Patent Application
20200060274
Embodiments disclosed herein relate to methods for treating photosynthetic organisms, such as fields of photosynthetic organisms, including farm crops, and/or enhancing their growth with formulations that comprise compositions of glycan composites comprising branched glycan deglycosylate components with coordination complex components. Moreover, in liquid compositions of matter, the foregoing formulations may include one or more preservatives, rendering the formulations suitable to ship and store. Embodiments disclosed herein include systems for delivery of glycan composites into photosynthetic organisms.
Embodiments disclosed herein relate to the application to photosynthetic organisms of glycan composite formulations comprised of one or more branched glycan deglycosylates and transition metal2+ coordination complexes.
The health of photosynthetic organisms in agricultural crops is dependent on their biological manufacture of photosynthates, especially, sugars; and compositions of the present embodiments enhance the availability of these photosynthates to promote crop health and growth. Recent major advancements for crop improvements include water-soluble glucosides as described in the patent literature. While these glucosides have proven effective at foliar rates ranging in the application of kilograms per hectare, there is a need for formulations that are more potent and effective in the range of grams per hectare as shown in the embodiments herein. The branched glycan deglycosylates of the present embodiments are of higher order potencies than conventional compositions while transition metal2+ coordination complex components of the glycan composites further improve activity. In addition, by treatment of sap nectar, glycan composites improve photosynthate flux capacity of a crop. Further still, methods and compositions of glycan composites may be customized for improvement of qualities and/or quantities of crops while sustaining potency of the glycan composite.
It is an object of embodiments disclosed herein to provide methods and formulations for treating photosynthetic organisms and enhancing qualities and quantities of crop yields. The formulations comprise one or more branched glycan deglycosylates combined with transition metal2+ coordination complexes, thereby forming a glycan composite. Embodiments provide methods and compositions for the formulation of glycan composites.
It is a further object of embodiments disclosed herein to provide liquid formulations comprising one or more formulations of glycan composites with preservatives for retention of high potency.
It is a further object of embodiments disclosed herein to provide methods and formulations for treating photosynthetic organisms such as plants, and for enhancing photosynthetic organismal growth, such as in plant crops, by applying a glycan composite formulation.
It is another object of embodiments disclosed herein to provide glycan composite formulations comprising metals2+ components of transition metal coordination complexes and providing anionic components of transition metal2+ coordination complexes.
It is a further object of embodiments disclosed herein to provide glycan composite formulations comprising soluble transition metals2+ preferably in coordination complexes.
It is a further object of embodiments disclosed herein to provide glycan composite formulations comprising one or more anionic components of the transition metal2+ coordination complexes.
It is a further object of embodiments disclosed herein to provide glycan composite formulations comprising one or more anionic components of the transition metal2+ coordination complexes, wherein the anionic component is selected from polydentate sequestering agents.
It is a further object of embodiments disclosed herein to provide a method for formulating a glycan composite composition that comprises one or more preservative components that maintains active liquid solutions through periods of shipping and storage.
It is a further object to provide methods for treatment of photosynthetic organisms, particularly plants, and growth formulations for photosynthetic organisms comprising glycan composite formulations rendered as liquid and/or dry packages that are available and penetrative consistent with facilitation of transcuticular, transepidermal and/or transmembrane transport; seed, foliar and root uptake of solutes; germination; and/or that maintain growth in low light intensity environments.
It is a further object to provide treatment and growth formulations for photosynthetic organisms and to their parts; and particularly applied to agricultural crops.
It is a further object to provide a plant seed treatment formulation comprising glycan composite compositions and methods for management of growth in green plants, particularly where applied to seeds to hasten germination and growth.
It is a further object to provide floral and/or fruit treatment formulations comprising glycan composite compositions for management of quality growth in photosynthetic organisms; particularly where applied to roots, foliage, flowers and/or fruit while attached to the plant before harvest; to detached flowers and/or fruit after harvest; and/or to improve flavor qualities.
It is another object to provide glycan composite compositions of matter.
It is another object to provide glycan composites comprised of branched glycan deglycosylates and soluble transition metals2+.
It is another object to provide glycan composites, comprised of branched glycan deglycosylates obtained from natural products.
It is another object to provide a glycan composite and/or its components as novel plant growth regulators selected to increase crop quantity and quality.
It is another object to provide novel glycan composite plant growth regulators for the management of respiration in photosynthetic organisms.
It is yet another object to provide a glycan composite environment in the presence of photosynthetic organisms under cultivation conducive of respiration in low light intensity shaded to dark conditions.
It is another object to provide an environment of respiratory acceleration in the presence of photosynthetic organisms under cultivation by treatments with glycan composites. In some embodiments, roots at any time of day or night, and/or shoots in the dark of night, are exposed to respiration accelerators that elevate ambient oxygen (O2) either through >25% O2 gas treatment of a photosynthetic organism, or by application of O2-generators. Furthermore, another object is to provide an environment conducive to respiration in the presence of the photosynthetic organisms under cultivation by treatments with glycan composites; wherein photosynthetic organisms under cultivation by treatments with glycan composites may be exposed to respiration accelerators.
It is yet another object to provide an environment conducive to the accumulation of photosynthates in the presence of photosynthetic organisms under cultivation by treatments with glycan composites. Photosynthetic organisms under cultivation by treatments with glycan composites may be exposed to respiration decelerators conducive to the accumulation of photosynthates.
It is another object to provide glycan composites and/or their components as natural product biostimulants selected to benefit nutrient use efficiency, improve tolerance to abiotic stress, and/or increase crop quantity and quality.
It is another object to provide glycan composite treatments for photosynthetic growth, comprising branched glycan deglycosylates and transition metal2+ coordination complexes. In certain embodiments, such as for convenient utilization in the field, the complex is rendered convenient to apply to photosynthetic organisms and readily safe for photosynthetic organisms by formulation in liquid and/or dry packages and with the option for admixture of crop treatments.
It is a further object to provide treatment methods and compositions for photosynthetic organisms comprising exogenous glycan composite compositions for control of the endogenous deconjugation of photosynthates that advance the quality and quantity of commercial harvests.
It is a further object to provide a treatment and growth formulation for photosynthetic organisms comprising exogenous glycan composite compositions for treatment of endogenous photosynthates that enhance the flavor qualities of commercial yields.
It is a further object to provide a treatment and growth formulation for photosynthetic organisms comprising glycan composite compositions that are preserved for storage with one or more phytobland preservatives.
It is a further object to provide a treatment and growth formulation for photosynthetic organisms comprising glycan composite systems for the improvement of crop growth in water culture.
It is a further object to provide a treatment and growth formulation for photosynthetic organisms comprising glycan composites as synergistic systems for improving crop aesthetic quality by reduction of the incidence of sun scorch.
It is a further object to provide a method for treatment of photosynthetic organisms that fortifies the nutritional qualities of sap nectar of flowering plant fields comprising applying glycan composites to flowering food plants for the vigorous health benefit of pollinators and grazers.
In certain embodiments, a glycan composite is formulated as a complex comprised of one or more methyl-α-D-Mannopyranosyl (Man)1-3 components and one or more citrate-Ca2+-Mn2+-coordination complex components and optionally with one or more D-block transition metals2+; and/or further formulated with one or more optional preservatives.
In certain embodiments, a glycan composite is formulated as a complex comprised of one or more methyl-α-D-Man1-3 components and one or more malate-Ca2+-Mn2+-coordination complex components and optionally with one or more D-block transition metals2+; and/or formulated with one or more optional preservatives.
In certain embodiments, a glycan composite is formulated as a complex comprised of one or more methyl-α-D-Man1-3 components and one or more glutarate-Ca2+-Mn2+-coordination complex components; and optionally with one or more D-block transition metals2+; and/or further with one or more optional preservatives.
In certain embodiments, a glycan composite is formulated as a complex comprised of one or more methyl-α-D-Man1-3 components and one or more succinate-Ca2+-Mn2+-coordination complex components and optionally with one or more D-block transition metals2+; and/or further with one or more optional preservatives.
In certain embodiments, a glycan composite is formulated as a complex comprised of one or more Man1-3N-linked-glycan components; one or more Ca2+-Mn2+-citrate coordination complex components; and optionally with one or more D-block transition metals2+; and/or further with one or more optional preservatives.
In certain embodiments, a glycan composite is formulated as a complex comprised of one or more Man1-3N-linked-glycan components; one or more Ca2+-Mn2+-coordination complex components; optionally with one or more D-block transition metals2+; and/or further with one or more optional preservatives
In certain embodiments, a glycan composite is formulated as a complex comprised of one or more methyl-α-D-Glc1 components; one or more citrate Ca2+-Mn2+-coordination complex components; optionally with one or more D-block transition metals2+; and/or further with one or more optional preservatives.
In certain embodiments, a glycan composite is formulated as a complex comprised of one or more deglycosylated glycan components; Ca2+-Mn2+-coordination complex components; optionally with one or more D-block transition metals2+; and/or further with one or more optional preservatives.
In certain embodiments, a glycan composite is formulated as a complex comprised of one or more deglycosylated O-linked-glycan components from botanical gums; Ca2+-Mn2+-coordination complex components; optionally with one or more D-block transition metals2+; and/or further with one or more optional preservatives.
In certain embodiments, a glycan composite is formulated as a complex comprised of one or more deglycosylated N-linked-glycan components from invertase; Ca2+-Mn2+-coordination complex components; optionally with one or more D-block transition metals2+; and/or further with one or more optional preservatives.
Unless otherwise defined, all technical and scientific terms employed herein have their conventional meaning in the art. As used herein, the following terms have the meanings ascribed.
“M” refers to molar concentration, “μM” refers to microMolar, and “mM” refers to milliMolar concentration.
“kD” refers to kiloDalton
“PGR” refers to a plant growth regulator.
“Percent” or “%” is percent by weight unless otherwise indicated.
“Ppb” refers to parts per billion by weight.
“Ppm” refers to parts per million by weight.
“Ppt” refers to parts per thousand by weight.
Nomenclature—Botanical names generally are given as common names that are referable to scientific nomenclature through abundant scholarly resources.
Statistics—Comparison of means by T-test (two-tailed) show significance at ρ≤0.05.
“Enhance growth” or “enhancing growth” refers to promoting, increasing or improving the rate of growth of a photosynthetic organism such as a plant; and/or increasing or promoting an increase in the size and/or yield; and/or enhancing the quality of the photosynthetic organism or its parts; regulating the flow of photosynthates; enhancing the flow of photosynthates to respiration; enhancing aesthetics; increasing hydrostatic pressure; improving fragrance; accumulating photosynthates within the photosynthetic organism; and/or improving the flavor of the photosynthetic organism, in particular, Brix (a measure of sugar content), of its seed, fruit, flower, nectar, root, stem, or its parts.
“Photosynthetic Organism” refers to life forms that synthesize photosynthates including C3, C4, and CAM plants; and photosynthetic Eukaryotes including, but not necessarily limited to, those of the following preferred supergroups: Archaeplastida such as Plantae, Chlorophyta and Rhodophyta; and Chromoveolata such as Phaeophyta. Photosynthetic organisms may also refer to botanicals; turf and ornamentals; crops, including food, fodder, fiber, feed, and agricultural crops; and harvests thereof; and plants, both higher and lower plants, and plant-like organisms. The systems, methods and formulations may be advantageously used with any species of photosynthetic life.
