Patent Application
20220400679
The present invention relates to a composition for enhancing plant growth. More particularly, the present invention relates to a composition including auxin, cytokinin, choline chloride, and γ-aminobutyric acid (GABA).
As world population reaches approximately 7.9 billion, food security has been a serious issue in many countries. According to the Food and Agricultural Organization of the United Nations (FAO), global food production will need to increase by 70% if the population reaches 9.1 billion by 2050.
Although there has been plant growth regulator (PGR) products for improving plant growth and enhancing yield of crops, stresses such as drought and herbicide drift affect performance of those PGR products. To help crops grow well under various conditions, advanced formulations for releasing the development potential of crops are in demand.
In one aspect, the present invention relates to a concentrate composition for enhancing plant growth. The concentrate composition comprises between about 0.8 g/L to about 80 g/L auxin, between about 0.18 g/L to about 18 g/L cytokinin, between about 0.5 g/L to about 50 g/L GABA, and between about 2.5 g/L to about 250 g/L choline chloride.
In another aspect, the present invention relates to a ready to use composition for enhancing plant growth. The ready to use composition comprises between about 0.8 mg/L to about 80 mg/L auxin, between about 0.18 mg/L to about 18 mg/L cytokinin, between about 0.5 mg/L to about 50 mg/L GABA, and between about 2.5 mg/L to about 250 mg/L choline chloride.
In another aspect, the present invention relates to a method for enhancing plant growth.
The present invention is illustrated but not limited by the following embodiments and drawings.
In some embodiments, the present invention provides a composition for enhancing plant growth. The composition comprises auxin, cytokinin, GABA, and choline chloride.
In some embodiments, the auxin is selected from indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), 2-phenylacetic acid (PAA), indole-3-propionic acid (IPA), and 1-naphthaleneacetic acid (NAA). In some embodiments, the auxin is IBA.
In some embodiments, the cytokinin is selected from N6-furfuryladenine (kinetin), 6-Benzylaminopurine (BA), zeatin (ZT), N6-(2-isopentenyl) adenine (2ip), diphenylurea (DPU). In some embodiments, the cytokinin is kinetin.
In some embodiments, the composition of the present invention is a concentrate composition, comprising between about 0.8 g/L to about 80 g/L auxin, between about 0.18 g/L, to about 18 g/L cytokinin, between about 0.5 g/L to about 50 g/L GABA, and between about 2.5 g/L to about 250 g/L choline chloride. A concentrate solution refers to a solution which is intended to be diluted with water to form a use solution prior to application to the plant.
In some embodiments, the concentration of auxin in the concentrate composition is between about 0.8 g/L to about 80 g/L, between about 1.6 g/L to about 40 g/L, between about 3 g/L to about 20 g/L, between about 6 g/L to about 10 g/L, and preferably is, but is not limited to, about 0.8 g/L, about 1.5 g/L, about 3 g/L, about 6 g/L, about 8 g/L, about 10 g/L, about 20 g/L, about 30 g/L, about 40 g/L, about 50 g/L, about 60 g/L, about 70 g/L, about 80 g/L, or any concentration between about 0.8 g/L to about 80 g/L, such as about 1.853 g/L, about 13.748 g/L, or about 62.739 g/L. In some embodiments, the concentration of auxin in the concentrate composition is about 0.8 g/L, about 8 g/L, or about 80 g/L.
In some embodiments, the concentration of cytokinin in the concentrate composition is between about 0.18 g/L to about 18 g/L, between about 0.5 g/L to about 9 g/L, between about 1 g/L to about 5 g/L, between about 1.5 g/L to about 3 g/L, and preferably is, but is not limited to, about 0.18 g/L, about 0.5 g/L, about 1 g/L, about 1.5 g/L, about 2 g/L, about 5 g/L, about 7.5 g/L, about 10 g/L, about 15 g/L, about 18 g/L, or any concentration between about 0.18 g/L to about 18 g/L, such as about 1.267 g/L, about 7.823 g/L, about 14.869 g/L. In some embodiments, the concentration of cytokinin in the concentrate composition is about 0.18 g/L, 1.8 g/L, or 18 g/L.
In some embodiments, the concentration of GABA in the concentrate composition is between about 0.5 g/L to about 50 g/L, between about 1 g/L to about 25 g/L, between about 2 g/L to about 12 g/L, between about 4 g/L to about 6 g/L, and preferably is, but is not limited to, about 0.5 g/L, about 1 g/L, about 5 g/L, about 10 g/L, about 15 g/L, about 20 g/L, about 25 g/L, about 30 g/L, about 35 g/L, about 40 g/L, about 45 g/L, about 50 g/L, or any concentration between about 0.5 g/L to about 50 g/L, such as about 1.097 g/L, about 16.062 g/L, about 28.502 g/L. In some embodiments, the concentration of GABA in the concentrate composition is about 0.5 g/L, 5 g/L, or 50 g/L.
