms are cultivated. Thus, for example, treatment with glycan composites was carried out in combination with exposure of cultures to breath accelerators, either by adding them or by incorporating glycans into the composites. Breath accelerators were selected from the following reagents: iP, such as, for example, salts of phosphoric acid, such as ammonium, potassium and sodium phosphates; glyphosphates such as Gly-phosphates such as Glc-phosphates and Man-phosphates, Glc ₂ -phosphates such as mannobiose-phosphates, sucrose-phosphates, trehalose-phosphates and xylobiose-phosphates; plant growth regulators such as auxins, and oxaloacetic, aconitic, citric, fumaric, glutaric, malic and succinic acids. The acids mentioned above also serve as anionic components of coordination compounds added at a minimum anion:cation ratio of 10:1.

Materials and methods

Responses were studied under the influence of various components of composite preparations on the germination and initial growth of dihybrid sweet corn Burpee Sweet Corn cv. Bi-Licious Hybrid. Rapid seedling growth studies were carried out using hydroponic cultivation,

- 9 040708 in which the aqueous media were sterilized and cooled. Before processing, the seeds were checked to exclude too large, small or damaged seeds. The plants were kept in the dark for breathing at 30°C. Exactly 48 seeds were sown in a 15 cm sterile disposable plastic Petri dish on Whatman paper discs moistened with control nutrient or treatment solutions. The number of replications was 8 per treatment (n=8). Germination was determined by detecting the appearance of primary roots in 50% of control seeds after 30 hours. Treatment and control solutions were prepared by dissolving nutrients in deionized ultrapure water. Instead of stainless steel vessels and stirrers, plastic laboratory glassware was used to prevent Ni ²⁺ and other metals from entering the solutions. Mutual contamination of some nutrients with other nutrients was prevented by disposing of plastic laboratory glassware immediately after use. Initial solutions were prepared from chemically pure compounds. The stock aqueous glycan solutions for the preparation of GC in this study contained 1-15% deglycosylated Mann glycans, which were obtained by acetolysis of phytelefas fruit flour in a mixture of acetic acid:acetic anhydride:sulfuric acid 25:25:1. Solutions of 0.0001-5% Ca ²⁺ and transition metals Fe ²⁺ , Mn ²⁺ , Zn ²⁺ , Co ²⁺ and Ni ²⁺ (abbreviated as Cat) were obtained by complexation in a mixture of 1 mM citrate, 5 mM malate and 1 mM succinate, abbreviated as CMS. GC was used at a concentration of 10 μM Mann. Ca- and Mn-EDTA salts are bound ions, abbreviated as EDTA. Other concentrations used were 1 μM Man ₃ and 100 μM Man1. Man1-CatCMS was prepared with ²⁺ Cat-CMS transition metal coordination compounds for comparison with Man1-EDTA and the like. Water was used as a negative control.

results

As shown in Table. 2, corn seeds treated with the GC glycan composite showed a highly significant acceleration of mean germination numbers (p=0.000) compared to the individual components, Cat, glycan, CMS, used alone. Germination numbers for CMS, glycans and Cat were the same as for water and no difference was found between water and Cat. The GC glycan composite with all its components showed a significant improvement over G-EDTA. The GC glycan composite showed a marginal significant improvement over using the glycan together with CaMn-CMS, indicating that all Cat increased GC performance to a greater extent than bound ions. Thus, both Cat and CMS in the GC glycan composite promoted germination. In addition, composite preparations in which glycans were replaced with Man1 or Man ₃ with Cat CMS showed a significant improvement in germination compared to Man1 or Man ₃ preparations with bound ions and EDTA salts, which did not increase respiration.

conclusions

In the case of the GC glycan composite with all its components, the components had a positive effect on respiration and growth, however, on the contrary, the individual components used separately did not give an effect. The complete set of ²⁺ transition metals in Cat, particularly in the ²⁺ transition metal coordination compounds CMS, significantly improved product performance over bonded ion formulations. The selection of suitable anionic components for the ²⁺ transition metal coordination compounds that facilitated breathing significantly improved the performance of the GC glycan composite compared to EDTA, which did not. It is noteworthy that the activity of the glycan and Man ₃ composites was an order of magnitude higher than the activity of the Man1 composite, which was manifested by seed germination at much lower doses of both composites compared to the Man1 composite. It was found that the use of composites leads to significant improvements in the Man1 and Man ₃ preparations, which is an unexpected result of the studies. Namely, the use of composites of glycan GC with alkylamide coordination compounds of transition metals ²⁺ after seed germination led to the accumulation of primary products of photosynthesis, in particular, in an environment with a reduced partial pressure of oxygen. For example, application of GCCaMnEDDHA 1 week prior to harvesting lettuce leaves resulted in higher Brix than a composite of GC glycan with all its constituents and controls.

Table 2. The effect of preparation components on corn germination showed that glycan composites with all their constituent components have the best functional characteristics. The statistical significance of the differences between the average values of germinated seeds grown when treated with various components, compared with treatment with a unified glycan composite, was the values shown in the table, where Cat=unchelated Ca ²⁺ , Fe ²⁺ , Mn ²⁺ , Zn ²⁺ , Co ^{2 +} and Ni ²⁺ ; CMS=citrate, malate, succinate; EDTA=Ca-EDTA salts+Mn-EDTA salts; GC=GC CatCMS; G-CaMnCMS=glycan and Ca ²⁺ -Mn ²⁺ -CMS; count=average number of germinated seeds; n=8 for all replications and p=statistical significance.

- 10 040708

Comparable processing methods Count R GC vs Glycan 46 vs 23 0,000 GC vs. CMS 46 vs 23 0,000 GC vs Cat 46 vs 23 0,000 Cat vs water 23 vs 23 0.601 GC vs. G-EDTA 46 vs 36 0.043 GC vs. G-CaMnCMS 46 vs 38 0.054 Man ₁ CatCMS vs. Man ₃ EDTA 39 vs 27 0.025 Man ₃ CatCMS vs Man ₃ EDTA 45 vs 29 0.016

Preservatives for liquid concentrates

It is often advantageous to produce glycan composite products in the form of 10 ppm - 30% concentrates that can be transported in dry or liquid form and additionally stored in cold, dry, dark conditions, but for organic compounds that are part of glycan composites, it is inherent that the complex was consumed by various and varied microbes, as well as plant cells. Therefore, care must be taken to protect compositions from spoilage, especially aquatic products. In the case of storage, especially liquid compositions, suitable preservatives may be included in the composition to improve the stability of the products. Commercially available preservatives include biocides and germicides such as, for example, peroxides, sodium hypochlorites, bleaches, acids, bases, oxidizing agents, formaldehyde releasing preservatives such as 1,3-dimethylol-5,5-dimethylhydantoin, quaternium-15, bronopol , diazolidinyl urea, Na-hydroxymethylglycinate, silver, copper acetate, permanganates, dinitromorpholines, phenolic compounds such as 4-chloro-3-methylphenol and 2-phenylphenol, thiazolinones and preferred isothiazolinones (IT) such as benzoisothiazolinones (BIT), methyl chloroisothiazolinones and methylisothiazolinones (MIT). IT is a herbal antimicrobial used in concentrations ranging from 1-800 ppm. Preservatives are recommended for incorporation into liquid concentrates of glycan composites in the range of concentrations indicated on the labels, from 1 ppm to 1%. For example, BIT has been safe and effective in liquid concentrates in the range of 50 to 750 ppm, preferably 100 to 300 ppm. Thus, the liquid preparation of the glycan composite can be mixed with any antimicrobial agent, and these agents must be selected from herbal preservatives in accordance with the embodiments disclosed in the invention. In a glycan composite product that is a composition concentrate containing 10 ppm to 30% glycan composite to be diluted prior to application to crops of photosynthetic organisms to increase their yield, a suitable concentrate comprises one or more deglycosylated glycans in a concentration range of 1 ppm to 20% and one or more transition metal coordination compounds ² * in the concentration range from 1 ppm to 10% and a preservative in the concentration range from 50 ppm to 1% by weight.

Taken as an example, the preservative preserves the activity of glycan composites

The effect of preservatives on the activity of glycan composites as plant growth regulators was compared. Root growth was quantified in experiments. The results showed that the activity of preparations with preservatives was maintained after storage for one month. In contrast, preparations without preservatives lost their activity.

Materials and methods

Root growth in early seedlings of leaf beet (Beta vulgaris subspecies cicla L., cultivar Fordhook® Giant) was studied for response to treatment with glycan composites (GC) supplemented with BIT preservative. In preliminary experiments, preservatives were excluded that had a narrow antimicrobial spectrum, were not suitable for use in foods, or were toxic to plants in the antimicrobial dose range. Roots of chard sprouts showed responses of increased growth in length during the week of GC application and this was consistent with increases in weight. A branched N-linked glycan, 100 ppm deglycosylated Man ₃ GlcNAc ₂ , was chosen to prepare a glycan composite mixture by stirring in water. In addition, a glycan composite was prepared by mixing an anionic component of 5 mM malic acid in an aqueous solution of glycan with water-soluble metal nitrate salts ² *, which resulted in a 5 mM transition metal malate coordination compound ² * containing 1-5 ppm Ca ² * and transition metals 1-3 ppm Fe ² *, 0.1-0.5 ppm Mn ² *, 0.2-1 ppm Zn ² *, 0.01-0.1 ppb Co ² * and 0.001-0.01 ppb Ni ² *. Proxel™ GXL preservative was selected from the IT antimicrobials and used in the preparation of liquid concentrate products from 1 ppm to 30% for storage in the range of preservative concentrations of 100-200 ppm. The preparations were stored for a month at 35°C before testing. Concentrated preparations were diluted in the required volume of water immediately before the treatment of seedlings. Chemically pure compounds of other required elements were introduced into the solutions.

- 11 040708 cops.

The rapid root growth studies were based on modified methods of the hydroponic cultivation mentioned above on moistened Whatman 598 Seed Culture paper mugs, and the treatment solutions and aqueous media were not sterilized prior to use. Root weights in milligrams were determined by weighing on a verified digital Mettler balance. The terminology used in the invention refers to the non-use or use of nutrients as follows: GC=10 μM glycan composite without IT, GC-IT=GC with IT; Man1=100 μM Man1-GC without IT, Man1IT=Man _r GC with IT; MG=375 mM methyl-D-GlcrGC without IT, MG-IT=MG with IT; Man ₃ =1 μM Man ₃ -GC without IT, Man ₃ -IT=Man ₃ -GC with IT.