The compositions may be applied to virtually any variety of live photosynthetic organisms. Photosynthetic organisms which may benefit include, but are not limited to, all Plantae particularly those in all crop groups recognized by the United States Environmental Protection Agency (2012: 40 CFR 180.41) as for example such as the following: alfalfa, allspice, amaranth, angelica, anise, annatto, arugula, back ciao, balm, barley, basil, bean, beet, borage, breadfruit, broccoli, Brussels sprouts, burdock, burnet, cabbage, cantaloupe, caper, caraway, cardamom, cardoon, carrot, cassava, castor, cauliflower, cavalo, broccolo, celeriac, celery, celtuce, cereals, chard, chayote, chervil, chickpea, chicory, chive, cilantro, cinnamon, clove, clover, coffee, collards, coriander, corn, cotton, cranberry, cress, cucumber, cumin, curry, daikon, daylily, dill, endive, euphorbia, eggplant, fennel, fenugreek, flax, forage, fritillaria, gherkin, gourd, grape, grain, garlic, guar, hay, hemp, horehound, hosta, hyssop, jackbean, jicama, jojoba, kale, kohlrabi, kudzu, kurrat, lablab bean, lavender, leafy greens, leek, legume, lemongrass, lentil, lespedeza, lettuce, lupin, mace, marjoram, melon, millet, mint, mizuna, Momordica, muskmelon, mustard, nasturtium, nutmeg, oat, onion, orach, parsley, parsnip, pasture, pea, peanut, pepper, peppermint, perilla, popcorn, potato, poppy, pumpkin, purslane, radicchio, radish, rape greens, rhubarb, rice, rosemary, rutabaga, rye, safflower, saffron, sage, sainfoin, salsify, skirret, sesame, shallot, sorghum, soybean, spinach, squash, stevia, strawberry, sunflower, sweet bay, sweet potato, sugar beet, sugar cane, Swiss chard, swordbean, tanier, taro, tarragon, tea, teosinte, thyme, tobacco, tomato, trefoil, triticale, turmeric, turnip, vanilla, vernonia, vetch, watermelon, wheat, wild rice, wintergreen, woodruff, wormwood, yam, zucchini and the like; fruit-bearing plants, such as, almond, apple, apricot, avocado, azarole, banana, beech nut, blackberry, blueberry, Brazil nut, breadfruit, butternut, cashew, cherry, chestnut, chinquapin, citrus, cocoa, cocona, coffee, currant, dragonfruit, elderberry, fig, filbert, goji, gooseberry, grapefruit, guava, hickory nut, huckleberry, kiwifruit, kumquat, lemon, lime, loganberry, loquat, macadamia nut, mango, mangosteen, martynia, mayhaw, naranjilla, nectarine, nopales, nut, okra, olive, orange, papaya, passion fruit, peach, pear, pecan, pepper, pistachio, plum, plumcot, prune, pummelo, quince, raspberry, roselle, tangelo, tangerine, tangor, tejocote, tomatillo, uniq fruit, walnut, spices, and the like; florals and ornamentals, such as achillea, adenium, agave, ageratum, aloe, alyssum, anemone, aquilegia, aster, azalea, begonia, bird-of-paradise, bleeding heart, borage, bromeliad, bougainvillea, buddlea, cactus, calendula, camellia, campanula, carex, carnation, celosia, chrysanthemum, clematis, cleome, coleus, cosmos, crocus, croton, cyclamen, dahlia, daffodil, daisy, dandelion, day lily, delphinium, dianthus, dietes, digitalis, dipladenia, dock, dusty miller, euonymus, forget-me-not, fremontia, fuchsia, gardenia, gazania, geranium, gerbera, gesneriad, gladiolus, hibiscus, hydrangea, impatiens, jasmine, lily, lilac, lisianthus, lobelia, marigold, mesembryanthemum, mimulus, myosotis, narcissus, New Guinea Impatiens, nymphaea, oenothera, oleander, orchid, ornamentals, oxalis, pansy, penstemon, peony, petunia, plumeria, poinsettia, polemonium, polygonum, poppy, portulaca, primula, ranunculus, rhododendron, rose, salvia, senecio, shooting star, snapdragon, solanum, solidago, stock, ti, torenia, tulip, verbena, vinca, viola, violet, yucca, zinnia, and the like; indoor garden and houseplants, such as African violet, Chinese evergreen, succulents, dieffenbachia, dracaena, ficus, hosta, peace lily, philodendron, pothos, rubber tree, sansevieria, chlorophytum, and the like; trees, such as Abies, Aspen, birch, cedar, Cinnamomum, Cornus, cycad, cypress, Dawn Redwood, elm, ficus, fir, ginkgo, gymnosperm, hardwood trees, Indian Rosewood, jacaranda, juniper, Laurel, legume, Liriodendron, magnolia, mahogany, maple, oak, palm, Picea, Pinus, Pittosporum, Plantago, poplar, redwood, rosewood, saguaro, Salix, sycamore, Taxus, teak, willow, yew, Christmas tree, sources of lumber, sources of paper, and the like; grasses, such as turf, sod, bluegrass, bent grass, Bermuda grass, bromegrass, calamogrostis, carex, creeping bent, elymus, fescue, festuca, helictotrichon, imperata, miscanthus, molina, panicum, paspalum, pennisetum, phalaris, poa, grass seeds, and the like; dwarfs; grafts; cuttings; hybrids; and the like. In addition to the aforementioned crops, the formulations are also suitable for application to photosynthetic organismal sources of secondary metabolites such as switchgrass, Jatropha, euphorbia, nicotiana, lichen, kelp, diatom, cyanobacteria, bacteria, dunaliella, nannochloropsis, chlorella, haematococcus, eucheuma; bryophytes such as moss and fern; and the like. This list is intended to be exemplary and is not intended to be exclusive. Other photosynthetic organisms that may benefit by application of the compositions and methods of the present embodiments will be readily determined by those skilled in the art. The methods and formulations disclosed herein may be used to enhance growth in juvenile and mature photosynthetic organisms, as well as cuttings, tissues, seeds, meristems, callus, cells, and micropropagation. Seed priming and coatings prior to sowing may be applied in the range of 10-1000 μg of glycan composites per seed, preferably in the range of 20-300 μg/seed.
Alternatively, seeds, corms, bulbs, stolons, and cuttings, may be treated in-furrow, simultaneously with sowing. Generally, the anatomical location to which the composition of the method is applied should have a surface area large enough to enable the photosynthetic organism to absorb the composition. For example, it is desirable to include the sprouted cotyledon (i.e., the “seed leaf”), potato stolon, bulb, corm, or other substantial surfaces that facilitate absorption, such as true leaves and roots. Fruit bearing plants may be treated before and after the onset of bud, fruit and seed formation. For plants such as annuals, perennials, trees, orchids, gesneriads, and cacti in which stems, roots and/or trunks may be treated, application methods include treatment of shoots with sprays and/or treatment of shoots and roots by sprench or dip application or by separate root and shoot applications. Commercial aqua- and mariculture crops such as spirulina, aonori, laver, kombu, macrocystis, nori and wakame, may be misted, sprayed, brushed or dipped in sterile aqueous freshwater or seawater solutions of 10 ppb-3% glycan composites, allowing 15-90 minutes to absorb.
The methods and formulations in accordance with certain embodiments disclosed herein are designed, for example, to treat any of the aforementioned photosynthetic organisms such as plants, and to enhance quality, increase growth and/or improve the quality and quantity of harvested yields. This can be achieved by applying glycan composite formulations comprised of the following: one or more branched glycan deglycosylates with certain transition metal2+coordination complexes. The formulations can be applied in a dry or liquid form directly to photosynthetic organisms. In certain embodiments, liquid formulations additionally may include a preservative for prevention of spoilage during shipping and storage periods. The methods disclosed herein make glycan composites readily available for uptake by photosynthetic organisms.
Certain embodiments disclosed herein provide branched glycan deglycosylates that are components of the glycan composite. Hereinafter, this branched glycan deglycosylate component will be referred to as the “glycan” or “deglycosylate” component of the glycan composite.
Glycans are rather expensive when chemically synthesized and, if not for certain of the embodiments of the invention, agricultural applications would not be economically justifiable.
Fortunately, embodiments disclosed herein provide a number of cost-effective products by means of deglycosylation of certain inexpensive macromolecules, and these products make suitable glycan components of the glycan composite. Thus, certain branched glycan deglycosylates render economically feasible farm crop treatments. Suitable glycans may originate by cleavage, i.e., deglycosylation of glycan subunits from their parent macromolecule. Generally, macromolecules greater than 1000 s kD are chemical structures too large for treatment and uptake by a photosynthetic organism; therefore, deglycosylates less than 10 s kD are preferred branched glycan deglycosylate components. Deglycosylates are typically from macromolecules such as proteins, glycoproteins, N-linked-glycan-macromolecules and/or O-linked-glycan-macromolecules. They may be products of hydrolysis or other processes known to the art, resulting from actions of acids, bases, enzymes and/or microbes breaking bonds. Biosynthesis of branched glycan components by a plant or yeast may be cost effective as compared to products of chemical synthesis and purification, the expense of which has proven prohibitive. For example, purchase of pure high mannan branched N-linked glycans may cost $1000 s/gram; whereas, suitable high mannan branched N-linked glycans, deglycosylated from proteins per embodiments disclosed herein, may cost pennies/gram.
Botanical sources of suitable glycan subunits include the following: Cyanaposis tetragonalobus and Cyanaposis psoraloides, guar gums, GalMan2; Caesalpinia spinosa, tara gums, GalMan3; Ceratonia siliqua, locust bean gums, GalMan1-8; Amorphophallus konjac, konjac gums, Glc2Man2; Canavalia ensiformis Jack Bean, N-linked glycans; Ivory nut, Mann; carob; coffee bean; fenugreek; barley; palms, lilies, irises, and legumes, endosperm tissues, Mann; soft wood and bark of various trees; birch; gymnosperms; Norway spruce; and Chlorophyta such as Dasycladales, Characeae, Codium fragile, Caulerpa and Acetabularia acetabulum Mannan Weed. Furthermore, branched mannan derivative structures such as exhibited in
The aforementioned gums, proteins and other macromolecules may undergo deglycosylation by commercial processes known in the art. For example, some branched glycan macromolecules may be microbially digested under tightly controlled fermentation and others may be subjected to various other enzymatic digestion processes known in the art; and whereby, branched glycan macromolecules may be partially hydrolyzed by cleaving >100,000 kD gums to average molecular weights of 0.2-10 kDa glycan deglycosylates. That permits uptake of the smaller deglycosylates by plants. By comparison with a variety of natural sources, branched glycan deglycosylates from invertases showed the highest feasibility, exhibiting low cost and high potency, making them suitable for commercial production; see Table 1.
Terminal ligands of glycans were key to their activity, rendering identification of this part of glycan structure critical. Suitable terminal ligands of a glycan were identified in glycopyranoses, such as, galactopyranoses, glucopyranoses, and preferably mannopyranoses; alkyl-, acyl-, and aryl-substitutions thereof; and acylglycosamines. It followed that suitable glycans were cationic, anionic and neutral polymers; aldosyls and/or ketosyls; and branched glycans with any of the above terminal ligands. The molecular weight sizes typically ranged from 0.1 to >500 kD, preferably between 0.2 to 10 kD, and most preferably in the range of 0.5 to 2 kD.