In some embodiments, the concentration of choline chloride in the concentrate composition is between about 2.5 g/L to about 250 g/L, between about 5 g/L to about 125 g/L, between about 10 g/L to about 60 g/L, between about 20 g/L to about 30 g/L, and preferably is, but is not limited to, about 2.5 g/L, about 5 g/L, about 10 g/L, about 25 g/L, about 50 g/L, about 100 g/L, about 150 g/L, about 200 g/L, about 225 g/L, about 250 g/L, or any concentration between about 2.5 g/L to about 250 g/L, such as about 6.978 g/L, about 24.234 g/L, about 182.607 g/L. In some embodiments, the concentration of choline chloride in the concentrate composition is about 2.5 g/L, 25 g/L, or 250 g/L.
In some embodiments, the concentrate composition for enhancing plant growth is diluted around 500 to 1,500 folds with water before use.
In some embodiments, the composition of the present invention is a ready to use composition, comprising between about 0.8 mg/L to about 80 mg/L auxin, between about 0.18 mg/L to about 18 mg/L cytokinin, between about 0.5 mg/L to about 50 mg/L GABA, and between about 2.5 mg/L to about 250 mg/L choline chloride. A ready to use solution is not diluted with water prior to application to the plant. A ready to use solution is a use solution when it is applied to the plant without further dilution.
In some embodiments, the concentration of auxin in the ready to use composition is between about 0.8 mg/L to about 80 mg/L, between about 1.6 mg/L to about 40 mg/L, between about 3 mg/L to about 20 mg/L, between about 6 mg/L to about 10 mg/L, and preferably is, but is not limited to, about 0.8 mg/L, about 1.5 mg/L, about 3 mg/L, about 6 mg/L, about 8 mg/L, about 10 mg/L, about 20 mg/L, about 30 mg/L, about 40 mg/L, about 50 mg/L, about 60 mg/L, about 70 mg/L, about 80 mg/L, or any concentration between about 0.8 mg/L to about 80 mg/L, such as about 1.853 mg/L, about 13.748 mg/L, or about 62.739 mg/L. In some embodiments, the concentration of auxin in the ready to use composition is about 0.8 mg/L, about 8 mg/L, or about 80 mg/L.
In some embodiments, the concentration of cytokinin in the ready to use composition is between about 0.18 mg/L to about 18 mg/L, between about 0.5 mg/L to about 9 mg/L, between about 1 mg/L to about 5 mg/L, between about 1.5 mg/L to about 3 mg/L, and preferably is, but is not limited to, about 0.18 mg/L, about 0.5 mg/L, about 1 mg/L, about 1.5 mg/L, about 2 mg/L, about 5 mg/L, about 7.5 mg/L, about 10 mg/L, about 15 mg/L, about 18 mg/L, or any concentration between about 0.18 mg/L to about 18 mg/L, such as about 1.267 mg/L, about 7.823 mg/L, about 14.869 mg/L. In some embodiments, the concentration of cytokinin in the ready to use composition is about 0.18 mg/L, 1.8 mg/L, or 18 mg/L.
In some embodiments, the concentration of GABA in the ready to use composition is between about 0.5 mg/L to about 50 mg/L, between about 1 mg/L to about 25 mg/L, between about 2 mg/L to about 12 mg/L, between about 4 mg/L to about 6 mg/L, and preferably is, but is not limited to, about 0.5 mg/L, about 1 mg/L, about 5 mg/L, about 10 mg/L, about 15 mg/L, about 20 mg/L, about 25 mg/L, about 30 mg/L, about 35 mg/L, about 40 mg/L, about 45 mg/L, about 50 mg/L, or any concentration between about 0.5 mg/L to about 50 mg/L, such as about 1.097 mg/L, about 16.062 mg/L, about 28.502 mg/L. In some embodiments, the concentration of GABA in the ready to use composition is about 0.5 mg/L, 5 mg/L, or 50 mg/L.
In some embodiments, the concentration of choline chloride in the ready to use composition is between about 2.5 mg/L to about 250 mg/L, between about 5 mg/L to about 125 mg/L, between about 10 mg/L to about 60 mg/L, between about 20 mg/L to about 30 mg/L, and preferably is, but is not limited to, about 2.5 mg/L, about 5 mg/L, about 10 mg/L, about 25 mg/L, about 50 mg/L, about 100 mg/L, about 150 mg/L, about 200 mg/L, about 225 mg/L, about 250 mg/L, or any concentration between about 2.5 mg/L to about 250 mg/L, such as about 6.978 mg/L, about 24.234 mg/L, about 182.607 mg/L. In some embodiments, the concentration of choline chloride in the ready to use composition is about 2.5 mg/L, 25 mmg/L, or 250 mg/L.
In some embodiments, the composition for enhancing plant growth of the present invention may include one or more adjuvants, such as a surfactant or a drift control agent. In other embodiments, the composition for enhancing plant growth of the present invention may not include an adjuvant. For example, the composition for enhancing plant growth may include a surfactant and/or a drift control agent. Exemplary surfactants include, but are not limited to, cationic surfactants, anionic surfactants, zwitterionic surfactants, and nonionic surfactants, preferably including but not limited to, Tween® 20, Tween® 40, Tween® 60, Tween® 65, Tween® 80, Tween® 85, Laureth-4, Ceteth-2, Ceteth-20, Steareth-2, PEG40, PEG100, PEG150, PEG200, PEG600, Span® 20, Span® 40, Span® 60, Span® 65, Span® 80. An exemplary drift control agent includes LI 700®, which is commercially available from Loveland Products (Loveland, Colo., USA).