Preservatives preserve the activity of stored drugs

When treated after germination of seedlings of leaf beet with a glycan composite without a preservative, its activity was two times less than the activity of the same preparation with IT. The results in the form of mean values±standard error (SE) are presented in table. 3. Doubling (2X) the concentration of composite preparations without IT resulted in higher root mass compared to control, but at a 20% weight reduction compared to 1X concentrations of glycan composite with IT. Preparations without preservative lost at least half of their activity after 1 month of storage. Maintaining the original potency of GC preparations with preservatives shows a clear improvement in all products that must be stored before they are sold to end users and before they are used.

Table 3. Effect of composites containing or not containing IT preservative on root growth of leaf beet seedlings is characterized in the table by root weight. Preparations were studied after 1 month of storage, and preparations with IT showed better results than preparations without IT. The mean values of the root mass in the case of preparations without IT were equivalent to the results in the case of water as a control. Values are the mean of root mass measured in milligrams (mg)±standard error (SE). Abbreviations: GC=GC without IT, GC-IT=GC with IT; and so on. Man1=Man _r GC; MG=methyl-D-GlCl-GC; Man3=Man3-GC.

Treatment Mean + SE (mg) GC IT 12+0.3 Man ₃ -IT 12+0.4 Map ₃ -1T 11 + 0.5 MG IT 11+0.6 2X GC 10 + 0.5 2X Man ₃ 10+0.6 GC 8 + 0.4 man ₃ 8 + 0.5 man ₃ 8 + 0.6 MG 8 + 0.6 Water 8 + 0.5

In the example shown in FIG. 1, a plant cell was exposed to a glycan composite solution that was delivered into the cell. In accordance with the affinity and specificity of glycoprotein binding, the glycan composite displaced the primary products of photosynthesis from storage, making them available for respiration, growth, and germination. This redirected energy flow resulted in consistent faster germination than control nutrients, along with other important characteristics.

A suitable method for preparing a glycan composite is as follows. In specific embodiments, the glycan composite was prepared with one or more of the ²⁺ transition metals mentioned above. To prepare the ²⁺ transition metal coordination compound, one or more appropriate anionic components, such as, for example, 0.1-5 mM citric acid, malic acid, succinic acid, and/or oxaloacetic acid, were added and dissolved in water, and then ppb - ppm amount of suitable metal components ²⁺ components of the coordination compounds of transition metals ²⁺ were stirred to dissolve in an aqueous preparation. Thus, for example, the added ²⁺ metal components were 1-10 ppm Ca ²⁺ and 0.1-1 ppm Mn ²⁺ , one or more of 1-10 ppm Mg ²⁺ , 1-3 ppm Fe ²⁺ and 0 ,2-1 ppm Zn ²⁺ , and 0.01-0.1 ppb Co ²⁺ and 0.001-0.01 ppb Ni ²⁺ . In order to form the appropriate ²⁺ transition metal coordination compounds, it is necessary to provide appropriate transition metal ²⁺ cation:anion ratios of at least 1:10, and preferably 1:25. The preparation of the glycan composite was completed by adding ²⁺ transition metal coordination compounds to the amount of glycan from 1 μM to 500 mM. Formulations that were to be stored for more than one day before being applied to plants included labeled amounts of a broad spectrum preservative selected from IT, BIT, MIT, hydantoin, and other similar preservatives. The method may also include the step of adding one or

- 12 040708 more surfactants/emulsifiers for agricultural purposes and/or other agronomic additives/auxiliaries in the quantities indicated on the label, which allow reaching at least critical micelle concentrations, in particular for foliar application.

Suitable surfactants and emulsifiers include anionic, cationic, nonionic and zwitterionic surfactants, e.g. amines, alkylphenol ethoxylates, phosphate esters, polyalkylene oxides, polyalkylene glycols, polyoxyethylene (POE) esters with fatty acids, POE with fatty diglycerides, POE polymers, POP polymers, PEG polymers, protein surfactants, sorbitan fatty acid esters, alcohol ethoxylates, sorbitol fatty acid ester ethoxylates, ethoxylated alkylamines, quaternary amines, sorbitol ethoxylated esters, substituted polysaccharides, alkyl polyglucosides (APG), APGcitrates , alkyl glycosides such as methyl glucosides, alkyl mannosides, methyl mannosides, ethyl acetoacetates, N-acetylglucosamines, meglumines, glucamides, dimethylglucamines, copolymers, siloxanes, amines from beef and mutton fat, and mixtures thereof. When the glycan composites are applied to the leaves, the preparation may additionally include one or more aqueous surfactants, and the resulting mixture is applied to the foliage of the plant by spraying, aerosol irrigation, fine spraying, or electrostatic method in an amount of from about 1 to 100 gallons (from 3 .78 to 378 liters) per acre (4047 m ² ), preferably from 10 to 80 gallons (37.8 to 302.8 liters) per acre (4047 m ² ).

GC-nutrient blends maintained plant viability, promoted root development, enhanced plant growth, improved flowering plant appearance, promoted fruiting, and improved fruit taste and odor, providing an impact that was especially important in nutrient-deficient soils and waters. The main primary elements include N-P-K. Major secondary nutrients include Ca, Mg and S. Major micronutrients include B, Cl, Co, Cu, Fe, Mn, Mo, Ni, Si, Na and Zn. Preferred nutrients are not chosen to exclude other elements, ions or salts and, depending on the situation, they may be available in soil and water, in particular in such an abundance that additional nutrition is not needed to increase yield, therefore, nutrient supplementation is applied in accordance with the norms agreed with the state regulatory and control authorities in agriculture, following the instructions on the labels. Suitable sources include generally well known salts and minerals. For example, the most preferred choice of micronutrients for compositions may include 0.5-5 ppm of chelated Fe and/or Zn and applying an appropriate amount of the resulting mixture to one or more plants.

The phosphorus-containing plant nutrient can be obtained from one or more of the following sources: iP, phosphate rock, phosphoric acids, phosphates, phosphites, pyrophosphates, Steric P, guano, manure, seaweed extracts, bird droppings, fish, poultry and livestock and other similar sources. Organic P sources are generally too expensive to apply in fields with concentrations in the 1-20% iP range, but at concentrations below 1% they are often effective as respiration accelerators. Organic sources of P, including, for example, glycerophosphates and the aforementioned sugar phosphates, were used in the concentration range from 0.1 ppm to 800 ppt.

Nitrogen can be obtained from one or more of the following sources: nitrate N, such as nitric acid and its salts, ammonium N, such as ammonia, ammonium urea nitrate (UAN), ammonium nitrate, ammonium sulfate; N urea such as methylene urea, formaldehyde urea, urea, urea with a low and preferably extremely low carbamyl urea content; N amines/amides/aminos such as alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, ornithine, proline, selenocysteine, taurine, tyrosine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan , valine; salts such as monosodium or potassium glutamate; derivatives; mixtures and other similar compounds; and mixtures of amino acids; proteins such as proteins from gluten, caseins, fish and blood; hexamines and combinations thereof. Nutrients are metabolized to all biochemical components, growth components, reproductive and structural components of photosynthetic organisms.

The use of glycan composites provides an opportunity to compensate for nutrient deficiencies in plants by using tank mixtures or by using glycan composite preparations with selected additives. Amounts of plant nutrients were applied in accordance with the data of the guarantee analysis carried out by the authorities on the label at known application rates. Feeding included the steps of dissolving plant nutrient components in an aqueous solution, resulting in a mixture comprising glycan composites. For example, in specific embodiments, 100-2000 ppm N, 50-250 ppm P, 50-2000 ppm K, 1-100 ppm Ca, 1-10 ppm Fe, 0.5-6 ppm Mn and/or or 0.5-5 ppm Zn. Various preparations of glycan composites can be classified as plant biostimulants, which can increase the efficiency of plant nutrient use, increase resistance

- 13 040708 plants to abiotic stress and increase yield. Biostimulants typically include natural products such as, for example, plants, algae, fermentation process metabolites and animal metabolites, humates, microbial waste products, biochemical products, and other similar natural products. Other glycan composite preparations may be referred to as elicitors, which typically include natural products such as, for example, algae, fermentation process and animal metabolites, proteins, enzymes, microbial waste products, and other similar natural products.

Embodiments disclosed in the invention in relation to agricultural crops are composites of glycans formulated in the form of compositions, the compounds of which themselves serve as agents for the control of photosynthetic organisms, including living photosynthetic eukaryotes, and, to a large extent, agricultural archeplastid plants. In this regard, the glycan composite can be suitably prepared with agrochemicals and used in the form of aqueous compositions that promote the growth of photosynthetic organisms, in particular cultivated flowering plants. The methods disclosed herein can be used to safely enhance the balanced metabolism of exogenous components that enhance the yield of photosynthetic organisms. However, at the same time, they can be methods of using glycan composite compositions for beneficial treatments of photosynthetic organisms, which, in addition, can be carried out to enhance normal growth and improve the quality of crops.

Without advocating any theory, the inventor nevertheless assumes that the glycan composites of the embodiments disclosed in the invention act according to a specific lock and key mechanism. A glycan with a suitable terminal ligand exhibits a high binding affinity for a specific receptor glycoprotein. The lock is a glycoprotein and the key is the terminal ligand. The switches of the glycoprotein-lock are the internal sections of the structures of calcium and manganese. When the lock is opened with a glycan key, the primary products of photosynthesis are displaced in order to accumulate or transfer them for participation in the process of respiratory metabolism, and, as a result, the possibility opens up for choosing respiration accelerators in the anionic components of the transition metal ²⁺ coordination compound. During the daytime, the roots consume these primary products of photosynthesis. As a result of respiration of the roots, carbon dioxide is formed, part of which (from 10 to 20%) is transferred up to the shoots, thereby improving the results of photosynthetic carbon fixation. Thus, the glycan composite can be used to reduce energy losses during photorespiration by conducting these treatments of photosynthetic organisms with the effect on the nectar of the juice, which modulates the quality of sugar content, measured by the Brix scale. In particular, the glycan composite can have a positive effect on yield when, for example, plants are grown in environmental conditions with photorespiratory stresses such as intense sunlight, drought and heat. In addition, embodiments provide glycan composite systems for regulating the accumulation of primary photosynthetic products by reducing the rate of respiration to improve the organoleptic and nutritional characteristics perceived by agricultural consumers, livestock, poultry, and nectar collected by pollinating insects.