Glycan abbreviations are as follow:
Gal means galactopyranosyl;
Glc means glucopyranosyl;
GlcNAc means N-acetylglucosaminosyl;
GalNAc means N-acetylgalactosaminosyl;
Gly means glycopyranosyl;
Lac means lactosyl;
Ara means arabinosyl;
Man means mannopyranosyl; and
Mann means poly-Man.
j, m, n subscripts mean corresponding chainlengths, where m=1−24 and n=1−24, unless otherwise noted. For example, GalGlcMann means GalactopyranosylGlucopyranosylMannopyranosyln and GlcmMann means GlucopyranosylmMannopyranosyln.
Hyphenated numerals denote the range of sizes. For example, Man8-14GlcNAc1-2 means the branched mannopyranosyl8-14N-acetylglucosamine1-2 in which Man8-14 means a range of 8 to 14 Man units in the branched chain.
Examples of preferred branched chains include MannGly with alkyl, acyl, aryl, polyacyl, polyalkyl, amine or no substitutions; aldosyls; ketosyls; GlcNAcn; alkylGlcn; methylGlcn; methylGlcGlyn; alkylMann; GalnMan2; pentoses; arabinoses; riboses; xyloses; hexoses; mannoses, mannosides, mannans; glucoses, glucosides, glucans; galactoses, galactosides, galactans, raffinoses; Gly2, for example, Glc2 sucroses, trehaloses, maltoses, gentiobioses, cellobioses, GalGlc lactoses, xylobioses, laminaribioses; GalmMann; GalmGlcjMann; GlcjMann; XyloMann; AraMann; AraGaln; fructofuranosylmGlcj; Lac; Maltopyranosyls; Man1-3, such as, Man1, methyl-α-D-Man, methyl-α-D-Man1-3, methyl-α-D-Man3Gal; triosyls, such as, Man3; and derivatives and combinations thereof.
Suitable glycans in a blend of deglycosylates of the glycan composite are, for example, Man1, Man3, methyl-D-Mann and methyl-D-Glcn. Glycan deglycosylates may be selected from short chains, such as, Mann, where n is from 1 to 8, preferably Man1-3; O-linked-branched-chains, such as, Manm-nGly, Manm-nGlc, Manm-nGal, and Manm-nGalGlc, where m is from 1-8 and n is from 1-8. Yet other suitable glycans are >1 kDa chains; such as, for example, GalmMann; GalnMann; GlcmMann; GaljGlcmMann, j=1−24, m=1−24, n=1−24; deglycosylates are preferred <1 kDa where j=1−8, m=1−8, n=1−8, such as for example, Gal2Man2; and in combinations and blends. Preferred glycan deglycosylates are O-linked glycans and N-linked glycans with Glym-n where m=1−8 and n=1−8. Further examples include the following Mann core structures such as α-mannobioses, mannotetraoses, mannopentaoses; amino-functionalized-mannoses, for example, glycylMann, alanylMann, and aminylMann, where n=1−24.
Preferred branched glycan deglycosylates may comprise one or more N-linked-glycans such as for example MannGlcNAc1-3 and Manm-nGlyNAc1-3, for example, Man8-15GlcNAc2. Preferred N-linked-glycans are selected from low molecular weight MannN-glycans, therefore, Man3GlcNAc1-3 and trimannopyranosyl-N-glycans are most highly preferred. Suitable derivatives may have higher orders of branching, such as, Manm-nGalNAc1-3 for example, Man8-15GlcNAc1-2 and Man9-20GlcNAc1-3; and derivatives, such as N-glycan, acyl, alkyl, and aryl-substitutions. Furthermore, suitable branched GalGlcManN-glycans include Glc:Gal:Man in ratios between 1:2:16-9:2:20; as, for example, Gal4Man10GlcNAc2. In addition, N-acetylglycosaminyl-terminal ligands such as, N-acetylgalactosamines, N-acetylglucosamines, and N-acetylneuramines, may be selected from GalNAc1-3; GlcNAc1-3; GlcNAc2; Man1-8GlcNAc1-3; derivatives and combinations, thereof.
Embodiments disclosed herein provide transition metal2+ coordination complex compositions of the glycan composite. In certain embodiments, the transition metal2+ coordination complex is comprised of the metals2+ component and one or more anionic components. Specific metals2+ are incorporated into the holoprotein structure for proper binding of glycans. In the absence of specific metals2+, the protein structure is incomplete, lacking the conformation to conjugate. Therefore, these preferred metals2+ include calcium (Ca2+) and manganese (Mn2+), and both applied together are preferred because Ca2+ and Mn2+ naturally occur in holoprotein binding sites. However, suitable transition metals2+ other than Mn2+ may be added, substituted or formulated including one or more D-block transition metals2+ selected from cobalt (Co2+), nickel (Ni2+), and zinc (Zn2+); and combinations thereof; and always in the presence of Ca2+. In addition, the presence of iron (Fe2+) and magnesium (Mg2+) and/or one or more of the aforementioned D-block transition metals2+ may further support the structural conformation of the holoprotein by Ca2+ and Mn2+. These metals2+ and/or their water-soluble salts may be measured into the glycan composite as liquids or solids; for example, applied in the ranges of 0.1-100 ppm Ca2+, 0.1-100 ppm Mg2+, 0.1-10 ppm Fe2+, 0.1-10 ppm Mn2+, 0.1-10 ppm Zn2+, 0.001-1 ppb Co2+, and 0.001-0.1 ppb Ni2+.
Preferred anionic components of the aforementioned transition metal2+ coordination complexes of glycan composites may be selected from sequestering anions that further function as respiration accelerators, as follow: oxaloacetates; acetates; aconitates; citrates, isocitrates; fumarates; glutarates, ketoglutarates; malates; and succinates. Suitable acid derivatives thereof, named herein without exclusion of others, were selected from aconitic, citric, fumaric, glutaric, malic, oxaloacetic, succinic, and like acids of transition metal2+ coordination complexes; and preferably at 10:1 anion:cation molar ratios or greater, within the range of 100 ppb to 30% w/w. Aconitic acids include aconitates, cis- and trans-aconitic acids, salts, and the like. Citric acids include citrates, citric, isocitric, and methylcitric acids; citric acid anhydrides; citric phosphates; salts, and the like. Fumaric acids include fumarates, fumaric acids, boletic acids, alkylfumarates, salts and the like. Glutaric acids include glutarates, ketoglutarates, glutaric acid, glutaric anhydride, alkylglutarates, 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; acetylphosphate, acetic anhydrides; alkylacetates, alkylacetoacetates, oxaloacetates, salts, and so forth. Succinic acids include succinates, succinic acids, Sprit of Amber, alkylsuccinic acids, succinyl anhydride, salts, and the like. The aforementioned salts include, one or more of Ca, Mg, Na, and transition metal2+ coordination complexes with the aforementioned acids. Anionic components of transition metal2+ coordination complexes may be preferably selected from their phosphates, such as, for example, malate-phosphate, citrate-phosphate, and the like. Generally, pure components of transition metal2+ coordination complexes are commercially available in bulk. Anionic components of transition metal2+ coordination complexes may be selected from suitable polydentate chelants, such as alkylamide chelants as follow: ammonium, sodium, and/or potassium salts of alkyl amide chelants such as, ethylene diaminetetraacetic acids (EDTA), N-hydroxyethylethylenediaminetriacetic acids (HeEDTA), ethylenediamine-N,N′-bis2-hydroxyphenylacetic acids (EDDHA), di(ortho-hydroxybenzyl)-ethylenediaminediacetic acids (HBED), diethylenetriaminepentaacetic acids (DTPA); methylglycine N,N-diacetic acids (MGDA); glutamic acid diacetic acids (GLDA); and the like. The anionic components are conventionally added to liquid solutions of the metals at a minimum of 7:1, and preferably at 10:1 anion:cation molar ratios or greater.
Preferred salts of the transition metal2+ coordination complexes may result from reacting metal2+ and anion components. Moreover, suitable commercially available salts include derivatives of N, P, K, S, C, H, O, Cl, secondary and micronutrients; and other agriculturally compliant combinations of compounds known to the art. For example, N as amines, amides, nitrates, polyacylamines; C as carbonates; Cl as chlorides; P as phosphates, phosphites; S as sulfates; H as acids; OH as bases; and the like.
Glycan composites (GC) are composed of several compounds having distinct chemical characteristics, each contributing desirable properties to the whole. It has been most elucidative to discover that when components of the glycan composite are applied separately to crops in the field, performance is inconsistent. Although direct application of each component to separate plant populations is possible, it is not preferred for lack of beneficial effect. Therefore, experiments were undertaken to verify functional exemplary glycan composites; and, at the same time, showing that each of the separate components did not function adequately alone.
Photosynthetic crops grow by means of respiratory metabolism of photosynthates to build, for maintenance and to reproduce. However, the ratio of respiration to photosynthesis is less than a third. Regulation of crop growth through more efficient transfer of photosynthates to respiration than before, by application of glycan composites in accordance with embodiments disclosed herein, were used to raise that ratio. This was expedited by efficient treatments with glycan composites in the dark (i.e., in an environment where sunlight does not reach); whether to seeds and roots underground during the day; or to roots and/or shoots at night, for example. It would be of benefit to agriculture to enhance productivity by optimizing treatments with glycan composites to seed, fruit, flower, sap nectar, photosynthate, root, stem and/or trunk; that is, via shoot and/or root applications, through these novel systems, as well. Taken together, application of glycan composites to achieve a positive effect is realized by embodiments disclosed herein.
In contrast to photosynthetic leaves, seeds are entirely respiratory. As a consequence, hastening germination has resulted from applications of exceedingly low doses of GC to seeds as compared to nutrient control. Early growth responses to separated components were compared to the unified GC and results were analyzed for statistically significant compared means. It was clearly indicated that separate components did not work; but together, they contributed to efficacy. Furthermore, the transition metal2+ coordination complexes of the embodiment that included the full set of D-block transition metals2+ improved performance of composite formulations.
Respiration is dependent on available oxygen (O2) and O2 was enhanced through co-application or otherwise exposing crops under GC treatment to elevated O2. Site-directed O2 enhancement, in particular to roots or seeds, was achieved more efficaciously in the field by application of O2-generating compounds such as peroxides. Suitable inexpensive peroxides include H2O2 and carbamide peroxides, while O2-generating granular compounds are known to the art, such as, CaO2 and/or MgO2 that slowly released O2 while crops were under GC treatments. CaO2 and MgO2 provided O2 enriched environments that supported respiration, particularly when applied to seeds or roots, as separate oxygen sources in conjunction with formulations of the embodiment. Peroxides are O2-generating components that may be formulated into dry products, but preferably are stored and applied separately to the crop before, during or after treatments with GC. Peroxides tend to destabilize and decompose GC-concentrates, thus, shortening shelf life.
An exemplary O2-generating co-application method follows: Prior to treatment with GC, liquid H2O2, 10-100 grams granular CaO2 and/or MgO2 was incorporated at 15-30 cm soil depth at a rate of 50 Kg/ha during the crop season; and/or potting media or planting hole soils were mixed with 10-20 g/L prior to transplanting. GC was applied as a side dressing or spray drench to the photosynthetic organism to the same acreage. Thereafter, while the plant was under GC-treatment, the peroxide slowly released O2, advancing respiration in conjunction with the actions of GC. Injecting O2 gas may be undertaken, primarily by bubbling into liquid media to saturation. Co-applications of O2-generators with glycan composites were synergistic, resulting in enhanced quantities of productivity.