In some embodiments, the concentration of the adjuvant in the ready to use composition for enhancing plant growth is between about 0.01 to 1% (v/v), and preferably is, but is not limited to, about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1% (v/v). In some embodiments, the concentration of the adjuvant in the ready to use composition for enhancing plant growth is about 0.1% (v/v).
In some embodiments, the composition for enhancing plant growth of the present invention may be applied as part of a tank mix, which may include additional nutrients, such as micro or macro nutrients and/or pesticides such as fungicides, herbicides, or insecticides.
In one example, the composition is applied with at least one fungicide, such as, but not limited to, azoles, benzimidazoles, chloronitriles, dithiocarbamates, phenylamides, strobilurins, and triazoles.
In one example, the composition is applied with at least one herbicide, such as, but not limited to, 2,4-D, acetochlor, acifluorfen-Na, alachlor, aminopyralid, amitrole, atrazine, benfluralin, bensulfuron-methyl, bensulide, bentazon, bromacil, carfentrazone-ethyl, chlorimuron-ethyl, chlorsulfuron, clethodim, clopyralid, dicamba, diquat, diuron, EPTC, fluazifop-P-butyl, flumetsulam, flumioxazin, fomesafen, glufosinate-ammonium, glyphosate, halauxifen-methyl, hexazinone, imazamox, imazapyr, imazethapyr, isoxaflutole, lactofen, MCPA, MCPB, metribuzin, metsulfuron-methyl, MSMA, nicosulfuron, norflurazon, oxadiazon, oxyfluorfen, paraquat, pendimethalin, penoxsulam, picloram, prometryn, propanil, prosulfuron, quinclorac, quinmerac, rimsulfuron, s-metolachlor, saflufenacil, sethoxydim, sulfentrazone, sulfosulfuron, thifensulfuron-methyl, trifluralin, and triflusulfuron-methyl. In one specific example, the composition is applied with glyphosate.
Suitable concentration ranges for the concentrate composition of the present invention are provided in Table 1, and suitable concentration ranges for the ready to use composition of the present invention are provided in Table 2. In some embodiments, the concentrate composition and the ready to use composition can consist of or consist essentially of the components listed in Table 1 and 2, respectively.
In some embodiments, the present invention provides a method for enhancing plant growth, comprising a step of applying a use solution composition to a plant, and the use solution composition comprising between about 0.8 mg/L to about 80 mg/L auxin, between about 0.18 mg/L to about 18 mg/L cytokinin, between about 0.5 mg/L to about 50 mg/L GABA, and between about 2.5 mg/L to about 250 mg/L choline chloride.
In some embodiments, the composition for enhancing plant growth of the present invention is applied to a plant during the vegetative phase. In some embodiments, the composition for enhancing plant growth of the present invention is applied to a plant during the reproductive phase.
The composition of the present invention can be applied to different plants, such as, but not limited to, asparagus, berry (such as blackberry, blueberry, caneberrry, kiwi, and raspberry), brassica vegetables (such as broccoli, cabbage, cauliflower, and mustard greens), bulb vegetable (such as garlic, leek, and onion), cereal grains (such as barley, corn, millet, oats, rice, sorghum, and wheat), citrus fruit (such as grapefruit, lemon, lime, sweet orange, and tangerine), coffee, cotton, cucurbit vegetables (such as cantaloupe, cucumber, honeydew, muskmelon, squash, and watermelon), forage, fodder, and straw of cereal grains, fruiting vegetables (such as eggplant, pepper, and tomato), grass forage, fodder, and hay, grass grown for seed (such as perennial ryegrass, tall fescue, or bentgrass), grape, herbs and spices (such as basil, dill, mustard, and sage), hemp, hops, leafy vegetable (such as celery, head and leaf lettuce, kale, and spinach), legume vegetables (such as bean, peas, and soybeans), mint, peppermint, spearmint, nongrass animal feeds (such as alfalfa, clover, hay, and vetch), oil seed crops (such as canola, flax, and sunflower), peanut, pome fruits (such as apple and pear), root and tuber vegetables (such as carrot, ginseng, horseradish, parsley, potato, radish, sugar beet, sweet potato, and turnip), stone fruits (such as apricot, cherry, peach, and plumcot), strawberry, sugarcane, tobacco, tree nuts (such as almonds, cashews, and pecans). In another example, the composition for enhancing plant growth is applied to corn, soybeans, wheat and cotton.
In some embodiments, the composition for enhancing plant growth of the present invention is applied to plant foliage (for example, leaves, stems, flowers and/or fruits), for example as a foliar application or foliar spray. In some embodiments, the composition for enhancing plant growth of the present invention is applied to plant roots, such as by a soil application or soil drench, and/or to seeds, such as by a seed treatment.