In accordance with the embodiments disclosed herein, a novel plant growth regulation system is provided that improves photosynthetic excretion to trigger the accumulation of primary photosynthetic products by photosynthetic organisms. This is initiated, for example, by creating novel crop exposures of glycan composites that can be applied to photosynthetic organisms. Composites of glycans may consist of branched glycans with ²⁺ transition metal compounds with polydentate anions. Preferred anions of this embodiment are alkylamides selected from salts of EDTA, EDDHA, HeEDTA, DTPA, HBED, MGDA, GLDA, and the like. In addition, oxygen-depleted environments with reduced oxygen partial pressure in the range of 0-10% O ₂ can be created using physical methods to reduce the respiration rate, such as flooding the roots with irrigation water, keeping plants or parts of them in a nitrogen atmosphere, or increasing concentrations _CO2 . Alternatively, the respiration rate-reducing agents may be selected from suitable plant growth regulators co-administered in accordance with known agriculturally approved methods, for example, plant growth regulators at doses of 1-750 ppm were selected from various suitable cytokinins, salicylic acids and/ or gibberellins and their derivatives and other similar compounds.

Photosynthetic organisms give an appropriate response to the use of components of glycan composites, preferably suitably prepared and converted into active preparations, whereby they promote the growth and quality of photosynthetic organisms, as well as provide a set of positive physiological effects, which increases their market attractiveness.

- 14 040708

In accordance with the embodiments disclosed in the invention, the component complexes of the embodiments may be applied separately, sequentially, or simultaneously. Moreover, especially under conditions of physiological stress, as a result of the impact of glycan composites on the nectar of the juice of a photosynthetic organism, the yield increased due to an increase in the flow of primary photosynthetic products in the direction of the respiratory metabolism in photosynthetic organisms in accordance with the above-mentioned lock and key mechanism according to the options disclosed in the invention. implementation.

In specific embodiments, glycan composite concentrate products in compositions comprising at least deglycosylated glycans in a concentration range of 0.1 ppm to 30% and one or more ²⁺ transition metal coordination compounds in a concentration range of 0.1 ppm to 20% can be easily prepared for use by dissolving the amount of glycan composites in the preferred carrier water. Alternative carriers include, for example, vegetable and mineral oils, alkyl acetoacetates or aliphatic alcohols. Therefore, it is convenient for the grower to mix the finished solution containing the glycan composites with water, which is the preferred carrier for the final dilution. In most cases, stirring and heating at 25-80° facilitates the dissolution of the dry product in the carrier. The glycan composite can be applied in aquatic agricultural systems such as hydroponic and aquatic cultivation, by metering with pumps into the medium, by root immersion in dilute glycan composites, or by foliar treatment.

The formulations used may include any of a wide variety of agricultural additives, excipients, or other agronomically acceptable ingredients and components (hereinafter referred to as additives) that may enhance, or at least not impair, the beneficial effects of the glycan composite at labeled rates. application expense. Common additives for agricultural use are periodically listed by the US Environmental Protection Agency. In particular, compositions for foliar application may contain distributing agents present in an amount sufficient to further promote wetting, emulsification, uniform distribution and penetration of active agents. The spreading agents are usually organic alkanes, alkenes or polydimethylsiloxanes which provide a coating effect on the phylloplane during processing. Suitable carrier materials include paraffin oils and the surfactants mentioned above. Penetrants include, for example, alkyl acetoacetates, sodium dodecyl sulfate, formamides, dimethyl sulfoxide, and alcohols.

The embodiments of the invention may be used when mixing or tank-mixing with various plant treatments such as agriculturally acceptable pesticides, insecticides, herbicides, plant growth regulators, fungicides, germicides, biocides, elicitors, biostimulants, antagonists, antitranspirants, synergists , systemic preparations, surfactants, spreaders, adhesive fillers, vitamins, minerals, salts, solvents, genetics, biologicals and other similar products. Herbicides that rely on ammonia metabolism, such as glufosinates, Ignite®, Rely®, and Liberty®, are made safer to use by using glycan composites that reduce phytotoxicity to herbicide-resistant GM crops when applied as indicated on the label. consumption rates.

Examples of suitable additives and adjuvants include minerals such as limestone, iron filings and the like; salts such as ammonium nitrate, ammonium sulfate, potassium phosphate, calcium permanganate, calcium phosphates, calcium acetates, calcium aconitates, calcium citrates, calcium phosphate citrates, calcium fumarates, calcium malate, calcium malonate, calcium maleate, calcium malate phosphate, calcium gluconates , calcium glutarates, CaO2, calcium succinates, calcium chelates, calcium nitrate, calcium glycerophosphate, manganese phosphates, manganese acetates, manganese citrates, manganese fumarates, manganese glutarates, rhodochrosite, manganese carbonates, manganese oxides, MgO ₂ , manganese malate, manganese malonate, manganese maleate, manganese succinates, manganese chelates and other similar salts; cosolvents such as alcohols, ketones, oils, lipids, water, and the like; genetically modified organisms and genetic materials such as Bt, genes, sequences, RNA, DNA, plasmids, genomes and other similar organisms and materials; bio-organisms such as microbes, yeasts, bacteria, viruses, vectors and the like; and colorants, dyes and pigments such as annatto, methylene dyes, cobalt blue and indican. Other components that may be added to the compositions include soil conditioners, antibiotics, plant growth regulators, genetically modified foods, gene therapies, and other such components. Among the plant growth regulators that can be added to the preparations of the present invention, mention should be made of auxins, brassinolides, cytokinins, gibberellins, salicylates, benzyladenine, amino acids, benzoates, carboxylic acids,

- 15 040708 vitamins, carbohydrates, herbicides such as phosphonomethylglycines, halosulfuron alkyls, selective herbicides such as setoxydimes and sulfonylureas, their salts, esters, phosphates, hydrates and derivatives, and genetic compositions.

The glycan composite treatment technology is intended for use on crops, but without limitation, in dark and shaded conditions during periods of maximum plant respiration, as well as in conditions of exposure to direct sunlight. Typically, glycan composites are easily applied directly to shoots and/or roots and/or seeds and/or parts thereof, including cuticle, skin, flower, fruit, sap, nectar, bark, trunk, leaves, needles, leaf blade, phylloplana, core of a tree, trichome, root hair, tap root, cotyledon, cone and other similar objects. The concentration of glycan composites in formulations applied to photosynthetic organisms should generally be from about 1 ppb to 1%, and more preferably from about 10 ppb to 500 ppm. For specific applications, the concentration at the site of application may be lower in the case of roots than in the case of shoots, for example, in the case of root application, concentrations are 1 ppb - 300 ppm. The glycan composites may be applied to the rooting medium and then shed with water, or may be first diluted in an aqueous vehicle and then applied to the medium. On foliage, treatment is typically carried out by applying mist, fogging, spraying, drip irrigation, jet spraying, dipping, layering, or co-spraying and soaking at glycan composite concentrations of 1 ppb to 1%. When diluted in an aqueous vehicle, the resulting diluted glycan composite is applied to the photosynthetic organism in an amount of about 1 to 500 gal/acre (3.78 to 1892 L/4047 m ² ).

The following are examples to illustrate the embodiments disclosed in the invention and should in no way be construed as limitations to the present invention. In these examples, purified water was obtained by reverse osmosis, the transition metal coordination compound ²⁺ components and surfactants were purchased from Brandt. The abbreviations used in the following examples are defined as follows: ° stands for °C; Sil is an organosiloxane/copolymer blend; 12-26-26 stands for Brandt 12-26-26 Micro, NPK with B, Cu, Fe, Mn, Mo and Zn; aManda is methyl-aD-Mann, n=1-3; GG denotes combinations of branched O-linked Gal1-12Man2 from partially hydrolyzed guar gum; Ag is GlcNAc1-3; Ethan denotes branched Man3GlcNAc2 (FIG. 2); Cat means a mixture of soluble 1 ppm Fe ²⁺ , 0.5 ppm Mn ²⁺ , 0.5 ppm Zn ²⁺ , 0.01 ppb Co ²⁺ and 0.01 ppb Ni ²⁺ ; CMS denotes a ²⁺ transition metal coordination compound with 0.1-5 mM citrate, malate, maleate and/or succinate; IT stands for isothiazolinone preservatives; MnCO3 is manganese carbonate; AMS stands for ammonium sulfate; MKP stands for monopotassium phosphate; DKP stands for dipotassium phosphate; MAP stands for monoammonium phosphate; DAP stands for diammonium phosphate; NH4OH is ammonium hydroxide; KOH stands for potassium hydroxide; Ca(OH) ₂ is calcium hydroxide; l stands for liter; ml stands for milliliter; mg stands for milligram; g stands for gram; kg stands for kilogram; mM stands for millimole; micronutrients refer to trace amounts of soluble B, Ca, Co, Cu, Fe, Mg, Mn, Mo, Ni, Zn and KOH, Ca(OH) ₂ , NH4OH, MnCO ₃ , MAP, DAP, MKP and DKP are nutrients for plants and buffer substances.

The following are examples of specific formulations that can be successfully used in methods to treat photosynthetic organisms such as plants and to enhance plant growth. The following examples are intended to facilitate the understanding of the present invention by those skilled in the art and do not represent an exhaustive list of formulations that are within the scope of the disclosed embodiments.

Example 1 Glycan Composite Preparation for Root Application

Ingredient Concentration range g/l Preferred concentration g/l

Ethan 10ppb to 1ppm 100ppb Aconitic acid 0.001-10 0.05 ppm concentration range Preferred ppm concentration Ca ²⁺ 1-10 5 cat 0.01-10 3

This formulation may be further supplemented with components of ²⁺ transition metal coordination compounds selected from citrates, fumarates, glutarates, malates, oxaloacetates, succinates and Mg.

Glycan composites for root application were dissolved in 1 L of water with stirring at room temperature from 25 to 35°C and the pH was adjusted to 5-7 by titration with KOH.

The formulation was applied as close as possible to the roots, either in row spacing or by drip irrigation, at a rate of 50 to 450 gal/acre (189 to 1703 L/4047 m ² ). When using irrigation, the treatment was carried out by spilling the soil in the direction of the roots in order to improve the quality and quantity of the primary products of photosynthesis.