Under circumstances in which O2 could not be elevated, alternate methods were applied to provide an environment conducive to respiration wherein photosynthetic organisms under cultivation. Thus, treatments with glycan composites were undertaken in conjunction with exposure of crops to respiration accelerators, either by addition to or incorporation with glycan composites. Respiration accelerators were selected from the following: iP, such as for example, salts of phosphoric acid, such as, ammonium, potassium and sodium phosphates; Gly-phosphates such as Glc-phosphates and Man-phosphates, Glc2-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 aforementioned acids also serve as anionic components of the coordination complex, added at a minimum of 10:1 anion:cation ratio.
Germination and early growth of Burpee Sweet Corn cv. Bi-Licious Hybrid was tested for response to various components of composite formulations. Rapid assays of seedling growth were undertaken with hydroponic cultures in which aqueous media were sterilized and cooled. Seeds were examined to exclude aberrantly large, small, or damaged seeds, prior to treatment. Plants were maintained in the dark for respiration at 30° C. Exactly 48 seeds were sown per 15 cm sterile disposable plastic Petri dish on Whatman paper circles moistened with nutrient control or treatments. Replicates numbered 8 per treatment (n=8). Germination was established when radicle emergence was observed for 50% of controls after 30 hours. Treatment and control solutions were prepared by dissolving nutrients in deionized ultrapure water. In place of stainless steel vessels and stirrers, plastic laboratory utensils were utilized to prevent intrusion of Ni2+ and other metals. Cross-contamination with nutrients was avoided by disposing of plastic utensils immediately after use. Stock solutions were from reagent grade compounds. The aqueous glycan stock solutions of the GC in this investigation were 1-15% Mann glycan deglycosylates that were obtained by acetolysis of ivory nut flour in acetic acid:acetic anhydride:sulfuric acid 25:25:1. The 0.0001-5% Ca2+ and transition metals Fe2+, Mn2+, Zn2+, Co2+, and Ni2+ (Cat) were prepared by sequestration in a blend of 1 mM citrate, 5 mM malate, and 1 mM succinate, abbreviated CMS. The GC was applied at 10 μM Mann concentration. Ca- and Mn-EDTA salts are limited ions, abbreviated EDTA. Other concentrations applied were 1 μM Man3 and 100 μM Man1. Man1-CatCMS was formulated with Cat-CMS transition metal2+ coordination complexes for comparison to Man1-EDTA, and so forth. Water was provided as a negative control.
As presented in Table 2, corn seeds treated with GC showed highly significant acceleration of germination mean counts (ρ=0.000) as compared to those of separate components, Cat, glycan, CMS alone. Counts of CMS, glycan, and Cat were the same as for water; and there was no difference between water and Cat. Whole GC showed significant enhancement as compared to G-EDTA. GC showed borderline significant improvement when compared to glycan with CaMn-CMS, indicating that the whole Cat improved efficacy of the GC over the contribution of limited ions. Thus, both Cat and CMS contributed to germination in the GC. Furthermore, composite formulations in which the glycan was substituted with Man1 or Man3 with Cat CMS, showed significant improvements of germination as compared to the Mani and Man3 formulations with limited ions and with EDTA salts that did not enhance respiration.
In the whole GC, the components contributed to respiration and growth; yet, in contrast, individual components applied separately did not work. The full complement of the transition metals2+ in Cat, in particular in CMS transition metal2+ coordination complexes, significantly improved the product as compared to limited ion formulas. Selection of suitable anionic components of the transition metal2+ coordination complexes that facilitated respiration contributed to the GC significantly as compared to EDTA that did not. Remarkably, the potencies of glycan and Man3 composites were orders of magnitude greater than Man1, both showing germination at far lower doses than Man1. Composites were found to be applicable toward significant improvements of Mani and Man3formulations, an unexpected outcome of the investigations. Notably, post-germination applications of GC with transition metal2+-alkyl amide coordination complexes resulted in a trend toward accumulation of photosynthates, particularly in environments of reduced oxygen tension. For example, applications with GC-CaMnEDDHA 1 week prior to lettuce leaf harvest resulted in higher Brix than whole GC and control.
It is often advantageous to provide glycan composite products as 10 ppm-30% concentrates that may be shipped in dry or liquid form while kept under cool, dry, dark storage conditions; but inherent to organic compounds that comprise glycan composites is that the complex was consumed by various and sundry microbes as well as by plant cells. Therefore, measures must be taken to preserve the compositions from spoilage, particularly of the aqueous products. For storage, especially of liquid compositions, suitable preservative agents may be incorporated to the formula to improve the stability of products. Commercial preservative agents include biocides and germicides, such as for example, the following: 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; phenolics, such as, 4-Chloro-3-methylphrnol and 2-phenylphenol; thiazolinones and the preferred isothiazolinones (IT), such as, benzoisothiazolinones (BIT), methylchloroisothiazolinones and methylisothiazolinones (MIT). IT is a phytobland antimicrobial in the range of 1-800 ppm. The preservatives are recommended for formulation into liquid glycan composite concentrates in the range of label rates, from 1 ppm to 1%. For example, BIT in the range between 50 to 750 ppm, preferably between 100-300 ppm in liquid concentrates was safe and effective. Thus, liquid formulation of the glycan composite may be blended with any antimicrobial, yet they must be selected from phytobland preservatives as per embodiments disclosed herein. In a glycan composite product present as a concentrated product composition in the range of between 10 ppm to 30% glycan composite for dilution prior to application to a crop of photosynthetic organisms for the enhancement of productivity, a suitable concentrate comprises one or more of glycan deglycosylates in the range of between 1 ppm to 20% and one or more transition metal2+ coordination complexes in the range of between 1 ppm-10% and a preservative in the weight range of 50 ppm to 1%.
The effects of preservatives on potencies of plant growth regulator glycan composites were compared. The experiments quantified root growth. The results showed that potency was retained after storage for one month with preservatives. In contrast, formulations without preservative lost activity.
Early post-germination Swiss Chard (Beta vulgaris subspecies cicla L., cultivar “Fordhook® Giant”) root growth was tested for response to glycan composites (GC) supplemented with the preservative, BIT. Preliminary experiments of preservatives that were narrow in antimicrobial effect, not suitable for food use, or that were phytotoxic at antimicrobial doses were excluded. Roots of Swiss Chard germlings showed responses of improved growth in length within a week of application of GC and this corresponded to weight increases. The branched N-linked glycan, 100 ppm Man3GlcNAc2 deglycosylate, was selected to initiate blending the glycan composite with agitation into water. The glycan composite was further formulated by stirring 5 mM malic acid anion component in the glycan-water solution with aqueous metal2+-nitrate salts; resulting in the 5 mM malate transition metal2+ coordination complex of 1-5 ppm Ca2+ and transition metals 1-3 ppm Fe2+, 0.1-0.5 ppm Mn2+, 0.2-1 ppm Zn2+, 0.01-0.1 ppb Co2+, and 0.001-0.01 ppb Ni2+. The preservative, Proxel™ GXL, was selected from IT antimicrobials and was applied to 1 ppm to 30% liquid product concentrates for storage in the range of 100-200 ppm. Formulations were stored for a month at 35° C. prior to testing. Concentrated formulations were diluted in water as needed immediately prior to treatment of seedlings. Solutions incorporated reagent grade compounds of other required elements.
Rapid assays of root growth were based on modified methods of the aforementioned hydroponic culture on moistened Whatman 598 Seed Culture paper circles and in which treatments and aqueous media were not sterilized prior to application. Root mg weights were taken with a calibrated Mettler digital balance. Terminology used herein indicates the omission or inclusion of nutrients, as follow: GC=10 μM glycan composite without IT, GC−IT=GC with IT; Man1=100 μM Man1−GC without IT, Man1−IT=Man1−GC with IT; MG=375 mM methyl-D-Glc1-GC without IT, MG−IT=MG with IT; Man1=1 μM Man3−GC without IT, Man1−IT=Man3−GC with IT.
Post-germination Swiss chard seedlings treated with glycan composite without preservative showed losses of potency of as much as half that of the same formulation with IT. Results of means ±SE are presented in Table 3. Doubling (2×) the concentration of composite formulations without IT resulted in higher root weights than control, yet at 20% lower yields than 1× concentrations with IT. Formulations without a preservative lost at least half their potencies after 1 month of storage. The retention of original efficacies of GC formulations with preservatives shows a distinct improvement of all products that must be stored until end-user sales and applications.
In the example of
A suitable synthesis to make a glycan composite is as follows. In certain embodiments, the glycan composite was formulated with one or more of the aforementioned transition metals2+. To make the transition metal2+ coordination complex, one or more of the appropriate anionic components, such as for example, 0.1-5 mM citric, malic, succinic, and/or oxaloacetic acids were added and dissolved in water; and then suitable ppb-ppm metal2+ components of the transition metals2+ coordination complexes were stirred in to dissolve in the aqueous formulation. Thus, for example, 1-10 ppm Ca2+ and 0.1-1 ppm Mn2+, one or more of 1-10 ppm Mg2+, 1-3 ppm Fe2+, and 0.2-1 ppm Zn2+, and 0.01-0.1 ppb Co2+ and 0.001-0.01 ppb Ni2+ were the added metal2+ components. Formation of proper transition metal2+ coordination complexes requires at least 1:10 and preferably 1:25 transition metal2+-cation:anion ratios. The glycan composite unit was completed by blending in 1 μM to 500 mM glycan. Formulations that were to be stored for more than a day before applications to plants included label quantities of a broad spectrum preservative selected from IT, BIT, MIT, hydantoin, and the like. The method may also comprise the step of blending one or more agricultural surfactant/emulsifiers, and/or other agricultural additives/adjuvants at label quantities that achieve at least critical micelle concentrations, in particular, for foliar applications.
Suitable surfactants and emulsifiers include anionic, cationic, nonionic, and zwitterionic detergents; for example, amine ethoxylates, alkyl phenol ethoxylates, phosphate esters, polyalkylene oxides, polyalkylene glycols, polyoxyethylene (POE) fatty acid esters, POE fatty diglycerides, POE polymers, POP polymers, PEG polymers, protein surfactants, sorbitan fatty acid esters, alcohol ethoxylates, sorbitan fatty acid ester ethoxylates, ethoxylated alkylamines, quaternary amines, sorbitan ethoxylate esters, substituted polysaccharides, alkyl polyglucosides (APG), APG-citrates, alkylglycosides such as methylglucosides, alkylmannosides, methylmannosides, ethylacetoacetates, N-acetylglucosamines, meglumines, glucamides, dimethylglucamines, copolymers, siloxanes, tallow amines, and blends. When applying glycan composites to foliage, the formulation may further comprise one or more aqueous surfactants and applying the resulting mixture by spraying, misting, fogging or electrostatics to the plant foliage in an amount between about 1 to 100 gallons per acre, preferably 10 to 80 gallons per acre.
Blending the GC with nutrients sustained vigor, expanded root systems, enhanced plant growth, enlarged floral displays, promoted fruiting and improved flavor; a rendering that was particularly important in nutrient-deficient soils and water. Essential primary elements include, N—P—K. Essential secondary nutrient elements include Ca, Mg, and S. Essential micronutrient elements include B, Cl, Co, Cu, Fe, Mn, Mo, Ni, Si, Na, and Zn. Preferred nutrients are not selected to the exclusion of other elements, ions, or salts, and, depending on the situation, may be available in the soil and water in particular abundance such that supplementation is unnecessary for productivity; therefore, nutrient supplementations are applied at rates compliant with government agricultural regulatory agencies following instructions on labels. Suitable sources include salts and minerals generally known to the art. For example, the most highly preferred micronutrient selections to the compositions may include 0.5-5 ppm chelated Fe and/or Zn; and applying a suitable amount of the resulting mixture to one or more plants.