In some embodiments, the composition of the present invention enhances plant growth by at least one of the methods selected from increasing shoot growth, increasing root growth, increasing photosynthesis in plants, increasing nutrient uptake and assimilation, increasing water management by plants, and reducing herbicide mediated phototoxicity in plants.
In some embodiments, the composition of the present invention increases shoot growth by at least one of the methods selected from increasing shoot fresh weight, increasing shoot dry weight, and increasing leaf area.
In some embodiments, the composition of the present invention increases root growth by at least one of the methods selected from increasing root length, increasing root dry weight, and up-regulating expression of genes involved in root growth and elongation.
In some embodiments, the composition of the present invention increases photosynthesis in plants by at least one of the methods selected from increasing electron transport rate (ETR) of plants and up-regulating expression of genes related to increasing growth and/or photosynthesis.
In some embodiments, the composition of the present invention increases nutrient uptake and assimilation by at least one of the methods selected from increasing activity of nitrate reductase (NR) in the plants, increasing activity of glutamine synthetase (GS) in the plants, increasing activity of glutamate synthase (GOGAT) in the plants, and increasing root growth.
In some embodiments, the composition of the present invention increases water management by plant via at least one of the methods selected from up-regulating expression of genes related to improving water use efficiency and increasing root growth.
In some embodiments, the composition of the present invention reduces herbicide mediated phototoxicity in plants by at least one of the methods selected from reducing electrolyte leakage in the plants, reducing 2-thiobarbituric acid reactive substances (TBARS) level in the plants, increasing activity of nitrate reductase (NR) in the plants, increasing activity of glutamine synthetase (GS) in the plants, increasing activity of glutamate synthase (GOGAT) in the plants, increasing auxin level in the plants, increasing gibberellin level in the plants, increasing cytokinin level in the plants, reducing abscisic acid level in the plants, and improving plant growth, for example as measured by shoot dry weight and leaf area.
It has been found that when auxin, cytokinin, GABA, and choline chloride are combined in the composition of the present invention, the plant growth regulating actions of the respective components are increased synergistically, and the combination of the components exhibits a marked synergistic effect not seen when the components are used individually.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control.
As used herein, the term “auxin” refers to a class of plant growth regulators that promote stem elongation, inhibit growth of lateral buds, and therefore maintain apical dominance. Naturally occurring (endogenous) auxins are produced by apical meristem, such as stem tips and root tips. Auxin moves to the darker side of the plant, causing the cells there to grow longer than corresponding cells on the lighter side of the plant, and this produces a curving of the plant stem tip toward the light. Examples of auxin include, but are not limited to, indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), 2-phenylacetic acid (PAA), indole-3-propionic acid (IPA), 1-naphthaleneacetic acid (NAA).
As used herein, the term “cytokinin” refers to a class of plant growth regulators that enhance cell division, cell differentiation, and axillary bud growth, and inhibit apical dominance. There are two types of cytokinins based on their chemical structures: adenine-type and phenylurea-type cytokinin. Most cytokinins are synthesized in root tip and transported to photosynthetic tissues through xylem. Although roots are the major site of cytokinin biosynthesis, they are not the only site. Cambium and possibly all actively dividing tissues, such as embryo, leaves, fruits, are responsible for the synthesis of cytokinin. Examples of cytokinin include, but are not limited to, N6-furfuryladenine (kinetin), 6-Benzylaminopurine (BA), zeatin (ZT), N6-(2-isopentenyl) adenine (2ip), and diphenylurea (DPU).
As used herein, the term “γ-aminobutyric acid (GABA),” also known as 4-aminobutanoic acid, refers to a non-protein amino acid having the formula of C4H9NO2 and the following chemical structure:
As used herein, the term “choline chloride” refers to an organic compound having the formula of ((CH3)3N(Cl)CH2CH2OH) and the following chemical structure:
As used herein, the term “glyphosate” refers to the active ingredient in Roundup herbicide and has the chemical formula of C3H8NO5P. Glyphosate inhibits 5-enolpyruvylshikimate-3-phosphate Synthase (EPSPS) in plants and disrupts synthesis of aromatic amino acids, such as phenylalanine, tyrosine, and tryptophan. Tryptophan is a precursor of indole-3-acetic acid (IAA), which is the main auxin in plants and plays an important role in regulating plant development via promoting cell division and elongation. When applied to plants, Glyphosate causes inhibition of protein synthesis and a decrease in IAA level in the plants, and leads to plant growth inhibition and death.
As used herein, the term “electrolyte leakage” refers to the phenomenon of efflux of electrolytes, such as potassium ion (K+), from a plant cell when the cell dies and loses the integrity of the cell membrane. Electrolyte leakage is an indicator of cell membrane stability of plants under stress conditions and a hallmark of stress response in plant cells. The greater electrolyte leakage, the less cell membrane integrity.