Example 2 Glycan Composite Formulation for Foliar Application

Ingredient Concentration range g/l Preferred concentration g/l Apple acid 0.7-50 3.5 GG 0.001-10 1 AMS 0.6-3 3 Sil 0.3-3 0.8 Concentration range Preferred ppm ppm concentration Ca ²⁺ 1-100 1-5 cat 0.1-6 1-3

Ca and Cat were dissolved together with malic acid in 1 liter of water. The other ingredients were added, dissolving individually in succession, and the pH of the solution was adjusted to 5-5.5 by titration with DKP/MAP, if necessary. The ²⁺ transition metal coordination compounds were selected from 0.01 ppb Ni and Co, and ^{the 2+} transition metal coordination compounds may include ppm to ppt aconitates, citrates, fumarates, glutarates, oxaloacetates and/or succinates. Foliar spray application was carried out until glossy at a rate of approximately 75-100 gal/acre (284-378 L/4047 m ² ), resulting in increased primary photosynthetic products, improved quality and yield.

Example 3 Glycan Composite Preparation for Application to Fields and Flowering Plants

Preferred concentration Upper concentration limit Ingredient g/l g/l 12-26-26 0.3 10 Hell 0.1 0.5 Ca ²⁺ 0.005 1 succinic acid 0.7 50 Preparation for application to fields and flowering plants The components were premixed in the form of dry concentrates to prepare a kit in which the dry components were stored and later dissolved in water. To prepare the mixture, the components were mixed in 1 liter of water until they were completely dissolved. The solution was applied directly to the roots. Top dressing with 0.5-3 g/l CMR or other agricultural wetting agents was carried out by

foliar application

The field formulation was preferably applied by spraying the leaves of seedling plants at a rate of 50 to 100 gallons/acre (189 to 378 liters/4047 m ² ) to increase primary photosynthetic products, improve quality and yield.

In addition, coordination compounds of transition metals ²⁴ selected from N1, Co; aconitic, citric, fumaric, glutaric, malic and oxaloacetic acids; EDTA, EDDHA, HeEDTA, DTPA, HBED, MGDA, GLDA; Mg and other similar components.

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Example 4 Foliar Concentrate

Ingredient Concentration range g/l Preferred concentration g/l aManda 1-20 5-15 cat 1-10 5 Ca ²⁺ 0.001-5 0.03 Lemon acid 1-50 5-15 MKR/DAP, pH 5 1-25 5-15 BIT preservative 0.01-0.8 0, 1

In addition, anionic components of ²⁺ transition metal coordination compounds, such as, for example, breath accelerators selected from aconitic, fumaric, glutaric, malic, oxaloacetic, succinic acids and other similar compounds, can be added to this preparation. Alternatively, for the accumulation of primary products of photosynthesis, anionic components were selected from polydentate chelating agents such as EDTA, EDDHA, HeEDTA, DTPA, HBED, MGDA, GLDA and other similar substances.

All components were mixed to homogeneity in an aqueous solution with rapid stirring until complete dissolution and brought the pH to 5-5.5 using MKP/DAP. For foliar application, 0.05% Sil was added to this formulation for a seedling treatment at a rate of 20-100 gal/acre (75.7-378 L/4047 m ² ) to enhance the formation of primary products of photosynthesis and respiration in crops.

The foliar application experiment in this example was carried out as follows. Suitable bell pepper seedlings were selected and kept in about 2 L plastic containers, in each of which the seedlings were divided into two equal groups, treated with the glycan composite preparation and treated with control nutrients. The glycan composite from this example was diluted to 1% with water and applied to seedlings in the treatment group by foliar irrigation, while seedlings in the control group were sprayed with an aqueous solution of mineral nutrients at the same concentration. In terms of all other parameters, the control and treatment groups were grown next to each other under the same field conditions. At harvest, in the treated group, the yield of bell pepper fruits was on average 35% higher than in the control group, which was confirmed by the statistical significance of p=0.001 at n=30. In addition, there were no fruits with sunburn in the treated group, while in the control group, the loss of fruits from sunburn was 1-5%, which reduced their marketability due to unattractive appearance. Thus, an increase in the flow of primary photosynthesis products led to an increase in the marketable yield due to an improvement in the aesthetic appearance of the final product as a result of treatment of the glycan composite with the drug.

Regulation of the flow of primary products of photosynthesis using glycan composites consisting of aManda, in addition, was carried out by optional co-application of a respiration accelerator at a rate of 10-100 g CaO ₂ in the soil near the roots to improve quality and yield.

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Example 5 Plant Growth Regulator (PGR) Example for Foliar Application

Component Concentration range % Preferred concentration % GG 0.01-10 1 Apple acid: maleic anhydride 0.7-50 1 Water 5-80 53 KOH pH 5-7 pH 5.5 Cat-malate 0.01-1 0, 1 Ca-glutarate 0.01-0.3 0, 1 Sil 0.3-3 0.8 Mixing Instructions For lysis in malic acid, GG and 1% sulfuric acid are added to a 50% aqueous solution of a mixture of malic acid:maleic anhydride 1:1 and heated to 70°C for 8-24 hours. Add the remaining dry crystals in 0.5 l of water with stirring, and after complete dissolution, add the liquid solution to the aqueous solution with rapid stirring, for example, stirring with a stirrer. Bring the total volume to 1 liter by adding water. Adjust to the required pH with NH ₄ OH or KOH. After mixing the components, they are diluted with water, if necessary, and applied to the foliage of seedling plants by spraying, preferably at a rate of 10-100 gallons/acre (37.8-378 liters/4047 m ² ). This solution can be supplemented with anionic components of transition metal coordination compounds ²⁴ selected from aconitates, citrates, fumarates, glutarates, malates, oxaloacetates, succinates and chelating polydentate alkylamides, to increase the primary products of photosynthesis, improve quality and yield.

Example 6 - System for accelerating germination

Ingredient Concentration range g/l Preferred concentration Glutaric acid 0.07-10 3 Ethan 10ppb - 1ppm 100ppb KOH pH 5-7.5 pH 5.5 cat 0.01-5 0, 1 Ca ²⁺ 0.01-1 0.05

In addition, anionic components of transition metal coordination compounds ²⁺ can be added to this solution, selected in the range of ppm-ppt amounts from aconite, citric, fumaric, malic, oxaloacetic and succinic acids, in the range of ppm-ppt amounts from chelating polydentate alkylamides and Mg in the ppm-ppt range.

Radish seeds, 25 seeds per cup, were sown in 16 Gosselin seed germination saucers on Whatman 598 Seed Culture paper mugs moistened with either a control nutrient solution or a glycan composite solution. The seeds were kept at a constant temperature of 27°C in the dark only for their respiration. The fact of germination was considered the point in time at which the appearance of the primary root was observed in 50% of the seeds, G ₅₀ . The results showed accelerated germination for the glycan composite with a mean _G50 =15 h compared to the mean of the control nutrient _G50 =22 h, n=8, p=0.001. Treatment of radishes by applying a layer of glycan composite to seeds at a rate of 20-50 µg/seed, based on the weight of the dry glycan composite, has proven to be highly effective, significantly accelerating germination compared to control nutrients, as a result of increasing the flow of primary products. photosynthesis. A similar acceleration in germination was observed for radish seeds that were pre-coated with glycan composite at a rate of 20-50 µg glycan composite/seed and dried compared to nutrient controls.

Example 7. System for increasing the mass of roots

Component Concentration range % Preferred concentration % Fumaric acid 0.001-20 0.03 GG 0.001-1 0, 1 cat 0.01-5 0.01 Ca ²⁺ 0.01-1 0.05

In addition, anionic components of ²⁺ transition metal coordination compounds selected from aconite, citric, fumaric, malic, oxaloacetic and succinic acids, chelating polydentate alkyl amides, and Mg in the ppm-ppt range can be added to this solution.

Bulgarian pepper seeds were sown on 12 plots with homogeneous loamy soil with the norms indicated on the label 12-26-26. A glycan composite in water was prepared and the pH of the solution was adjusted to 5.5 with DAP, and the control solution was also adjusted to an equivalent P content with DAP/MAP. Two weeks after germination, six randomly selected plots were subjected to simultaneous impregnation and spraying of the glycan composite to plantings, with all 12 bell pepper plots receiving the same treatment to maintain plant growth. In order to avoid crowding, each plot contained 20 plants spaced 50 cm apart. After 2 weeks, the plants were harvested along with intact roots, then the roots were cut off from the shoots, thoroughly washed from soil residues, and dried in an oven. The dry weight of the roots of each plant was determined. The results showed that the mean dry weight of the roots when treated with the glycan composite was 1.2 g compared to the mean 1.0 g when treated with the control nutrient solution, n=6, p=0.02. Glycan composite treatment significantly increased root dry weight compared to control treatment. In the case of the control treatment, fruits with sunburn were present, while they were absent in the case of the treatment of plants with the glycan composite, indicating an increase in the primary products of photosynthesis and an increase in the quality of the harvested crop.

Example 8 Example of Glycan Composite Increasing Hydrostatic Pressure

Plumeria flower fields are traditionally cultivated at high light intensity ~1500-1700 microeinstein/m ² /s and low to moderate humidity levels of ~20-30%. Under these environmental conditions, daily hydrostatic pressure readings were recorded in the afternoon. Usually early morning flowering plants have high hydrostatic pressure, but by mid-afternoon the leaves begin to drop. This midday wilt cycle, characterized by a high to low hydrostatic pressure change, was visually distinguishable as leaf elevation changed from top to bottom by approximately 5 to 20 millimeters (mm). An increase in hydrostatic pressure is a necessary prerequisite for growth, and hydrostatic pressure was measured by the change in leaf uplift, especially at noon. The aim of the study was to record the change in hydrostatic pressure in plumeria by measuring the change in leaf uplift in millimeters and comparing the effect of a control aqueous nutrient solution with the effect of a separate root treatment with 1-25 ml of a glycan composite of 0.1-5 ppm Ethan. The composition of the glycan composite was that of Example 6 above, but other exemplary glycan composites may optionally be used to increase hydrostatic pressure. For example, an effective amount of 1-200 ml of 0.1-5 ppm partially hydrolyzed deglycosylated invertases was applied to the roots at 8 am, and then at noon the amount of leaf uplift indicative of increased hydrostatic pressure was recorded, while the results of exposure to the control solution indicated reduction of hydrostatic pressure. Obtuse plumeria (Plumeria obtusa L.), a blunt plant, was kept in 4 L plastic containers for a week to acclimatize under environmental conditions under direct sunlight, and then the relationship of daily changes in hydrostatic pressure with evening watering once a week was studied. . In the middle of the week between watering, the initial position of the leaves was measured in the late morning and noted on the measuring rulers, and later, compared with the values of the rise of the same leaves 5 hours after treatment.