Plant nutrient phosphorus may be obtained from one or more of the following sources: iP, phosphorus rock, phosphoric acids, phosphates, phosphites, pyrophosphates, steric P, guano, manures, seaweed extracts, bird droppings, fishery, poultry and livestock waste and the like. Organic sources of P tend to be far too expensive to apply to fields in the 1-20% ranges of iP, however, at ranges below 1% concentration they often proved effective as respiration accelerators. Organic sources of P included, for example, glycerophosphates and the aforementioned sugar-phosphates were utilized in the range of between 0.1 ppm to 800 ppt.
Nitrogen may be obtained from one or more of the following sources: nitrate N, such as, nitric acid and salts, thereof; ammoniacal N, such as ammonia, UAN, ammonium nitrate, ammonium sulfate; urea N such as methylene urea, urea-formaldehyde, urea, low biuret and preferably ultralow biuret urea; amine/amide/amino N, 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 sodium or potassium glutamate; derivatives; blends; and the like; and mixtures of amino acids; proteins, such as from, glutens, caseins, fish, and blood; hexamines; and combinations, thereof. Nutrient elements were metabolized to all biochemical, growth, reproductive and structural components of photosynthetic organisms.
Applications of glycan composites provide opportunities to supplement plant nutrient deficiencies by use of tank mixes or by applications formulated with a selection of additives. The amounts of plant nutrients were applied in accordance with labeling of guaranteed analysis by governance boards at rates known to the art. Supplementation comprised the steps of dissolving plant nutrient components into aqueous solution, resulting in a mixture comprising glycan composites. For example, in certain embodiments, glycan composites were supplemented with 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 0.5-5 ppm Zn. Various glycan composite formulations may be classified as plant biostimulants that may benefit nutrient use efficiency, improve tolerance to abiotic stress, and increase yields. Biostimulants generally include natural products, such as for example, plant, algal, fermentation and animal metabolites; humates; microbials; biochemicals; and the like. Other glycan composite formulations may be classified as elicitors that generally include natural products, such as for example, algal, fermentation and animal metabolites; proteins; enzymes; microbials; and the like.
Embodiments disclosed herein as applied to crops are of glycan composites made into compositions of which the compounds themselves serve as resources for control of photosynthetic organisms, including live photosynthetic Eukaryotes; and, for the most part, agricultural Archaeplastida plants. As such, the glycan composite may be appropriately formulated with agrochemicals and are rendered into aqueous compositions that are capable of facilitating the growth of photosynthetic organisms, particularly cultivated flowering plants. The methods disclosed herein may be applied to safely enhance the balanced metabolism of exogenous components that contribute to the productivity of photosynthetic organisms. While, at the same time, they may be methods for applications of compositions of the glycan composite in the beneficial treatments of photosynthetic organisms, that, further, may be made to enhance the healthy growth and quality of crops.
Although the present inventor is not to be bound by any theory, the glycan composites of the embodiments disclosed herein feature a “lock and key” mechanism. A glycan with a suitable terminal ligand shows high binding affinity to a specific receptive glycoprotein. The lock is the glycoprotein and the key is the terminal ligand. The “tumblers” of the glycoprotein-lock are the internal structural sites of calcium and manganese. When the glycan key opens the lock, photosynthates are displaced to accumulate or to move on to respiratory metabolism; and thus, opening the option for selection of respiration accelerators in the anionic components of the transition metal2+ coordination complex. During daylight hours, roots consume those photosynthates. Respiring roots produce carbon dioxide, a portion (10% to 20%) of which is transported up to the shoots; and enhanced photosynthetic carbon fixation results. Thus, the glycan composite may be useful to reduce energetic losses to photorespiration by these treatments of photosynthetic organisms through their reach into sap nectar that modulate quality of sweetness measured as Brix. In particular, when, for example, plants are cultivated under photorespiratory environmental stresses such as saturated sunlight intensities, droughts and heat, the glycan composite may benefit yields. Moreover, the embodiments provide glycan composite systems for regulating the accumulation of photosynthates through the deceleration of respiration for flavor and nutritional enhancement by human consumers, livestock, poultry, and as well as, in robust nectar for pollinators.
In accordance with embodiments disclosed herein, a novel plant growth regulator system is introduced that advances photosynthetic flux to drive a photosynthetic organism to accumulate photosynthates. This is initiated, for example, by creation of novel crop inputs of glycan composites that may be applied to photosynthetic organisms. Glycan composites may be comprised of branched glycans with transition metal2+-polydentate anions. The preferred anions of this embodiment are alkyl amides selected from salts of EDTA, EDDHA, HeEDTA, DTPA, HBED, MGDA, GLDA, and the like. In addition, oxygen-starved environments under reduced oxygen tension, in the range from 0-10% O2, may be induced physically by respiration decelerators, such as by flooding roots with irrigation water, by storing plants or their parts in nitrogen gas, or by elevating CO2 concentrations. Alternatively, respiration decelerators may be selected from suitable plant growth regulators, co-applied according to agriculturally labeled methods known to the art; for example, plant growth regulators at 1-750 ppm dosages were selected from various suitable cytokinins, salicylic acids, and/or, gibberellins; and derivatives and the like.
Photosynthetic organisms respond consistently to glycan composite components when applied, preferably suitably formulated and rendered into potent formulations such that they facilitate the growth and quality of photosynthetic organisms as well as provide an array of physiological benefits that enhance their marketable qualities.
In accordance with embodiments disclosed herein, the complexes of embodiments may be applied separately, serially, or simultaneously. Indeed, particularly during physiological stress, by action of glycan composites on sap nectar of a photosynthetic organism, productivity was enhanced by the increased flow of photosynthates to respiratory metabolism in photosynthetic organisms in accordance with the aforementioned lock and key mechanism of the embodiments disclosed herein.
In certain embodiments, concentrated glycan composite products in compositions comprising at least glycan deglycosylates in the range of between 0.1 ppm to 30% and one or more transition metal2+ coordination complexes in the range of between 0.1 ppm to 20%, are made ready for application by dissolving an 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 made convenient for the grower to stir the final solution containing glycan composites into water as the carrier of choice for final dilution. In most instances, agitation and 25-80° heat facilitates the dissolution of the dry product in the carrier. The glycan composite is amenable to water-borne agricultural systems, such as, hydroponic and water cultures, by metered application with pumps into the medium, immersion of roots in diluted glycan composites, or as a foliar treatment.
The formulations employed may include any of a wide variety of agronomically suitable additives, adjuvants, or other agriculturally compliant ingredients and components (hereinafter “additives”) that can improve or at least do not hinder the beneficial effects of the glycan composite when applied at label rates. Generally accepted additives for agricultural application are periodically listed by the United States Environmental Protection Agency. In particular, foliar compositions may contain spreaders present in an amount sufficient to further promote wetting, emulsification, even distribution and penetration of the active substances. Spreaders are typically organic alkanes, alkenes or polydimethylsiloxanes that provide a sheeting action of the treatment across the phylloplane. Suitable spreaders include paraffin oils and the foregoing surfactants. Penetrants include, for example, alkyl acetoacetates, sodium dodecylsulfate, formamides, DMSO, and alcohols.
Embodiments herein are useful when blended or tank mixed with various plant treatments such as agriculturally compliant pesticides, insecticides, herbicides, plant growth regulators, fungicides, germicides, biocides, elicitors, biostimulants, antagonists, antitranspirants, synergists, systemics, surfactants, spreaders, stickers, vitamins, minerals, salts, solvents, genetics, bioagents, and the like. Herbicides that are based on ammonia metabolism, for example the glufosinates, Ignite®, Rely®, and Liberty®, are safened by application of glycan composites, reducing phytotoxicity in related herbicide-resistant GMO crops; and per application at label rates.
Examples of suitable additives and adjuvants include the following: 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 citrate-phosphate, calcium fumarates, calcium malate, calcium malonate, calcium maleate, calcium malate-phosphate, calcium gluconates, calcium glutarates, CaO2, calcium succinates, calcium chelants, calcium nitrate, calcium glycerophosphate, manganese-phosphates, manganese acetates, manganese citrates, manganese fumarates, manganese glutarates, rhodochrosite manganese carbonates, manganese oxides, MgO2, manganese malate, manganese malonate, manganese maleate, manganese succinates, manganese chelants, and the like; co-solvents 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 the like; bioagents 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 constituents that may be added to the compositions include soil conditioners, antibiotics, plant growth regulators, GMO, gene therapies and the like. Among the plant growth regulators which may be added to the formulations of the present invention are auxins; brassinolides; cytokinins; gibberellins; salicylates; benzyladenine; amino acids; benzoates; carboxylic acids, vitamins; carbohydrates; herbicides, such as, phosphonomethylglycines, halosulfuron alkyls; selective herbicides, such as, sethoxydims and sulfonyl ureas; salts, esters, phosphates, hydrates and derivatives thereof; and genetic compositions.
Glycan composite technology is appropriate for, but not limited to, crop application in the dark or shade, as during periods of maximum respiration; as well as under direct sunlight. In general, glycan composites are readily applied directly to shoots and/or roots and/or seeds; and/or parts, thereof, including cuticle, epidermis, flower, fruit, sap, nectar, bark, stem, foliage, needle, blade, phylloplane, spine, trichome, root hair, tap root, cotyledon, cone, and the like. The concentration of glycan composites in the formulations as applied to photosynthetic organisms should generally be between about 1 ppb to 1% and more preferably between about 10 ppb to 500 ppm. For specific applications, the concentration at the point of applications may be lower for roots than for shoots; thus, between the concentrations of 1 ppb-300 ppm for root application. Glycan composites may be applied to rooting media and then watered in or may be diluted first in an aqueous carrier and then applied to the media. On foliage, treatments generally are applied in a mist, fog, spray, drip, stream, dip, coating, or sprench between 1 ppb to 1% concentrations of the glycan composite. When diluted in an aqueous carrier, the resulting diluted glycan composite is applied to a photosynthetic organism in an amount of about 1 to 500 gallons/acre.
The following examples are provided to illustrate the embodiments disclosed herein and should not be construed as limiting. In these examples, purified water was obtained through reverse osmosis; transition metal2+ coordination complex components and surfactants were obtained from Brandt. Abbreviations used in the following examples are defined as follows: “° ” means ° C.; “Sil” means organosiloxane/copolymer blend; “12-26-26” means Brandt 12-26-26 Micro, N—P—K with B, Cu, Fe, Mn, Mo, and Zn; “αManda” means methyl-α-D-Mann, n=1-3; “GG” means combinations of branched O-linked Gal1-12Man2, from partially hydrolyzed guar gum; “Ag” means GlcNAc1-3; “Ethan” means branched Man3GlcNAc2 (
The following are examples of specific formulations that may advantageously be employed in methods to treat photosynthetic organisms such as plants and to enhance growth in the same. The following examples are intended to provide guidance to those skilled in the art and do not represent an exhaustive list of formulations within the scope of the embodiments disclosed.
This formulation may be further supplemented with components of transition metal2+ coordination complexes selected from citrates, fumarates, glutarates, malates, oxaloacetates, succinates; and Mg. Root glycan composites were dissolved into 1 L of water with stirring at room temperature, 25 to 35° C.; and adjusted by titrating KOH to pH 5-7. 50 to 450 gallons/acre applied as close to the roots as possible either by side dressing and/or through drip irrigation. With irrigation, the treatment was watered into the soil, toward the roots for enhanced photosynthates, quality and quantity.