As used herein, the term “2-thiobarbituric acid reactive substances (TBARS)” refers to substances that are formed as a byproduct of lipid peroxidation and can be detected by the TBARS assay using thiobarbituric acid (TBA) as a reagent. Malondialdehyde (MDA) is one of several end products formed through the decomposition of lipid peroxidation products, and fatty peroxide-derived decomposition products other than MDA are TBA positive. TBARS is an indicator of lipid peroxidation of cell membrane. Higher TBARS levels indicate lower cell membrane integrity.
As used herein, the term “nitrogen assimilation” refers to the formation of organic nitrogen compounds, such as amino acids and proteins, from inorganic nitrogen compounds present in the environment. In nitrogen assimilation in plants, nitrate (NO3−) and nitrite (NO2) are first reduced to ammonium (NH4+) by nitrate reductase (NR) and nitrite reductase (NiR), respectively, and then ammonium (NH4+) is incorporated into amino acid via the glutamine synthetase (GS)-glutamate synthase (GOGAT) pathway. Therefore, increasing activities of enzymes involved in nitrogen assimilation, such as nitrate reductase (NR), glutamine synthetase (GS), and glutamate synthase (GOGAT), in plant cells indicate that the plant synthesizes more amino acids and proteins.
As used herein, the term “electron transport rate (ETR)” refers to transport rate of electrons released by water splitting during photosynthesis. Since energy is generated during electron transportation, the faster the electron transport rate is, the more energy (ATP) is generated, which helps plants synthesize more sugar from CO2.
As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains,” “containing,” “characterized by” or any other variation thereof, are intended to cover a non-exclusive inclusion, subject to any limitation explicitly indicated. For example, a composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus.
The transitional phrase “consisting of” excludes any element, step, or ingredient not specified. If in the claim, such would close the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith. When the phrase “consisting of” appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.
The transitional phrase “consisting essentially of” is used to define a composition, method or apparatus that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting essentially of” occupies a middle ground between “comprising” and “consisting of”.
Where applicants have defined an invention or a portion thereof with an open-ended term such as “comprising,” it should be readily understood that (unless otherwise stated) the description should be interpreted to also describe such an invention using the terms “consisting essentially of” or “consisting of.”
Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).
As used herein, “around”, “about” or “approximately” shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range. Numerical quantities given herein are approximate, meaning that the term “around”, “about” or “approximately” can be inferred if not expressly stated.
The term “a,” “an,” or “the” disclosed in the present invention is intended to cover one or more numerical values in the specification and claims unless otherwise specified. For example, “an element” indicates one or more than one element.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention.
1. Plant Growth and Treatment
Glyphosate-tolerant corn seeds (D40SS48, Dyna-Gro®) were seeded in pots containing culture medium (peat soil:vermiculite=3:1) and placed in a phytotron, which was operated at 25/23° C. day/night temperature and on a 16/8 light/dark cycle. One seed was seeded in one pot. Corn plants were applied with the reagents listed in Table 3 at the stage of V1 (first leaf collar) once at a rate of 15 mL/12 pots using a foliar spray treatment. There were 11 groups in this test, 1 Control group, 1 Glyphosate only group, and 9 Test groups.
Glyphosate-tolerant soybean seeds (S29RY05, Dyna-Gro®) were seeded in pots containing culture medium (peat soil:vermiculite=3:1) and placed in a phytotron, which was operated at 25/23° C. day/night temperature and on a 16/8 light/dark cycle. One seed was seeded in one pot. Soybean plants were applied with the reagents listed in Table 3 at the stage of V1 (one set of unfolded trifoliolate leaves) once at a rate of 10 mL/12 pots using a foliar spray treatment. There were 11 groups in this test, 1 Control group, 1 Glyphosate only group, and 9 Test groups.
2. Analyses
2.1 Shoot dry weight: Seven (7) days after the application of reagents, leaves and stems of each plant were dried at 50° C. overnight, and then the dry weight was measured.
2.2 Statistics: Unpaired Student's t-test was applied to assess numerical data statistical significance. Statistical significance was set at p-value less than 0.05.
3. Results
A. Corn
As shown in
B. Soybean
As shown in
1. Plant Growth and Treatment
Glyphosate-tolerant corn seeds (D40SS48, Dyna-Gro®) were seeded in pots containing culture medium (peat soil:vermiculite=3:1) and placed in a phytotron, which was operated at 25/23° C. day/night temperature and on a 16/8 light/dark cycle. One seed was seeded in one pot. Corn plants were applied with the reagents listed in Table 4 at the stage of V1 (first leaf collar) once at a rate of 15 mL/12 pots using a foliar spray treatment. There were 11 groups in this test, 1 Control group, 1 Glyphosate only group, and 9 Test groups.
Glyphosate-tolerant soybean seeds (S29RY05, Dyna-Gro®) were seeded in pots containing culture medium (peat soil:vermiculite=3:1) and placed in a phytotron, which was operated at 25/23° C. day/night temperature and on a 16/8 light/dark cycle. One seed was seeded in one pot. Soybean plants were applied with the reagents listed in Table 4 at the stage of V1 (one set of unfolded trifoliolate leaves) once at a rate of 10 mL/12 pots using a foliar spray treatment. There were 11 groups in this test, 1 Control group, 1 Glyphosate only group, and 9 Test groups.