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Results.

Leaf uplift served as a visually distinguishable sign of increased hydrostatic pressure. In the case of treatment with the glycan composite, the mean leaf lift increased by 15 mm and was statistically significant (n=6, p=0.003), while when the foliage was treated with the control nutrient solution, the mean leaf lift decreased by 5 mm. After treatment with glycan composites ranging from 10 ppb to 800 ppm, leaf uplift was higher in plants treated late in the afternoon after 15:00, while foliage treatment with control solution showed the most pronounced loss of hydrostatic pressure and a decrease in leaf uplift, caused by the midday wilt cycle.

In conclusion, it should be noted that the plumeria responded positively to treatment to increase hydrostatic pressure, while treatment with control solution showed a decrease in hydrostatic pressure. A similar increase in the quality of crops with increasing hydrostatic pressure occurred in the case of bell peppers, ornamental cabbages, pumpkins, pome fruits and root crops treated with glycan composites prepared with suitable anionic components of transition metal ²⁺ coordination compounds. For example, the anionic components were selected from one or more of aconitic, fumaric, glutaric, malic, oxaloacetic and succinic acids at 10-900 ppm and chelating polydentate alkyl amides such as EDTA, EDDHA, HeEDTA, DTPA, HBED, MGDA, GLDA and other similar, with their content of 10-900 ppm. The growth and development of all photosynthetic organisms depend on cell growth initiated by increased hydrostatic pressure. When hydrostatic pressure was increased over a long period of time as a result of using the glycan composite systems of the embodiments disclosed herein, the results were characterized by a significant increase in growth and development of the treated photosynthetic organism. Glycan composite systems used in conjunction with breath accelerators have increased crop yields.

Example 9 Example of a Glycan Composite System for Low Light Intensity Conditions

The effect of control nutrients on canola vegetative growth in shade was compared to shade-grown canola groups treated with branched N-linked glycan composite and further treated with glycan composite and control nutrients, canola groups were grown in unshaded conditions to determine if there was an opportunity to increase yields under relatively low light conditions. Canola seeds were sown in 36-well plastic trays and after 1 month they showed growth even when plants treated with control solution and glycan composite were placed under shade conditions under 50% light shading fabrics. Plants that were not shaded were exposed to natural midday light with an intensity in the range of 1500-1800 microeinstein/m ² /s, and under a 50-85% light-shading cloth, low light intensity was in the range of 100 to 900 microeinstein/m2 ² /s or was less than half of the total light intensity. Conducted foliar treatment using surfactants by spraying or spraying at a rate of 100 gallons/acre (378.5 liters/4047 m ² ). The results obtained under full daylight and treatment with control nutrients were compared with the results obtained when the glycan composite was applied to canola leaves under the same conditions. The glycan composite composition was dissolved in water in the following order: 1-100 μM Ethan; 0.1-25 mM citrate; 0.1-2 ppm Mn-CMS Mn-CMS; 1-25 ppm Ca-CMS and adjusted to pH 5.5 with DAP, DKP or KOH. The plants were harvested two weeks later and dried at low temperature in an oven at 70°C for 48 hours. groups.

In conclusion, the shade control canola group showed reduced yields compared to the full daylight control groups and shade groups treated with glycan composites. Moreover, when glycan composites were treated with shaded plants, the quality and yield improvements compared to the control group under shaded conditions were statistically significant.

Control Processed Control group in composite group in conditions glycana full shading group in illumination conditions

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shading Average dry weight (g) 0.214 0.256 0.270 Weight gain compared to the control group in shading conditions 19.5% 20% Statistical Significance p=0.000

Example 10 Example of Process for Producing Deglycosylated Glycans from Glycoproteins

Many glycoproteins contain glycans, which can be obtained from the seeds of leguminous plants, such as swordfish, or by deglycosylation from a number of enzymes. Invertase is a commercially available enzyme from baker's yeast with suitable glycan components of the inner core or outer protrusions, which can be up to three-quarters of the total glycoprotein mass. Invertase contains preferred branched glycans with terminal Man ligands such as GalMan proteins; Mann such as Man _1-6 , mannotrioses, mannotetroses and mannopentoses; GalnMann such as Gal2Man and Gal ₂ Man ₂ ; GalnMann-N-glycans such as Gal ₂ Man ₄ GlcNAc and Gal ₄ Man ₁₀ GlcNAc ₂ ; Mann-N-glycans such as Man ₃ GlcNAc, and preferred Man _1-15 GlcNAc ₂ and other similar glycans. The 270 kDa glycoprotein was too large to penetrate the foliage. Therefore, in order to establish a baseline for comparing enzymatic degradation, a laboratory experiment was first performed to release deglycosylated glycans from glycoproteins, preferably from invertase.

Invertase was denatured in dilute 0.2N NH4OH, pH 12, 80° for 10 min and neutralized by titration with 0.2N HCl, prepared for digestion by treatment with 3% trypsin at 37° overnight, and then denatured by boiling for 10 minutes. These protease-treated samples showed moderate 50 μM sprouting activity in the glycan composite. Samples were deglycosylated by incubation with 200 mU of endoglucoaminidase H at 37° for a day and denatured in dilute NH4OH at pH 12 at 80° for 10 min. The remaining protein and peptides were precipitated and removed.

Deglycosylated glycans typically included mixtures of O-linked and N-linked mannans and, as glycan components of the glycan composite, glycan isolates or mixtures exhibited activity in the range of 0.01-1 ppm by weight.

The aforementioned deglycosylation with protolytic and glucolytic enzymes was relatively mild and, moreover, involved the use of expensive biochemical reagents. As a result of these experiments, it was found that the initial denaturation of glycoproteins with base significantly reduces the duration of subsequent heating in acid, and, from the point of view of energy saving, is preferable. Therefore, the preferred process has been the hydrolysis of glycoproteins in media that are commercially available in large quantities at relatively low cost. Preferred methods have been tested which can be used such as acid/base treatment, hydrolysis, hydrazinolysis and/or fermentation. For example, acetolysis (acetic acid:acetic anhydride:sulfuric acid 25:25:1) of invertase gave active deglycosylated glycans that were active at a fairly low concentration of 1-100 ppb. Optionally, a new lysis in maleic acid was used according to an embodiment by incubating invertase in a mixture of maleic acid: acetic anhydride: nitric acid 25:25:10 at 60-80° for 1-24 hours. Alternatively, invertase was incubated in citric acid:phosphoric acid mixtures 25:1; in a saturated solution of citric and/or succinic acid and/or in a 0.1-3N solution of a selected mineral acid such as sulfuric acid, and preferably deglycosylated by direct nitrolysis in a 1-3N nitric acid solution and incubated with stirring for 1 -24 h at 40-80°.

The preferred method for partial hydrolysis of invertase was as follows. 10-30% invertase was dissolved in an alkaline aqueous solution such as 0.2-1 N KOH and/or 0.2 N NH4OH by heating to 40-80° for 1-24 hours; mixed with 50-60% citric acid and incubated for 1-24 h at 40-80°. After incubation with acid, the pH of the solution was adjusted to 3-6. A preparation of partially hydrolysed invertase, now deglycosylated, was prepared for field use with at least 1-5 ppm Ca ²⁺ , 0.5-1 ppm Mn ²⁺ and preferably 0.01-6 ppm d- transition metals ²⁺ selected from Fe ²⁺ , Ni ²⁺ , Co ²⁺ , Zn ²⁺ , and with 1-5 ppm Mg ²⁺ . The anionic components of ^{the 2+} transition metal coordination compounds were selected from one or more of aconitates, citrates, fumarates, glutarates, oxaloacetates, and succinates. Alternatively, the anionic components may be selected from the polydentate chelators mentioned above. For storing solutions

- 22 040708 added one or more preservatives. An increase in the quality and yield of cultures of photosynthetic organisms was due to the treatment of these cultures with the above-mentioned solutions containing from 1 ppb to 10 ppm of deglycosylated invertase. For example, when a glycan composite containing deglycosylated invertase was applied to photosynthetic organisms at 100 ppb of partially hydrolyzed invertase, the dose was 0.1 mg/l.

All the above methods of deglycosylation made it possible to obtain composites of glycans with the same activity. The exclusion of any one of the components of the glycan composite reduced the activity. Composites of glycans, consisting of deglycosylated invertase, were characterized by an order of magnitude higher activity than those obtained from vegetable gums according to embodiments. In addition, the processes for obtaining glycans by glycosylation of invertase were simpler and more cost-effective than processes for obtaining glycans from other sources. Regulation of the flux of primary products of photosynthesis with glycan composites consisting of deglycosylated invertase was also carried out by optional co-administration with ppm breath retardants to improve palatability or with breath accelerators to increase yield.

Example 11 Example of Glycan Composite Concentrate Used in the Presence of Insect Pollinators

Invertase was denatured in an aqueous base solution by stirring 200 g of invertase in 1 liter of an aqueous 0.2 N KOH solution and steam sterilizing for 4 hours. 50% citric acid was added with stirring and the solution was heated to 80°C for 12 hours. After cooling the solution was titrated to pH 6 with NH ₄ OH and then 1% Ca, 1% Mg, 1% Cat and water were added to a total volume of 2 liters. The final 10% partially hydrolyzed invertase solution contained a mixture of 3-7% branched O-linked and N-linked deglycosylated glycans, including the glycans shown in FIG. 2. Adjust the pH of this concentrate to 4-8, preferably pH 5, by adding appropriate volumes of bases and/or acids selected from nutrients such as KOH, NH4OH, Ca(OH) ₂ , MnCO ₃ , calcium carbonate , sea shell meal, HCl, H ₂ SO ₄ , phosphoric acid, MAP, DAP, DKP, MKP and the like, and preferably oyster shell meal. The rapid growth of agricultural crops was supported by the presence of nutrients that can be introduced into the preparation in the following preferred amounts: main plant nutrients NPK, each at 1-25%, minor nutrients 0.1-1% Ca, 0.05 -0.5% Mg, 0.1-1% S, and/or micronutrients 0.0001-0.02% B, 0.0001-0.1% Cl, 0.0001 ppb - 0.005% Co, 0.001 -0.05% Cu, 0.01-0.3% Fe, 0.02-0.1% Mn, 0.01 ppb - 0.005% Mo, 0.001-0.05% Zn, 0.001-0.1% Na and 0.0001-1 ppb Ni. The glycan composites may preferably be supplemented with ²⁺ d-transition metals selected from one or more of Fe, Mn, Ni, Co, Zn, and anionic components selected from breath accelerators and polydentate chelating agents, and the like. In view of the need to store liquid concentrates, an important component of the liquid concentrated glycan composite preparation was a preservative, such as, for example, selected from the above IT in the range of 1 to 800 ppm, preferably BIT in the range of 100-200 ppm.