Ca2+ and Cat were dissolved with malic acid in 1 Liter of water. Other ingredients were added, dissolving each, one at a time; and the solution was adjusted within a range of pH 5 to pH 5.5 by titration with DKP/MAP, as needed. Transition metal2+ coordination complexes were selected from 0.01 ppb Ni and Co; and transition metal2+ coordination complexes may include ppm to ppt aconitates, citrates, fumarates, glutarates, oxaloacetates, and/or succinates. Foliar sprays were applied to glisten, approximating 75-100 gallons/acre resulting in enhanced photosynthates, quality and quantity of harvests.
This formulation may be further supplemented with anionic components of transition metal2+ coordination complexes, such as for example, respiration accelerators selected from aconitic, fumaric, glutaric, malic, oxaloacetic, succinic acids, and the like. Alternatively, for accumulation of photosynthates, anionic components were selected from polydentate chelants such as, EDTA, EDDHA, HeEDTA, DTPA, HBED, MGDA, GLDA, and the like.
All components were blended to homogeneity in aqueous solution with rapid agitation until completely dissolved and adjusted to pH 5-5.5 with MKP/DAP. For foliar application, this formulation was supplemented with 0.05% Sil for shoot treatments at 20-100 gallons/acre for enhancement of photosynthates and respiration in a crop.
An exemplary foliar system follows: Bell pepper sprouts were matched and maintained in half-gallon plastic containers each, separated into equal populations of Treated and Nutrient Controls. The glycan composite from this example was diluted to 1% with water and applied to shoots of the Treated population as a foliar spray, while the shoots of the Control population was sprayed with the same concentrations of mineral nutrients in water. In all other ways, Control and Treated populations were cultivated side-by-side under identical field conditions. At harvest, the Treated population averaged 35% bell pepper fruit mean weight yield increase over the control population that proved statistically significant ρ=0.001; n=30. In addition, sun scorched peppers were absent from treated fruit as compared to controls that showed 1-5% loss from scorched fruit that were not marketable due to unattractive appearance. Thus, enhanced flow of photosynthates resulted in an increase of marketable yields attributable to enhanced aesthetic quality of the final product by treatment with the glycan composite system.
The regulation of flow of photosynthates by glycan composites comprised of aManda was further managed by optional coapplications of the respiration accelerator, 10-100 g CaO2 to soil near roots, for high qualities and quantities of yields.
This solution may be further supplemented with anionic components of transition metal2+ coordination complexes selected from ppm-ppt aconitic, citric, fumaric, malic, oxaloacetic and succinic acids; ppm-ppt polydentate alkylamide chelants; and ppm-ppt Mg.
Radish seeds, 25 per dish, were sown in 16 replicate Gosselin germination dishes on Whatman 598 Seed Culture paper circles moistened with Nutrient Control or glycan composite. Seeds were maintained a constant temperature of 27° in the dark for respiration only. Germination was established at the time at which radicle emergence was observed for 50% of the seeds, G50. Results showed accelerated glycan composite mean G50=15 hours as compared to Nutrient Control mean G50=22 hours; n=8; p=0.001. Treatments of radish by coating seed with 20-50 μg/seed dry weight glycan composite proved highly potent, significantly accelerating germination as compared to Nutrient Controls as a result of enhanced flow of photosynthates. Similar acceleration of germination was observed for radish seeds that were pre-coated with 20-50 μg glycan composite/seed and dried, as compared to nutrient controls.
This solution may be further supplemented with anionic components of transition metal2+ coordination complexes selected from aconitic, citric, fumaric, malic, oxaloacetic and succinic acids; polydentate alkylamide chelants; Mg; and in ppm-ppt amounts. Bell Pepper seeds were sown in 12 replicate plots of uniform sandy loam with label rates of 12-26-26. The glycan composite was formulated in water and adjusted to pH 5.5 with DAP and Control was also adjusted with equivalent P with DAP/MAP. Two weeks after germination, six randomly selected plots were sprenched with glycan composite, targeting the plantings; and otherwise all 12 plots of bell peppers received identical growth maintenance. In order to eliminate crowding, each plot contained 20 plants, spaced 50 cm apart. Plants were harvested with roots intact after 2 weeks; followed by clipping roots from shoots, thoroughly washing off soil and oven-drying. Root dry weights of individual plants were taken. Results showed glycan composite root mean dry weight 1.2 grams as compared to Nutrient Control root mean dry weight 1.0 gram; n=6; ρ=0.02. Treatment significantly enhanced root dry weights as compared to controls. Sun scorched fruit, though present in controls, were absent from treated plants indicative of enhanced photosynthates and quality of the harvest.
Fields of Plumeria flowers are conventionally cultivated under high ˜1500-1700 μEinstein/m2/sec light intensity and low to moderate ˜20-30% humidity. Under these environmental conditions, daily hydrostatic pressure responses were observed in the afternoon. Typically, in the early morning, flowering plants are high in hydrostatic pressure, but by mid-afternoon, leaves begin to droop. This cycle of midday wilt showing high to low hydrostatic pressures was visually distinguishable as the elevation of leaves changed from pointing upward to downward, approximately 5 to 20 millimeters (mm). An increase of hydrostatic pressure is prerequisite to growth and was measured according to the change of elevation of foliage, especially during midday. The purpose of the trial was to record changes in hydrostatic pressure of Plumeria by measuring mm changes of elevation of leaves and comparing the responses of aqueous nutrient controls against plants to which a single treatment of 1-25 ml of 0.1-5 ppm Ethan glycan composite was applied to roots. The glycan composite formula was from the aforementioned Example 6; and optionally, other exemplary glycan composites may be applied for enhancement of hydrostatic pressure. For example, an effective amount of 1-200 ml of 0.1-5 ppm partially hydrolyzed invertase deglycosylates was applied to roots at 8 AM and a subsequent rise in elevation of foliage showing enhanced hydrostatic pressure was observed by noon when controls, at the same time, showed reduced hydrostatic pressure. Plumeria obtusa L. variety obtusa plants in 4 L plastic containers were allowed a week to acclimate to environmental conditions of direct sunlight and were further observed for consistency of diurnal changes in hydrostatic pressure with evening irrigation once per week. Mid-week between watering, the baseline elevations of leaves were measured late in the morning and marked against rulers; and later, compared against mm elevations of the same leaves 5 hours after treatment.
Results: Foliar elevation was a visually discernible sign of enhanced hydrostatic pressure. The mean +15 mm rise of leaves treated with glycan composite was significant (n=6; ρ=0.003) as compared to a corresponding mean −5 mm drop in elevation of nutrient control foliage. After treatment with 10 ppb to 800 ppm glycan composites, the rise of foliage was greater in plants treated late in the afternoon, after 3 PM; while at the same time, control foliage showed the most pronounced loss of hydrostatic pressure and drop in elevation caused by midday wilt.
In conclusion, Plumeria responded to treatments with increased hydrostatic pressure when controls showed decreases. Similar increases of quality of crop hydrostatic pressure occurred in bell pepper, brassicas, curcubits, pomes, and root crops treated with glycan composites formulated with suitable anionic components of transition metal2+ coordination complexes. For example, anionic components were selected from one or more of 10-900 ppm aconitic, fumaric, glutaric, malic, oxaloacetic, and succinic acids; and 10-900 ppm polydentate alkylamide chelants such as EDTA, EDDHA, HeEDTA, DTPA, HBED, MGDA, GLDA, and the like. Growth and development of all photosynthetic organisms are dependent on cell expansion initiated by increased hydrostatic pressure. When hydrostatic pressures increased over the long duration by application of the glycan composite systems of embodiments disclosed herein, the results showed significantly enhanced growth and development of the treated photosynthetic organism. Glycan composite systems coapplied with respiration accelerators improved quantity yields of crops.
Canola vegetative growth of nutrient controls in the shade was compared to shaded populations of Canola treated with branched N-linked glycan composite; and furthermore, treated and controlled populations were cultivated without shade to determine if there was a beneficial productivity enhancement in relatively reduced light environments. Canola seeds were sown in 36-cell plastic flats and showed even growth after 1 month, when shade control and glycan composite-treatments of shaded plants were placed under 50% shade cloth. Plants that were not shaded were under natural midday full light intensity in the range of 1500-1800 μEin/m2/sec; and under 50-85% shade cloth, the low light intensity was in the range of 100 to 900 μEin/m2/sec or less than half full light intensity.
Foliar treatments with foliar surfactants were applied, spray to drip ˜100 gallons/acre. Full daylight and shaded nutrient controls were compared against foliar glycan composite applied to Canola plants 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; 1-25 ppm Ca-CMS; and with adjustment to pH 5.5 with DAP, DKP or KOH. Plants were harvested after two weeks and dried at low temperature in a 70° oven for 48 hours. Dry shoots of individual plants were weighed and mean dry weights and T-tests of equality of means of populations are summarized in the table, below.
In conclusion, the population of Canola under Shade Control showed reduced productivity as compared to Full Daylight Control and Shade populations that were treated with glycan composites. Moreover, when shaded plants were treated with glycan composites, benefits of the enhanced quality and quantity of productivities as compared to Shade Control were statistically significant.
Many glycoproteins contain glycans that may be processed from seeds of legumes, such as Jack Bean; or by deglycosylation from a number of enzymes. Invertase is a commercially manufactured enzyme from bakers yeasts with suitable glycan components of the interior core or exterior protrusions that may constitute up to three quarters of the total glycoprotein weight. Invertase has preferred branched glycans with terminal Man-ligands from such as GalManProteins; Mann such as Man1-6, Mannotrioses, Mannotetraoses, and Mannopentaoses; GalnMann, such as, Gal2Man and Gal2Man2; GalnMann-N-glycans, such as, Gal2Man4GlcNAc and Gal4Man10GlcNAc2; Mann-N-glycans, such as, Man3GlcNAc and the preferred Man1-15GlcNAc2; and the like. At 270 kDa, the glycoprotein was too large to penetrate foliage. Therefore, to establish a baseline for comparisons to enzymatic digestions, a laboratory process was undertaken primarily to release glycan deglycosylates 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; predigested with 3% trypsin at 37° overnight; and further denatured by boiling for 10 minutes. These protease-treated samples showed moderate 50μM germination activity in the glycan composite. The samples were deglycosylated by incubation with 200 milliunit endoglucosaminidase H at 37° for a day; and denatured in dilute NH4OH pH 12 80° for 10 min. Residual protein and peptides were precipitated and removed. Deglycosylates typically comprised blends of O-linked and N-linked mannans and as glycan components of the glycan composite glycan isolates or blends, thereof, exhibited a range of 0.01-1 ppm w/w activity.
The aforementioned deglycosylations by means of proteolytic and glycolytic enzymes were relatively mild, yet involved costly biochemicals. It was found by these experiments that the initial denaturation of glycoproteins with base substantially shortened the subsequent heating duration in acids, and in consideration of energy savings, is preferred. Therefore, the preferred process was by hydrolyzing glycoproteins in components that were commercially available in bulk quantities at relatively low costs. Preferred methods were tested that may be utilized, such as, treatments with acid/base, hydrolysis, hydrazinolysis, and/or fermentations. For example, acetolysis (acetic acid:acetic anhydride:sulfuric acid 25:25:1) of invertase resulted in potent deglycosylates that showed activity as low as 1-100 ppb. Optionally, a novel malolysis of the embodiment was applied by incubation of invertase in maleic acid:acetic anhydride:nitric acid 25:25:10 at 60-80° for 1-24 h. Alternatively, invertase was incubated in citric:phosphoric acids 25:1; saturated citric and/or succinic acids; and/or selected from 0.1-3N mineral acids, such as sulfuric acid, and preferably, was deglycosylated by direct nitrolysis in 1-3 N nitric acid; and incubated with stirring for 1 to 24 h at 40-80°.