2. Analyses
Methods for analyzing shoot dry weight and statistics are the same as described in Example 1.
3. Results
A. Corn
As shown in
B. Soybean
As shown in
1. Plant Growth and Treatment
Glyphosate-tolerant corn seeds (D40SS48, Dyna-Gro®) were seeded in pots containing culture medium (peat soil:vermiculite=3:1) and placed in a phytotron, which was operated at 25/23° C. day/night temperature and on a 16/8 light/dark cycle. One seed was seeded in one pot. Corn plants were applied with the reagents listed in Table 5 at the stage of V1 (first leaf collar) once at a rate of 15 mL/12 pots using a foliar spray treatment. There were 4 groups in this test, 1 Control group, 1 Glyphosate only group, and 2 Test groups.
Glyphosate-tolerant soybean seeds (S29RY05, Dyna-Gro®) were seeded in pots containing culture medium (peat soil:vermiculite=3:1) and placed in a phytotron, which was operated at 25/23° C. day/night temperature and on a 16/8 light/dark cycle. One seed was seeded in one pot. Soybean plants were applied with the reagents listed in Table 5 at the stage of V1 (one set of unfolded trifoliolate leaves) once at a rate of 10 mL/12 pots using a foliar spray treatment. There were 4 groups in this test, 1 Control group, 1 Glyphosate only group, and 2 Test groups.
2. Analyses
2.1 Analyses of Leaves: Seven (7) days after the application of reagents, plant height was measured, and leaves were collected. The leaf area of the fourth leaf of each corn plant and the total leaf area of each soybean plant were measured by a leaf analyzer (WinFOLIA™ Regent Instruments Inc., Québec, Canada). Leaves and stems of each plant were dried at 50° C. overnight, and then the dry weight was measured.
2.2 Analyses of Roots: Seven (7) days after the application of reagents, length of plant root was measured by a root analyzer (WinRHIZO™, Regent Instruments Inc., Québec, Canada). In addition, roots of each plant were dried at 50° C. overnight, and then the dry weight was measured.
2.3 Electrolyte Leakage: Three (3) days after the application of reagents, sample leaves were cleaned with deionized water three times. Six (6) leaf disks (7.5 mm diameter) were cut from the sample leaves and immersed in 10 ml of deionized water at 25° C. for 24 hours. After that, the first electrolytic conductivity (EC1) of the sample solution was analyzed with an electrical conductivity meter (SevenCompact™ 5230 Conductivity Meter, Mettler-Toledo, Columbus, Ohio, USA). The sample leaf disks were then kept in the deionized water at 95° C. for 2 hours. After the temperature of the sample solution decreased to 25° C., the second electrolytic conductivity (EC2) of the sample solution was analyzed with the electrical conductivity meter again. Ten (10) milliliters of deionized water without any sample were used as control. Electrolyte leakage of the sample leaves was analyzed with the following equation.
Electrolyte leakage (%)=(Sample EC1−Control EC1)×100/(Sample EC2−Control EC2)
2.4 Content of TBARS: 2-thiobarbituric acid reactive substances (TBARS) are a byproduct and an indication of lipid peroxidation. Three (3) days after the application of reagents, 30 grams of fresh sample leaf were ground in liquid nitrogen and mixed with 1 ml of 20% (v/v) Trichloroacetic acid (TCA). The mixture was centrifuged at 10,000×g for 5 minutes. Two-hundred (200) microliters of the supernatant were then mixed with 800 μl of TBARS solution [20% (v/v) TCA containing 0.5% (w/v) 2-thiobarbituric acid], incubated at 95° C. in a water bath for 30 minutes, cooled on ice, and then centrifuged at 2,000×g for 20 minutes. Absorbance at an optical density (O.D.) of 532 nm (A532) and 600 nm (A600) was measured by a microplate reader (Infinite® M200 pro, TECAN, Männedorf, Switzerland). Concentration of TBARS in a sample was calculated with the following equation.
TBARS(nmol/gFW)=(A532−A600)×N×1.8×1000/155×W
N: dilution factor
W: Fresh weight of a sample (g)
2.5 Activity of Nitrate Reductase (NR): Three (3) days after the application of reagents, 0.5 grams of fresh sample leaf were ground in liquid nitrogen. One (1) ml of potassium phosphate buffer (100 mM, pH7.4) containing 7.5 mM cysteine, 1 mM EDTA, and 1.5% casein was added to the sample, mixed with the sample, and then the mixed sample was centrifuged at 4° C., 13,000 relative centrifugal field (rcf) for 30 minutes. Zero-point one (0.1) ml of the supernatant and 0.9 ml of reaction solution were incubated at 30° C. in a water bath for 30 minutes, and the reaction was stopped by adding 0.05 mL zinc acetate (1 M). The sample was centrifuged at room temperature, 3,000 rcf. The supernatant was transferred to a new tube and mixed with 0.5 mL sulphanilamide (5.8 mM) and 0.5 mL N-(1-naphthyl)ethylenediamine (0.8 mM). The mixture was incubated for 30 minutes. The NR activity is determined by NO2− production through measuring absorbance at an optical density (O.D.) of 540 nm (A540). KNO2 is used as a NO2− concentration standard.