For crops, not only is the availability of a full range of plant nutrients important, but also the health of pollinators, such as honey bees, butterflies, moths, beetles, birds and bumblebees, to provide them with a full spectrum of nutrients as they collect nectar containing vitamins and minerals from plants. In cultures of photosynthetic organisms, Co ²⁺ is a d-transition metal ²⁺ of an embodiment that is metabolized to vitamin B ₁₂ . Correction of this vitamin deficiency by foliar application of glycan composites containing, for example, Co-CMS, had a positive effect on health, in particular, pollinators and herbivores consuming photosynthetic organisms and juice nectar enriched in B ₁₂ and other nutrients from healthy photosynthetic crops. For example, when 0.0005% Co was introduced into the preparation as a suitable transition metal and the preparation was applied to a flowering crop, its enriched metabolites became available from flower to bee and to its colony. Immediately prior to application to the crop, the concentrate is diluted with water or other agricultural vehicles in the range of 1 ppb to 100 ppm, preferably 10 ppb to 10 ppm. The resulting dilute aqueous solution was applied by spraying or applied to the root system. For foliar application, one or more agricultural surfactants and/or additives have been incorporated into the glycan composite. During the season, one or more applications were carried out, preferably 1-2 times a month. To further optimize the flow of primary photosynthetic products in order to increase the sweetness of photosynthetic organisms and increase their production of nectar, glycan composites were used during the week of flowering and harvest in combination with respiration retardants, with ppm cytokinins and ppm gibberellins, for endogenous flavor enhancement. and nutrient enrichment.

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Example 12 An example of an invertase-based glycan composite (IGC)

Composites of glycans based on mixtures of glycans obtained by deglycosylation of invertase

Ingredient Concentration range % Preferred concentration % Deglycolized invertase 1ppb - 100ppm 0.1-10ppm cat 0.01-10 0.05 Lemon acid 0.001-20 0.001-15 Ca ²⁺ 0.001-1 0.005 Urea 0.001-25 1-15 Preservative IT 0.001-0.2 0.01 Water, balance up to 100%

²⁺ transition metal coordination compounds selected from d-transition metals, anionic components and trace amounts of Mg can be added to the IGC. The breath accelerators may be selected, for example, from one or more of aconitic, fumaric, glutaric, malic, oxaloacetic and succinic acids. Optionally, the anionic components may be selected from one or more polydentate chelating agents such as EDTA, EDDHA, HeEDTA, DTPA, HBED, MGDA, GLDA and the like.

IGC increases the yield and quality of tomatoes

Under stressful drought conditions for growing tomatoes, IGC was applied to plants and compared with application of control nutrients. IGC significantly improved the overall growth, yield, and quality of treated tomatoes compared to control applications. Tomato seeds treated with IGC by applying a layer of it on the seeds showed fast germination.

Materials and methods

Seeds of the Steak Sandwich (Burpee®) hybrid tomato variety were germinated under automatically controlled environmental conditions at 32° in the dark. One week after germination, the seedlings were transferred outdoors to grow in an arid environment of 45°:32° LD (temperature during daylight and darkness), relative humidity 10%, daylight hours (LD) 16:8 h and at mostly sunny days with photosynthetically active radiation (PAR) up to 1800 µmol photons per meter ² per second at noon. During the study, the solutions were simultaneously applied to the test plants and control plants, and all plants were under the same conditions, consistent with good laboratory practice. Fertilization with irrigation water was maintained at a sufficient level so that the roots were evenly moistened, but also dry enough without signs of water damage. Replicate plant groups were grown in TLC Pro 606 plastic trays, each with 36 125 ml wells, then transferred to 33 cm diameter plastic pots with soil-free media. Control and treated tomatoes were selected for size and growth activity, and undergrowths, damaged or diseased plants, and seeds were discarded prior to study in order to obtain uniform replicates. The application rates for the three foliar applications were 200 l/ha with a mixture of 0.1-1 ppm of invertase-derived glycans in the applied glycan composite. Sweetness, as an indication of the level of taste, was measured in Brix scale in juice nectar squeezed from individual tomatoes using a calibrated digital refractometer (Reichert).

IGC was obtained in accordance with the methods described in example 11, modified for the composition of the IGC concentrate shown in the table above. For foliar application, 0.030.05% Sil foliar surfactant was added according to label instructions. All plants received the same amounts of nutrients as a result of the use of the same solution sprayers for watering from above.

Seeds were germinated on water-moistened Whatman 598 paper in Gosselin saucers, 20 seeds/dish and 5 replications per treatment. Treated seeds were coated with 0.1 mg IGC, air dried for 48 hours, and the seeds were sown. Control seeds were treated with the same nutrients but without IGC. Germination was determined by the appearance of roots in 50% of the seeds. After 7 days, the seedlings were transplanted onto pallets.

For statistical analysis, each data set contained results from parallel experiments. Differences between treated groups and control groups in each experiment were statistically significant, unless otherwise indicated, and error bars were characterized by 95% confidence intervals.

results

Tomato seeds coated with IGC were characterized by accelerated germination. In all

In 24040708 replications, treated seeds showed 50% germination after 60 hours. In contrast, control seeds showed 50% germination in all saucers after 72 hours.

The purpose of the experiments was to determine the effect of IGC on fruit quality and yield under dry stress conditions. Field treatments under the conditions mentioned above, involving the use of a breath retarder 2 days before harvest, were compared with treatments with control nutrients to determine the effect on sweetness. Regardless of fruit color, 2 days after treatment, twelve of the largest tomatoes >50 mm in diameter were harvested and their body weight recorded. Half of the control treated tomatoes were red while all treated tomatoes were red. In table. 4 shows that the appropriate treatment of endogenous primary photosynthetic products of juice nectar resulted in an increase in fruit quality, expressed as a mean Brix value of 5.5, which was significantly higher (p=0.012), expressed as an improvement in taste, than a mean value of 4.9 Brix when treated with control nutrients.

Table 4. Tomato palatability enhancement (average Brix values)

Average Brix Average Brix Control IGC n=12 4.9 5, 5 p=0.012

The number of tomatoes per plant is an indicator of fruit yield, and in table. 5 shows that the IGC treatment resulted in a mean fruit count of 3.2 per plant that was significantly (p=0.000, n=12) greater than the mean fruit count of 1.6 per treated plant. control nutrients. As a result of the treatment with IGC, the yield was increased, the amount of nectar in the fruit juice increased, and the sweetness and taste improved when growing tomatoes in this arid environment.

Table 5. Average number of tomato fruits per plant increased as a result of IGC treatment

Average number of fruits per plant Average number of fruits per plant Control IGC n=12 1, b 3.2 p=0.000

The average total weight of tomato fruits per plant was analyzed as a measure of yield in cases where plants were treated with control nutrients and with IGC. In table. 6 shows that IGC treatment resulted in an average fruit yield per plant of 277 g wet wt/14 g dry wt, which was significantly (p=0.002/p=0.004) higher than the average fruit yield per plant of 124 grams wet weight/7 g dry weight when treated with control nutrients.

Table 6. Tomato fruit weight per plant increased by IGC treatment.

Average wet weight of fruit Control IGC n=10 124 grams/plant 277 grams/plant p=0.002 Average dry weight of fruits Control IGC n=10 7 grams/plant 14 grams/plant p=0.004

The effects of IGC treatment on various plant species were studied and the results are shown in Table 1. 7. The application has been effective in plants known to have C ₃ and C ₄ metabolism.

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Table 7. List of plants studied that benefited from IGC treatment

Plant Processing method Geranium Root Petunia Spraying and soaking lantana Root Green tea Root Balsam foliar bell pepper foliar Radish Root A coffee tree Root Lawn grass Spraying and soaking Corn Applying a layer of the drug to the seeds

conclusions

Rapid seed germination after IGC treatment indicated increased respiration and, since all plants respire, explained why CAM, C3, and C4 species responded positively to glycan composites. Endogenous modulation of the flow of primary photosynthetic products under the influence of the glycan composite caused an increase in tomato juice nectar, estimated on the Brix scale, with an improvement in palatability. Under environmental stress in the arid zone, cultivation of tomatoes by treatment with glycan composites showed an increase in quality and yield compared to control treatment. Regulation of the flow of primary photosynthetic products with IGC was supplemented by optional co-administration with respirators, with cytokinin at 10-200 ppm, to enhance flavor, or with respiration accelerators to increase yield. In addition, under conditions of oxygen starvation, the addition of an O ₂ generator, 30-100 ml of H ₂ O ₂ per plant, maintained a healthy root system, which ensured the consistency of high quality and high crop yields.

Example 13 Example of Partially Hydrolyzed Guar Gum (GG)

Ingredient Concentration range % Preferred concentration % GG 1-90 3-20 cat 1-10 5 Ca ²⁺ 0.01-3 1 Malic acids 0.1-50 5-25 Urea 1-25 5-15 bit 0.001-0.2 0.05 Water, balance up to 100% By balance

In addition, ppb-ppm amounts of components of d-transition metal ²⁺ coordination compounds selected from Zn, Co, Ni, aconite, citric, fumaric, glutaric, oxaloacetic and succinic acids, chelating alkylamides and Mg ²⁺ can be added to this preparation.

Photosynthetic organisms treated with glycan composites consisting of deglycosylated GGs were characterized by an increased flux of primary photosynthetic products, improving crop quality and yield. Regulation of the flux of primary photosynthetic products with glycan composites composed of GG was complemented by optional co-administration with breath retardants to enhance flavor or with breath accelerators to increase yield.