The preferred method to partially hydrolyze invertase was as follows: 10-30% invertase was dissolved in alkaline aqueous solution, such as, 0.2-1 N KOH and/or 0.2 N NH4OH with heating to 40-80° for 1-24 h; 50-60% citric acid was stirred in and incubated 1-24 h, 40-80°. After acid-incubation, the solution was adjusted to between a range of pH 3-6. The partially hydrolyzed invertase, now deglycosylated, was formulated to achieve field application with at least, 1-5 ppm Ca2+, 0.5-1 ppm Mn2+ and preferably with 0.01-6 ppm D-block transition metals2+ selected from Fe2+, Ni2+, Co2+, Zn2+; and 1-5 ppm Mg2+. Anionic components of transition metal2+ coordination complexes were selected from one or more of aconitates, citrates, fumarates, glutarates, oxaloacetates, and succinates. Alternatively, anionic components may be selected from the aforementioned polydentate chelants. For storage of solutions, one or more preservatives were added. Enhancement of quality and quantity of crops of photosynthetic organisms resulted from treatment of said crops with the aforementioned solutions containing in the range of 1 ppb to 10 ppm invertase deglycosylates. For example, when a glycan composite containing invertase deglycosylates was applied to photosynthetic organisms at 100 ppb partially hydrolyzed invertase the dose was 0.1 mg/L.
All of the aforementioned deglycosylation methods rendered similar potencies in their glycan composites. Elimination of any one of the components of the glycan composite decreased activity. The glycan composites comprised of invertase deglycosylates showed orders of magnitude higher potency than the processed botanical gums of the embodiments. Moreover, the manufacturing processes for invertase deglycosylates were simpler and more cost effective than those of other sources. The regulation of flow of photosynthates by glycan composites comprised of invertase deglycosylates was further instituted by optional coapplications of ppm respiration decelerators for flavor enhancement or respiration accelerators for enhancement of quantity yields.
Invertase was denatured in aqueous basic solution by blending 200 grams invertase into 1 Liter aqueous 0.2 N KOH and steamed for 4 h. With stirring, 50% citric acid was added and the solution was heated to 80° for 12 h. After cooling, the solution was titrated to pH 6 with NH4OH; and this was followed by addition of 1% Ca, 1% Mg, 1% Cat; and QID 2 L with water. The final 10% partially hydrolyzed invertase solution contained a blend of 3-7% branched O-linked and N-linked deglycosylates including those shown in
Not only was availability of the full body of plant nutrients important to the crop, it was also essential to the vigorous health of pollinators, such as honey bees, butterflies, moths, beetles, birds and bumble bees for provision of their full spectra of nutrients when they drew nectar containing vitamins and minerals from plants. In crops of photosynthetic organisms, Co2+ is a D-block transition metal2+ of the embodiment that is metabolized to Vitamin B12. Correction of this deficiency by foliar applications of glycan composites containing, for example, Co-CMS, conferred health benefits, particularly as pollinators and grazers consumed photosynthetic organisms and sap nectar fortified with B12 and other nutrients from healthy photosynthetic crops. For example, when 0.0005% Co was formulated as a suitable transition metal and applied to a flowering crop, its fortified metabolites became available from a flower to a honeybee and to its colony. Immediately prior to application to a crop, the concentrate was diluted in water or other agriculturally approved carriers in the range of 1 ppb to 100 ppm, preferably between 10 ppb to 10 ppm. The resultant diluted aqueous solution was applied as a spray drench or root application. For foliar application, the product was supplemented with one or more selections of agriculturally approved foliar surfactant and/or additives. One or more applications were applied per season, preferably 1-2× per month. For further optimization of the flow of photosynthates to sweetness and nectar production, glycan composites were applied within a week of blossoming and harvesting in conjunction with respiration decelerators, ppm cytokinins and ppm gibberellins, for endogenous enhancement of flavor and fortified plant foods.
The IGC may be further supplemented with components of transition metal2+ coordination complexes selected from D-block transition metals; anionic components; and Mg in trace quantities. Respiration accelerators may be selected from, for example, one or more of aconitic, fumaric, glutaric, malic, oxaloacetic, and succinic acids. Optionally, anionic components may be selected from one or more of polydentate chelants such as EDTA, EDDHA, HeEDTA, DTPA, HBED, MGDA, GLDA, and the like.
Under stressful arid conditions for cultivation of tomato, IGC was applied to plants and compared against nutrient controls. IGC significantly enhanced general growth, yield and quality of treated tomatoes over controls. Tomato seeds treated with IGC seed coats showed speedy germination.
Tomato cultivar Steak Sandwich Hybrid seeds (Burpee®) seeds were germinated under automatically controlled environmental conditions at 32° in the dark. A week after germination, sprouts were transplanted out of doors for culture in an arid environment of 45° :32° LD; 10% relative humidity; 16:8 h LD; and mostly sunny days with photosynthetically active radiation (PAR) up to 1800 μmol photons m−2 s−1 at midday. Application of solutions to test plants and control plants under study were made simultaneously and all plants were subjected to identical conditions consistent with good laboratory practices. Chemigations were maintained at sufficiency to keep roots uniformly moistened and drained without water damage. Replicate populations of plants were cultured in plastic TLC Pro 606 trays, each of the 36 cells with 125 ml capacity, until transfer to 33 cm diameter plastic pots containing soil-less media. Control and treated tomatoes were matched for size and vigor; and runts, damaged or diseased plants and seeds were discarded prior to onset in order to produce uniform replicates. Volumes for the three foliar applications were calibrated to 200 L/Ha with 0.1-1 ppm Invertase Deglycosylate glycans blend content in the applied Glycan Composite. Sweetness as an indicator of the level of flavor was measured as Brix of sap nectar squeezed from individual tomatoes using a calibrated digital refractometer (Reichert).
IGC was made according the methods described in Example 11 modified to the composition in the IGC Concentrate Table, above. For foliar applications, 0.03-0.05% Sil foliar surfactant was supplemented at label specifications. By application of identical overhead sprays of solution, the same quantities of nutrients were given to all plants.
Seeds were germinated on water-moistened Whatman 598 paper in Gosselin dishes, 20 seeds/dish, and 5 replicates per treatment. Experimental seeds were coated with 0.1 mg IGC, air-dried 48 h and sown. Controls were treated with the same nutrients without IGC.
Germination was determined by radicle emergence in 50% of the seeds. After 7 days, sprouts were transplanted into flats.
Each survey pool held replicates for statistical analyses. The differences between treated and control populations were statistically significant in each experiment unless otherwise noted; error bars show 95% confidence intervals.
IGC-coated tomato seeds showed accelerated germination. All replicates of treated seeds showed 50% germination in 60 h. In contrast, Controls showed 50% germination in all dishes after 72 h.
Experiments were designed to determine fruit quality and yield responses to IGC under stressful arid conditions. Field treatments under the aforementioned conditions consistent with decelerated respiration 2 days prior to harvest were compared to nutrient controls to determine effects on sweetness. Regardless of color, twelve of the largest >50 mm diameter tomatoes were harvested 2 days after treatment and live shoot weights were recorded. Half of the controls were red, whereas, all of the treated tomatoes were red. Table 4 shows that appropriate treatment of endogenous photosynthates of sap nectar resulted in enhanced fruit quality, expressed as mean Brix 5.5 that was significantly (ρ=0.012) improved as greater flavor than nutrient control Brix 4.9.
The count of tomatoes per plant is a measure of fruit yield and Table 5 shows that IGC treatment resulted in 3.2 mean fruit count per plant that was significantly (ρ=0.000; n=12) greater than the mean fruit count of 1.6 per nutrient control plant. The results of treatment with IGC were improved yield, enhanced fruit sap nectar and increased sweetness and flavor qualities in this arid environment.
Average total fruit mean weights per tomato plant were analyzed for Nutrient Control and IGC as measures of yield. Table 6, shows IGC treatment resulted in 277 grams wet/14 grams dry weight fruit mean yield per plant that was significantly (ρ=0.002/p=0.004) higher than control 124 grams wet/14 grams dry mean fruit weight per plant.
Responses of various species were surveyed with results displayed in Table 7. Applications were effective on plants known for C3 and C4 metabolism.
Geranium
Petunia
Lantana
Rapid germination after treatment of seeds with IGC was an indication of improved respiration and explained why species of CAM, C3 and C4 responded to glycan composites because all plants respire. Endogenous modulation of photosynthates flux by action of the glycan composite was consistent with tomato Brix elevation of sap nectar to improve flavor quality. Under environmental stress of the arid zone, tomato cultivation by treatment with glycan composites showed enhanced quality and quantity as compared to control. The regulation of flow of photosynthates by IGC was further instituted by optional coapplications of the respiration decelerator, 10-200 ppm cytokinin, for flavor enhancement or respiration accelerators for enhancement of quantity yields. Furthermore, under conditions of O2-starvation, supplementation with the O2-generator, 30-100 ml H2O2 per plant maintained root health for consistency of high qualities and quantities of yields.
This formulation may be further supplemented with ppb-ppm D-block transition metal2+ coordination complex components selected from Zn, Co, Ni; aconitic, citric, fumaric, glutaric, oxaloacetic, and succinic acids; alkylamide chelants; and Mg2+.
Photosynthetic organisms thus treated with glycan composites comprised of GG-deglycosylates resulted in enhanced flow of photosynthates for improvement of the quality and quantity of harvests. The regulation of flow of photosynthates by glycan composites comprised of GG was further established by optional coapplications of respiration decelerators for flavor enhancement or respiration accelerators for enhancement of quantity yields.
Tara gums are commercially available in bulk quantities and this species contains large polymers of branched GalMan3 units suitable for deglycosylation by the aforementioned acetolysis processes. That is, glycan deglycosylates were derived from food grade 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) yielding partially hydrolyzed branched GalMan3-deglycosylates. Glycans as partially Hydrolyzed Tara Gum (HTG) deglycosylates showed high potency in glycan composites between a range of 50-200 ppm concentrations. As a glycan component, HTG was investigated showing activity only in the presence of the glycan composite components, utilizing the methods as follow:
Cabbage was cultivated in environmentally controlled conditions as described above in plastic flats with 36 plants; 125 cc/cell. All aqueous foliar treatment solutions contained 0.5 g Sil/L, pH 6, and included Metals2+ in separate formulations of the following: Metals2+ 1 ppm manganese sulfate, 1 ppm ferrous sulfate, and 10 ppm calcium nitrate dissolved in water with 50 PPM DAP; Anionic component of the transition metal2+ coordination complex—300 ppm potassium α-ketoglutarate, abbreviated aKG; and 100 ppm HTG. Glycan composite components and Metals2+ were applied separately and blended together to test plant growth response to the components as compared to the holo-glycan composite. The solutions of aKG, HTG, and aKG+HTG were dissolved in the aqueous Metals2+ solution; and, therefore, transition metal2+ coordination complexes comprised of aKG, Ca2+ and Mn2+ were present in the treatment solutions. Metals2+ served as the control.