2.6 Activity of Glutamine Synthetase (GS): Three (3) days after the application of reagents, 0.5 grams of fresh sample leaf was ground in liquid nitrogen. The leaf powder and 1 ml of extraction solution, containing 10 mM Tris-HCl (pH7.6), 1 mM MgCl2, 1 mM EDTA, and 10 mM 2-Mercaptoethanol, were centrifuged at 4° C., 13,000 rcf for 30 minutes. Zero-point one (0.1) ml of the supernatant and 0.4 ml of reaction solution were incubated at 30° C. in a water bath for 30 minutes, and the reaction was stop by adding 1 mL of stop solution, containing 2.5 g FeCl3, 5.0 g TCA in 100 mL HCl (1.5 N). The sample was centrifuged at room temperature, 3,000 rcf. Absorbance at an O.D. of 540 nm (A540) of the supernatant was measured.
2.7 Activity of Glutamate Synthase (GOGAT): Three (3) days after the application of reagents, 0.5 gram of fresh sample leaf was ground in liquid nitrogen. The leaf powder and 1 ml of extraction solution, containing 10 mM Tris-HCl (pH7.6), 1 mM MgCl2, 1 mM EDTA, and 10 mM 2-Mercaptoethanol, were centrifuged at 4° C., 13,000 rcf for 30 minutes. Zero-point-one-five (0.15) ml of the supernatant was mixed with the reaction solution containing 0.2 mL of 20 mM L-glutamine, 0.25 mL of 2 mM 2-oxoglutarate, 0.05 mL of 10 mM KCl, 1 mL of 25 mM Tris-HCl (pH7.6), and 0.1 mL 3 mM NADH to initiate the reaction. The GOGAT activity is determined by NADH reduction through measuring fluorescence intensity at excitation/emission wavelengths of 340/445 nm. NADH concentration is determined by reference to a NADH standard curve.
2.8 Content of Plant Hormones: Twelve (12) days after the application of reagents, 1 g of fresh sample leaf was ground in liquid nitrogen and then mixed with 10 g extraction solution (80% (v/v) methanol, 1% (v/v) acetic acid) by a vortex mixer. The sample was then sonicated for 1 hour and centrifuged at 2,500 rpm for 10 minutes. The supernatant was collected, concentrated, dissolved in 1 ml of 100% methanol, and then analyzed for content of plant hormones with a tandem quadrupole mass spectrometer (Waters® Xevo® TQ MS, Waters Corporation, Milford, Mass., USA) and ACQUITY UPLC System (Waters Corporation, Milford, Mass., USA).
2.9 Statistics: Methods of statistics are the same as described in Example 1.
3. Results
A. Corn
A-3.1A composition of the present invention increases crop growth after glyphosate treatment.
As shown in
A-3.2 A composition of the present invention alleviates plant cell membrane damage after Glyphosate treatment.
As shown in
As shown in
A-3.3 A composition of the present invention improves activities of key enzymes in nitrogen assimilation in plants after Glyphosate treatment.
As shown in
A-3.4 A composition of the present invention increases growth hormone levels and reduces a stress hormone level in plants after glyphosate treatment.
As shown in Table 7, corn plants treated with Radiate® without glyphosate and corn plants treated with Sample 1 (8 g/mL IBA, 1.8 mg/L kinetin, 5 mg/L GABA, 25 mg/L choline chloride and 0.1% v/v Tween®80) (a composition of the present invention) without glyphosate both have higher content of indole-3-acetic acid (IAA), gibberellin A1 (GA1), and cytokinin (Zeatin+iPAs) and lower content of abscisic acid (ABA) than Control group. In addition, corn plants treated with Sample 1 without glyphosate have higher content of gibberellin A1 (GA1) and cytokinin (Zeatin+iPAs) and lower content of abscisic acid (ABA) than corn plants treated with Radiate® without glyphosate. The results show that a composition of the present invention (Sample 1) increases growth hormone (auxin, gibberellin, and cytokinin) levels and reduces a stress hormone (abscisic acid) level in plants without glyphosate treatment.
Furthermore, as shown in Table 7, corn plants treated with glyphosate and Radiate® (Test group 1) and corn plants treated with glyphosate and a composition of the present invention (Test group 2) both have higher content of IAA, GA1, and cytokinin (Zeatin+iPAs) and lower content of ABA than corn plants treated with glyphosate only. In addition, plants in Test group 2 have higher content of IAA and cytokinin (Zeatin+iPAs) and lower content of ABA than plants in Test group 1. The results show that a composition of the present invention increases growth hormone (auxin, gibberellin, and cytokinin) levels and reduces a stress hormone (abscisic acid) level in plants with glyphosate treatment.
B. Soybean
B-3.1 A composition of the present invention alleviates soybean growth inhibition after glyphosate treatment.