Example 14 Example of Partially Hydrolyzed Tara Gum (HTG)

Tara gums are industrially produced in large quantities and this plant contains

- 26 040708 large polymers of branched GalMan ₃ units suitable for deglycosylation by the acetolysis processes mentioned above. Namely, deglycosylated glycans were produced from edible tara gum by acetolysis (acetic acid:acetic anhydride:sulfuric acid 25:25:1 v/v, 60-80°C, 24-96 h) or nitrolysis (citric acid:acetic anhydride:nitric acid 20:20:10) to give partially hydrolyzed deglycosylated branched GalMan ₃ . Glycans in the form of partially hydrolyzed deglycosylated tara gum (HTG) showed high activity in glycan composites in the concentration range of 50-200 ppm. Studies of HTG as a glycan component have shown that HTG is active only in the presence of components of the glycan composite using the following methods.

Cabbage was grown under the controlled environmental conditions described above in 36-well plastic boxes for plants with volumes of 125 ^cc /well. All aqueous solutions for foliar application contained 0.5 g Sil/l, pH 6, and the following components were added to individual preparations: metals ²⁺ - 1 ppm manganese sulfate, 1 ppm iron (II) sulfate and 10 ppm calcium nitrate, dissolved in water with 50 PPM DAP; anionic component of coordination compounds of transition metals ²⁺ - 300 ppm potassium α-ketoglutarate, abbreviated as aKG; and 100 ppm HTG. The components of the glycan-metal ²⁺ composite were applied separately and in a mixed form with each other to study the effect on plant growth of the individual components compared to the full composition of the glycan composite. Solutions of aKG, HTG, and aKG+HTG were dissolved in aqueous solutions of ²⁺ metals, and as a result, transition metal ²⁺ coordination compounds consisting of aKG, Ca ²⁺ and Mn ²⁺ were present in the treatment solutions. Metals ²⁺ served as controls.

When the first well-defined leaves appeared, a foliar treatment was applied at a rate of 10 ml/box, n=36. Sprouts with leaves were collected, dried and weighed.

The test results shown in Table HTG below are presented in the form of a comparison of the average dry weight of cabbage sprouts during treatment with the average dry weight of cabbage sprouts when treated with the ²⁺ metal solution alone, the stock solution in which all other treatment solutions were prepared. Sprouts treated with aKG were not significantly different (n=36; p=0.057) in terms of yield from ²⁺ metal control; HTG solutions with ²⁺ metals had a significant (n=36, p=0.001) positive effect compared to solutions of ²⁺ metals alone. Finally, aKG+HTG+ ²⁺ metals showed the highest significant (n=36, p=0.000) increase in yield compared to ²⁺ metals. In addition, the overall composition of the glycan composite showed highly significant (aKG+HTG versus aKG, p=0.000; aKG+HTG versus HTG, p=0.000) yield increases compared to all other treatments.

HTG table

Average dry weight, grams R Metals ² * 0.56 aKG 0.59 0.057 HTG 0.61 0.001 aKG+HTG 0.63 0,000

In conclusion, the glycan composite showed the most significant improvements when co-prepared as a complete glycan composite when applied to photosynthetic organisms to target primary photosynthetic products. In addition, the positive effects of exposure to the full compositions of glycan composites were confirmed after the additional introduction of d-transition metals ²⁺ , Ca ²⁺ and Mg ²⁺ . Regulation of the flux of primary photosynthesis products with complete glycan composite formulations was further complemented by optional co-administration with breath retardants to enhance flavor or with breath accelerators to increase yield.

Example 15 Example of Partially Hydrolyzed Locust Bean Gum (PHLB)

Ingredient Concentration range % Preferred concentration % PHLB 0, 1-5 1-3 cat 1-10 5

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Ca ²⁺ 0.01-3 1 CMS 0.1-50 5-25 AMS 1-25 5-15 bit 1-750ppm 75-100ppm Water, balance up to 100%

One or more d-transition metals ²⁺ , anionic components and/or Mg ²⁺ can be additionally added to this PHLB preparation. Treatment of photosynthetic organisms with PHLB resulted in increased flux of primary photosynthetic products, which improved crop quality and yield. Flux regulation of PHLB glycan composites was further controlled by optional co-administration of a respiration retardant, 10-200 ppm salicylic acid, to improve palatability.

Example 16 Methods and compositions for accelerating germination

Range Preferred

Ingredient concentrations, g/l concentration, g/l

Ethan 1ppb - 1ppm 10-100ppb Glutaric acid 0.0007-1 0.003 KOH pH 5-7.5 pH 5.5 cat 0.01-5 0, 1 Ca ²⁺ 0.01-1 0.05

For use in order to improve seed germination, anionic components of ²⁺ transition metal coordination compounds, which are respiration accelerators, selected from one or more of aconitic, fumaric, glutaric, malic, oxaloacetic and succinic acids and their phosphate salts can be added to this preparation. .

Radish seeds, 25 seeds per saucer, were sown in 16 replications in Gosselin seed germination dishes on Whatman 598 Seed Culture mugs soaked in nutrient control solution or glycan composite solution. The seeds were kept at a constant temperature of 27°C in the dark only to maintain respiration. Germination was determined at the moment when roots appeared in 50% of the seeds, G ₅₀ . The results showed accelerated germination in the case of glycan composite treatment, mean G ₅₀ =15 h compared to mean G ₅₀ =22 h, n=8, p=0.001 in the case of nutrient control treatment. Treatment of radishes by layering seeds at a rate of 20-50 μg/seed based on the dry weight of the glycan composite proved to be highly active and significantly accelerate germination compared to treatment with nutrient control solution as a result of increased flux of primary photosynthetic products. Similarly, germination was accelerated in radish seeds pre-coated and dried with 20-50 µg glycan/seed composite compared to nutrient control treatment.

Example 17 N-linked Glycan Composites

Ingredient Concentration range, ppm Preferred concentration, ppm Ethan 0.001-1 0, 1 Lemon acid 0.001-5 0.05 Ca2 ⁺ 1-10 5 cat 0.01-10 3

Anionic components of coordination compounds of transition metals ²⁺ can be additionally introduced into this preparation. For example, anionic components can be selected from breath accelerators and polydentate chelating agents.

Composites of glycans for root application were prepared in 1 liter of water with stirring at room temperature from 25 to 35°C.

The formulation was titrated with KOH and/or NH4OH to a pH of 5-7.5 and applied at a rate of 500 to 750 gal/acre as close to the roots as possible by soaking, spray-soaking, inter-row application and/or by irrigation application. water. During irrigation, the treatment was carried out by

- 28 040708 soaking the soil with a solution of the glycan composite preparation in the direction of the roots to improve the quality and yield of an agricultural crop. The treatment was carried out from once a week to once a month, as needed, during the growing season.

Example 18 Liquid Invertase Concentrate

Invertase glycoproteins are composed of core proteins and Mann surface polymers. Accordingly, these glycoproteins were subjected to partial hydrolysis to obtain deglycosylated Mann. When preparing glycan composites from deglycosylated invertase and applying them to photosynthetic organisms, deglycosylated invertase modulated the energy flow from photosynthesis to respiration. Concentrated glycan composite preparations containing deglycosylated N-linked branched chains from invertase were diluted with water to field doses of 10 ppb - 5 ppm for photosynthetic organisms and used in germination studies where they were highly active at levels as low as 10 ppb of deglycosylated invertase.

Glycan composites were further prepared to control the flux of primary photosynthesis products with an optional set of respiration retarders to enhance palatability and respiration accelerators to increase yield. The ²⁺ transition metal coordination compounds for deglycosylated invertase can be further selected from anionic components that are respiration accelerators such as ppm amounts of aconitic, fumaric, glutaric, malic, oxaloacetic and succinic acids. Optionally, the anionic components may be selected from polydentate chelating compounds such as ppm amounts of EDTA, EDDHA, HeEDTA, DTPA, HBED, MGDA, GLDA and the like.

Treatment with the glycan composite resulted in improved quality and yield of crops such as blueberries, leafy vegetables, cotton, cereals, tomatoes, cherries, onions, coffee, bananas, citrus fruits, melons, green leafy vegetables, nuts, pome fruits, berries, fruit trees, food crops, flowers, trees, lawn grasses and ornamental crops.

Methods

To 40 ml of a strong base, preferably selected from 0.2 N KOH, NH ₄ OH and Ca(OH) ₂ , was added with stirring 10-12 g of dry invertase powder, preferably with an activity of 200,000 Sumner units/g, and the aqueous alkaline solution was heated steam to 60-80°C for 2-24 hours. Then 20 g of citric acid was added with stirring and the solution was heated with steam at 60-80°C for 3-12 hours. The lower the heating temperature, the longer should be incubated, and this process was followed by cooling to room temperature. A suitable base, preferably selected from KOH, NH ₄ OH and Ca(OH) ₂ , was added with stirring to adjust the pH to 4-6. The volume was adjusted to 100 ml to obtain 10% deglycosylated invertase. A stock solution of 1-6 ppm Cat was prepared and 1 ml of 10% deglycosylated invertase was diluted in 1 L of stock solution of Cat to give a 100 ppm deglycosylated invertase liquid concentrate. Added with stirring 100 ppm BIT to obtain the finished liquid concentrate of deglycosylated invertase.

Dilutions of deglycosylated invertase liquid concentrate for field use: 10 ml of deglycosylated invertase liquid concentrate at 100 ppm was diluted in 1 liter of water to give 1 ppm of deglycosylated invertase, then serial dilutions were made at 1, 10, and 100 ppb, as needed. Foliar application required the addition of one or more agricultural surfactants such as 0.5 g/l Sil.

The use of low concentrations of deglycosylated invertase in the range of 10 ppb - 5 ppm was effective and was characterized by significantly earlier germination and an increase in the quality and yield of photosynthetic organisms compared with the control treatment. Regulation of the flux of primary photosynthetic products with glycan composites consisting of deglycosylated invertase was supplemented by the combined use of a respiration retardant, 1-100 ppm gibberellin, to improve palatability. In addition, under conditions of oxygen starvation, the addition of 1 liter of the medium with 10 g of CaO2 contributed to the achievement of high quality and high yields of crops.