Foliar treatments were applied in a volume of 10 ml/flat, n=36, at expansion of the first true leaves. Shoots were harvested, dried and weighed.
Results of the trials in Table HTG, below, compared the mean dry weights of cabbage shoots to that of Metals2+, the stock solution in which all of the treatments were dissolved. The aKG shoots were not significantly (n=36; ρ=0.057) different in yield than the Metals2+ control; HTG with Metals2+ showed a significant (n=36, ρ=0.001) enhancement as compared to Metals2+ alone. Finally, aKG+HTG+Metals2+ showed the most highly significant (n=36; ρ=0.000) enhancement of yield as compared to Metals2+. Furthermore, the holo-glycan composite showed highly significant (aKG+HTG v aKG, p=0.000; aKG+HTG v HTG, ρ=0.000) enhancements of yields as compared to all of the other treated populations.
In conclusion, the glycan composite showed the most highly significant improvements when formulated together as the holo-glycan composite applied to photosynthetic organisms, targeting photosynthates. Moreover, beneficial responses to holo-glycan composites were confirmed after supplementation with D-block transition metals2+; Ca2+; and Mg2+. The regulation of flow of photosynthates by holo-glycan composites was further instituted by optional coapplications of respiration decelerators for flavor enhancement or respiration accelerators for enhancement of quantity yields.
This PHLB formulation may be further supplemented with one or more additional D-block transition metals2+; anionic components; and/or Mg2+. Treating photosynthetic organisms with PHLB resulted in enhanced flow of photosynthates for improvement of the quality and quantity of harvests. The regulation of flow of photosynthates by glycan composites comprised of PHLB was further controlled by optional coapplications of the respiration decelerator, 10-200 ppm salicylic acid, for flavor enhancement.
For germination, this formulation may be further supplemented with anionic components of transition metal2+ coordination complexes that are respiration accelerators selected from one or more of aconitic, fumaric, glutaric, malic, oxaloacetic, and succinic acids; and phosphates, thereof.
Radish seeds, 25 per dish, were sown in 16 replicate Gosselin germination dishes on Whatman 598 Seed Culture paper circles moistened with Nutrient Control or glycan composite. Seeds were maintained at a constant temperature of 27° C. in the dark for respiration only. Germination was established at the time at which radicle emergence was observed for 50% of the seeds, G50. Results showed accelerated glycan composite mean G50=15 hours as compared to Nutrient Control mean G50=22 hours; n=8; ρ=0.001. Treatments of radish by coating seed with 20-50 μg/seed dry weight glycan composite proved highly potent and significantly hastened germination as compared to Nutrient Controls as a result of enhanced flow of photosynthates. Similarly, germination was hastened in radish seeds that were pre-coated and dried with 20-50 μg glycan composite/seed, as compared to Nutrient Controls.
This formulation may be further supplemented with anionic components of transition metal2+ coordination complexes. For example, the anionic components may be selected from respiration accelerators and polydentate chelants.
Root glycan composites were made in 1 L of water with stirring at room temperature, 25 to 35° C. The formulation was titrated with KOH and/or NH4OH to pH 5-7. 5; and 500 to 750 gallons/acre were applied as close to the roots as possible either by drench, spray-drench, sidedressing and/or through chemigation. With irrigation, treatments were watered into the soil toward the roots for enhanced quality and quantity. Applications were applied weekly to monthly as needed during the growing season.
Invertase glycoproteins are comprised of proteins with core and surface Mann polymers. As such, these glycoproteins were partially hydrolyzed to yield Mann deglycosylates. When formulated in glycan composites and applied to photosynthetic organisms, invertase deglycosylates modulated the flow of energy from photosynthesis to respiration. Concentrated glycan composite formulations containing N-linked branched chain deglycosylates from invertase were diluted with water to 10 ppb-5 ppm field doses for photosynthetic organisms and were applied to germination assays, showing strongly potent activity at levels as low as 10 ppb invertase deglycosylates.
Glycan composites were further formulated for regulation of flow of photosynthates with optional selections for flavor enhancement from respiration decelerators and enhancement of quantity yields by respiration accelerators. Transition metal2+ coordination complexes of invertase deglycosylates may be further selected from anionic components that are respiration accelerators such as from ppm aconitic, fumaric, glutaric, malic, oxaloacetic, and succinic acids. Optionally, anionic components may be selected from polydentate chelants such as ppm EDTA, EDDHA, HeEDTA, DTPA, HBED, MGDA, GLDA, and the like.
Enhanced quality and quantities of crops yields resulted from glycan composite treatments of, for example, blueberry, leafy vegetables, cotton, cereals, tomato, cherry, onion, coffee, banana, citrus, melon, leafy greens, nuts, pomes, berries, tree fruit, food crops, florals, trees, turf and ornamental crops.
To 40 ml of strong base, preferably selected from 0.2 N KOH, NH4OH, and Ca(OH)2, 10-12 g dry invertase powder with preferred activity 200,000 Sumner Units/g was added with agitation and the aqueous alkaline solution was steamed to 60-80° C. for 2-24 hours. Next, 20 g citric acid was added with stirring and the solution was steamed at 60-80° C. for 3-12 hours. The lower the heating temperature, the longer the incubation; and this process was followed by cooling to room temperature. Suitable base, preferably selected from KOH, NH4OH, and Ca(OH)2 was added with agitation to adjust to pH 4-6. Volume was brought up to QID 100 ml to make 10% invertase deglycosylates. 1-6 ppm Cat stock solution was prepared and 1 ml 10% invertase deglycosylates was diluted in 1 L Cat stock solution for 100 ppm invertase deglycosylates liquid concentrate. 100 ppm BIT was added with agitation to complete invertase deglycosylates liquid concentrate.
Invertase deglycosylates liquid concentrate field dilutions: 10 ml invertase deglycosylates 100 ppm liquid concentrate was diluted in 1 L water to yield 1 ppm invertase deglycosylates, followed by serial dilutions to 1, 10, and 100 ppb, as needed. Foliar applications required addition of one or more agricultural surfactants, such as, 0.5 g/L Sil. Applications of low concentrations, in the range of between 10 ppb-5 ppm invertase deglycosylates were effective and showed significantly earlier germination and enhancement of the qualities and quantities of crops of photosynthetic organisms as compared to control. The regulation of flow of photosynthates by glycan composites comprised of invertase deglycosylates was further adapted by coapplication of the respiration decelerator, 1-100 ppm gibberellin, for flavor enhancement. Furthermore, under conditions of O2-starvation, supplementation of 1 L media with 10 g CaO2 maintained high qualities and quantities of yields.
Botanical gums were selected for branched chain high mannan contents that were partially hydrolyzed according to the following methods: Guar, konjac, locust bean, tara and ivory nut gums were separately dissolved in 60-80° water with agitation as 1% w/w gums. To each gum solution, 1-3 N nitric acid and 5-25% acetic anhydride were added. The solutions were maintained at 70-80° for 1-8 h. The solutions were cooled to 30-40° and titrated to pH 3.5-4 with Ca(OH)2, KOH and/or NH4OH. To the solutions, 30-50% citric acid was added to saturation and heated to 60-80° C. for 1-24 h. Solutions were allowed to cool to room temperature and titrated to pH 5-6 with Ca(OH)2, KOH, MnCO3 and/or NH4OH. 10× Cat was dissolved into the partially hydrolyzed gum solution to make 0.1% gum. Serial dilutions with Cat provided samples of various doses and were compared with the nutrient control solution.
Exemplary components for glycan composites comprised of partially hydrolyzed botanical gums are given in Table of Components, below.
Radish seeds, 30 per dish, were sown in 20 replicate Gosselin germination dishes on moistened Whatman 598 Seed Culture paper circles. Seeds were maintained at a constant temperature of 27° in the dark to maintain only respiration. Germination was established at the time at which radicle emergence was observed for 50% of the seeds, G50.
Results showed faster glycan composite mean G50 at 15 hours as compared to Nutrient Control mean G50 at 22 hours; n=10; p=0.001. Potencies of various partially hydrolyzed (PH) gums showed general correspondence of concentrations necessary for G50 activity to their contents of branched mannans. Potencies were observed from low activity in PH gums of guar, konjac, locust bean, tara, and to high activity by PH ivory nut, as detailed in the Table of Active Doses (ppm) of Partially Hydrolyzed (PH) Gums.
Treatments of radish seed with glycan composites from different sources of gums showed different levels efficacy and significantly hastened germination as compared to Nutrient Controls. Speedy germination was a result of endogenously enhanced flow of photosynthates. Preferred respiration accelerators included citric and malic acids. Furthermore, under conditions of O2-starvation, root supplementation of each 1 L media with 20 g CaO2 maintained root vigor for high qualities and quantities of yields.
Materials: Maxlnvert 200 invertase powder (DSM)
Citric Acid, anhydrous (Brandt)
A highly concentrated liquid solution of Invertase Glycan Composite was prepared by denaturing and deglycosylating active invertase with heating to 80° in saturated citrate solution. A saturated 50% citric acid was first prepared in water. To 500 ml saturated citric acid solution, 100 g Maxlnvert 200 invertase dry powder was slowly added into the agitated (300-800 rpm) acid solution to avoid clumping. After the invertase was dispensed, stirring was reduced to 100 rpm to minimize foaming. The solution was heated to 80° ; and maintained at 75-85° for hours with slow 100 rpm stirring that resulted in deglycosylates, a blend of Glycan components of the Glycan Composite, from this process that partially hydrolyzed invertase. After cooling to room temperature, the preservative, 100 ppm BIT was added with stirring and the total volume was brought to 500 ml with water to make a 20% Glycan solution.
The Metal+2 concentrate stock was prepared, containing the following: ultra low biuret urea; Ca; Mn; Fe; Zn; Mg; random block copolymer emulsifier, such as, Pluronic L-62; lower aliphatic alcohols, preferably, propanols; preservatives, such as, BIT; concentrations of each component per Table Invertase Glycan Composite Concentrate. A measure of 5 ml 20% Glycan was diluted per 10 L Metal+2 concentrate stock for 100 ppm Glycan Composite;
and titrated with citric acid, KOH, and/or NH4OH between pH 5-8. This 100 ppm Glycan Composite product was prepared for field dilutions to 0.01-1% in water for applications to photosynthetic organisms. For example, 10 ml Glycan Composite in 1 L water yielded 1 ppm Glycan; and similarly, 1 gallon/100 gallons per acre or 3 fluid ounce/1000 foot2. Serial dilutions also were applied, as needed. The field-diluted Glycan Composite from invertase deglycosylate was effective in a range of 0.01 to 10 ppm Glycan on roots; and/or with label rates of foliar surfactant additives, 0.1 to 10 ppm Glycan proved effective when applied to shoots.
Although specific features of the embodiments disclosed herein are described with respect to one example and not others, this is for convenience only as some feature of one described example may be combined with one or more of the other examples in accordance with the methods and formulations disclosed herein.
Other permutations of the methods and formulations of the embodiments disclosed herein will occur to those skilled in the art and are within the following claims:
This application is a divisional of U.S. patent application Ser. No. 15/492,110 filed Apr. 20, 2017, which claims priority of Provisional Application Ser. No. 62/329,226 filed Apr. 29, 2016, the disclosures of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62329226 | Apr 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15492110 | Apr 2017 | US |
| Child | 16666466 | US |