Example 19 Example of Partially Hydrolysed Vegetable Gums

Vegetable gums were selected from products with a high content of branched mannan chains, which were partially hydrolyzed by the following methods: guar gum, konjac gum, locust bean gum, tara gum and fitelefasa gum were separately dissolved in water at 60-80° with stirring in the form of 1% by weight. mass of gum. To each gum solution was added 1-3 N nitric acid and 5-25% acetic anhydride. The solutions were kept at 70-80° for 1-8 hours. The solutions were cooled to 30-40° and titrated to pH 3.5-4 with Ca(OH) ₂ , KOH and/or NH4OH. 30-50% citric acid was added to the solutions until saturation and heated to 60-80°C for 1-24 hours. The solutions were cooled to room temperature and titrated to pH 5-6 with Ca(OH) ₂ , KOH, MnCO ₃ and/or NH ₄ OH.

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10X Cat was dissolved in the partially hydrolysed gum solution to give 0.1% gum. Serial dilutions with Cat gave samples at various doses and compared their performance to that of a nutrient control solution. Examples of ingredients for glycan composites containing partially hydrolyzed vegetable gums are shown in the ingredient table below.

Component table

Component Concentration range % Preferred concentrations % vegetable gum 0.01-3 0, 1 HNO3 : acetic anhydride : CMS 5:5:5-25:25:60 10:10:50 NH ₄ OH, KOH, Ca (OH) ₂ pH 3-6 pH 5.5 Transition metal coordination compound ²⁴ 0.01-1 0, 1 coordination compound Sa 0.01-3 0, 1

Biological Activity Study

Radish seeds, 30 pieces per saucer, were sown in 20 replications in Gosselin saucers for seed germination on Whatman 598 Seed Culture paper mugs. The seeds were kept at a constant temperature of 27°C in the dark to maintain only respiration. Germination was determined at the moment when roots appeared in 50% of the seeds, G ₅₀ .

The results showed faster germination with a mean G ₅₀ =15 h for the glycan composite compared to a mean G ₅₀ =22 h, n=10, p=0.001 for the control nutrient. The activities of various partially hydrolyzed (PH) gums were characterized by general dependences of the concentrations necessary to achieve the G ₅₀ activity value on the content of branched mannans in them. The observed activities varied from low values for partially hydrolyzed guar gum, konjac gum, locust bean gum, tara gum to high values for partially hydrolyzed fitelefasa gum, as detailed in the table of active doses (ppm) of partially hydrolyzed (PH) gums.

Treatment of radish seeds with gum glycan composites from various sources was characterized by different levels of efficiency and significantly accelerated germination compared to control nutrients. Rapid germination was due to an endogenous increase in the flow of primary products of photosynthesis. Preferred breath accelerators included citric and malic acids. In addition, under conditions of oxygen starvation, additional treatment of each root with 1 liter of medium with 20 g of CaO ₂ promoted root growth, resulting in high quality and high crop yields.

Table of active doses (ppm) of partially hydrolyzed (PH) gums

100 ppm GG

[30 ppm partially hydrolyzed konjac ppm partially hydrolyzed locust bean gum ppm partially hydrolyzed tara gum

Claims (30)

ultra-low biuret, Ca, Mn, Fe, Zn, Mg, random block copolymer emulsifier such as Plutonic L-62, lower aliphatic alcohols, preferably propanols, preservatives such as BIT, the concentrations of each component are given in the concentrate composition table glycan composite from invertase. A 5 ml aliquot of 20% glycan solution was diluted in 10 L stock metal ⁺² stock solution to give a 100 ppm glycan composite solution and titrated with citric acid, KOH, and/or NH4OH to pH 5-8. This 100 ppm glycan composite product was prepared for field application dilutions to 0.01-1% in water for application to photosynthetic organisms. For example, 10 ml of glycan composite in 1 liter of water gave a solution of 1 ppm of glycan and likewise, 1 gal/100 gal/acre or 3 fl oz/1000 ft ² . In addition, serial dilutions were applied, if necessary. The diluted for field use deglycosylated invertase glycan composite exhibited efficacy in the range of 0.01 to 10 glycan at root application, and/or together with additives at labeled application rates of surfactants at foliar application from 0.1 to 10 ppm, glycan has proven to be effective in root applications. Composition table of invertase glycan composite concentrate Although the features of the embodiments disclosed herein are described in relation to one example and not to other examples, however, this is done only for reasons of convenience and therefore some features of one described example may be combined with one or more features. signs of other examples in accordance with the methods and preparations disclosed in the invention. For specialists in this field is obvious the possibility of existence of other combinations of methods and drugs, disclosed in the invention of embodiments, which are included in the scope of the following claims. CLAIM 1. A preparation containing a glycan composite for improving the viability of agricultural crops and the growth of photosynthetic organisms, including a branched deglycosylated glycan with a terminal ligand from glycopyranose or acylglycosamine mixed with a coordination compound Ca ²⁺ and one or more coordination compounds of D-transition metals ² *. 2. The drug according to claim 1, where the specified branched deglycosylated glycan is a high molecular weight polymer mannan 3. The drug according to claim 1, where the specified branched deglycosylated glycan is selected from the group consisting of O-linked glycans with Gly _mn where m=1-4 and n=1-4 and N-linked glycans with Gly _mn where m =1-4 and n=1-4. 4. The preparation according to claim 1, where the branched deglycosylated glycan is a high molecular weight mannan-N-glycan. 5. The preparation according to claim 1, further comprising one or more preservatives. 6. The formulation of claim 5, wherein the one or more preservatives are selected from the group consisting of benzoisothiazolinones, methylchloroisothiazolinones, methylisothiazolinones, and combinations thereof. 7. The drug according to claim 1, where the specified branched deglycosylated glycan is selected from the group consisting of O-linked glycans with Gly _mn where m=1-4 and n=1-4 and N-linked glycans with Gly _mn where m =1-4 and n=1-4, and wherein said one or more ² * D-transition metal coordination compounds include both ² * metal components and one or more anionic components selected from the group consisting of chelating polydentate alkyl amides, aconitates, citrates, fumara- - 31 040708 commodities, glutarates, malates, oxaloacetates, succinates, their acids, salts and esters. 8. The formulation of claim 1, wherein said branched deglycosylated glycan is trimannopyranosyl-N-glycan. 9. The formulation of claim 1, wherein said glycan composite is present in an amount of 1 ppm to 1% by weight and said one or more ²⁺ transition metal coordination compounds are present in an amount of 0.1 ppb to 100 ppm by weight. 10. The formulation of claim 1, wherein said glycan composite is present in the form of a concentrated composition product with glycan composite concentrations ranging from 100 ppm to 10% for dilution prior to application to photosynthetic crop organisms to enhance respiration; where the specified glycan composite product of the concentrated composition includes one or more deglycosylated glycans in the concentration range from 1 ppm to 20% and one or more coordination compounds of transition metals ²⁺ in the concentration range of 1 ppm-10% and a preservative in the concentration range from 50 ppm to 1 %. 11. A method for improving the viability of crops and the growth of photosynthetic organisms as a result of enhancing the respiratory metabolism of the primary products of photosynthesis, including applying to the specified organism from 1 ppm to 1% of the drug according to claim 1. 12. The method of claim 11 wherein said deglycosylated glycan is selected from the group consisting of O-linked glycans with Gly _m _ _n where m=1-4 and n=1-4 and N-linked glycans with Gly _m _ _n , where m=1-4 and n=1-4. 13. The method of claim 11 wherein the one or more ²⁺ D-transition metal coordination compounds comprise both ²⁺ metal components and one or more anionic components selected from the group consisting of polydentate alkyl amides, aconitates, citrates, fumarates, glutarates, malates, oxaloacetates, succinates, their acids, salts and esters. 14. The method of claim 11 wherein said branched deglycosylated glycan has a mannopyranosyl terminal ligand. 15. The method of claim 11 wherein said branched deglycosylated glycan is selected from the group consisting of O-linked glycans with Gly _m _ _n where m=1-4 and n=1-4. 16. The method of claim 11 wherein said branched deglycosylated glycan is selected from the group consisting of N-linked glycans with Gly _m _ _n where m=1-4 and n=1-4. 17. The method of claim 15, wherein said O-linked-glycan in said glycan composite is selected from the group consisting of aryl (Gly) _n , acyl (Gly) _n , alkyl (Gly) _n , where n=1-4. 18. The method of claim 11, wherein said branched deglycosylated glycan is selected from N-acetylglycosaminyl-terminal ligands. 19. The method of claim 11, wherein said one or more coordination compounds comprise calcium, Ca ²⁺ , and wherein said one or more ²⁺ transition metal coordination compounds of said ²⁺ transition metal coordination compounds comprise one or more ²⁺ D-transition metal compounds selected from the group consisting of Mn ²⁺ , Fe ²⁺ , Co ²⁺ , Ni ²⁺ and Zn ²⁺ . 20. The method of claim 11, wherein said formulation further comprises one or more preservatives. 21. The method of claim 11 wherein said branched deglycosylated glycans are selected from one or more glycoprotein enzymes, invertases, denatured invertases, partially hydrolyzed invertases, and deglycosylated invertases, and mixtures thereof. 22. The method of claim 11 wherein the preparation comprises one or more Ca ²⁺ Mn ²⁺ coordination compounds and one or more branched deglycosylated Glc-NAc^. 23. The method of claim 11, wherein said photosynthetic organism produces nectar, and wherein said drug is applied to increase nectar. 24. The method of claim 11 wherein said photosynthetic organism is a flowering plant. 25. The method of claim 21 wherein said partially hydrolysed invertases are deglycosylated with citric acid. 26. The method of claim 11, wherein said photosynthetic organism produces primary products of photosynthesis, and wherein said drug is applied to said photosynthetic organism to endogenously improve palatability. 27. The method of claim 13 wherein said one or more components are used in conjunction with O ₂ generating agents selected from the group consisting of H ₂ O ₂ , CaO ₂ and MgO ₂ . 28. The method of claim 11 wherein said drug is applied to said photosynthetic organism in an amount of 10 ppb to 800 ppm to increase the hydrostatic pressure of the photosynthetic organism. 29. The method of claim 11, wherein said branched deglycosylated glycan is selected from one or more partially hydrolysed plant materials selected from phytelfas, guar gum, seaweed, mannan-containing algae, yam, carob, tare, swordfish, carob wood, palm tree endosperm tissue and mixtures thereof. 30. The method of claim 21 wherein said partially hydrolyzed invertases are deglycosylated with acids selected from breath accelerators. -