As shown in
B-3.2 A composition of the present invention alleviates plant cell membrane damage after glyphosate treatment.
As shown in
As shown in
B-3.3 A composition of the present invention improves activities of key enzymes in nitrogen assimilation in plants after Glyphosate treatment.
As shown in
B-3.4 A composition of the present invention increases growth hormone levels and reduces a stress hormone level in plants after glyphosate treatment.
As shown in Table 9, soybean plants treated with Radiate® without glyphosate and soybean plants treated with Sample 1 (8 g/mL IBA, 1.8 mg/L kinetin, 5 mg/L GABA, 25 mg/L choline chloride and 0.1% v/v Tween®80) (a composition of the present invention) without glyphosate both have higher content of IAA, GA1, and cytokinin (Zeatin+iPAs) and lower content of ABA than Control group. In addition, soybean plants treated with Sample 1 without glyphosate have higher content of IAA and cytokinin (Zeatin+iPAs) and lower content of ABA than soybean plants treated with Radiate® without glyphosate. The results show that Sample 1 increases growth hormone (auxin, gibberellin, and cytokinin) levels and reduces a stress hormone (abscisic acid) level in plants without glyphosate treatment.
Furthermore, as shown in Table 9, soybean plants treated with glyphosate and Test group 1 and soybean plants treated with glyphosate and a composition of the present invention (Test group 2) both have higher content of IAA, GA1, and cytokinin (Zeatin+iPAs) and lower content of ABA than soybean plants treated with glyphosate only. In addition, plants in Test group 2 have higher content of IAA, GA1, and cytokinin (Zeatin+iPAs) and lower content of ABA than plants in Test group 1. The results show that a composition of the present invention increases growth hormone (auxin, gibberellin, and cytokinin) levels and reduces a stress hormone (abscisic acid) level in plants with glyphosate treatment.
1. Plant Growth and Treatment
Corn seeds (White Pearl, Known-You Seed Co., Ltd., Kaohsiung, Taiwan) were seeded in pots containing culture medium (peat soil:vermiculite=3:1) and placed in a phytotron, which was operated at 25/23° C. day/night temperature and on a 16/8 light/dark cycle. One seed was seeded in one pot. Corn plants were applied with the reagents listed in Table 10 at the stage of V1 (first leaf collar) once at a rate of 10 mL/12 pots using a foliar spray treatment. There were 6 groups in this test, 1 Control group and 5 Test groups.
Soybean seeds (Kaohsiung 10, Kaohsiung District Agricultural Research and Extension Station, Kaohsiung, Taiwan) were seeded in pots containing culture medium (peat soil:vermiculite=3:1) and placed in a phytotron, which was operated at 25/23° C. day/night temperature and on a 16/8 light/dark cycle. One seed was seeded in one pot. Soybean plants were applied with the reagents listed in Table 10 at the stage of V1 (one set of unfolded trifoliolate leaves) once at a rate of 10 mL/12 pots using a foliar spray treatment. There were 6 groups in this test, 1 Control group and 5 Test groups.
2. Analyses
Methods for analyzing shoot dry weight and statistics are the same as described in Example 1.
3. Results
A. Corn
As shown in
B. Soybean
As shown in
Corn seeds (White Pearl, Known-You Seed Co., Ltd., Kaohsiung, Taiwan) were seeded in pots containing culture medium (peat soil:vermiculite=3:1) and placed in a phytotron, which was operated at 25/23° C. day/night temperature and on a 16/8 light/dark cycle. One seed was seeded in one pot. Corn plants were applied with the reagents listed in Table 11 at the stage of V1 (first leaf collar) once at a rate of 10 mL/12 pots using a foliar spray treatment. There were 3 groups in this test, 1 Control group and 2 Test groups.
Soybean seeds (Kaohsiung 10, Kaohsiung District Agricultural Research and Extension Station, Kaohsiung, Taiwan) were seeded in pots containing culture medium (peat soil:vermiculite=3:1) and placed in a phytotron, which was operated at 25/23° C. day/night temperature and on a 16/8 light/dark cycle. One seed was seeded in one pot. Soybean plants were applied with the reagents listed in Table 11 at the stage of V1 (one set of unfolded trifoliolate leaves) once at a rate of 10 mL/12 pots using a foliar spray treatment. There were 3 groups in this test, 1 Control group and 2 Test groups.
2. Analyses
Methods for analyzing leaves, roots, activities of NR, GS, and GOGAT, and statistics are the same as described in Example 3.
2.1 Electron Transport Rate (ETR): Four (4) days after the application of reagents, leaves were analyzed with photosynthesis system (LI-6400XTQ, LI-COR Biosciences, Lincoln, Nebr., USA) under lights (1500 mol photons m−2s−1).
2.2 Gene Expression Analysis: One (1) or 3 days after the application of reagents, all trifoliolate leaves were collected for RNA extraction with LabPrep™ RNA plus mini kit (LabTurbo Biotech Co., Taipei, Taiwan). Reverse transcription polymerase chain reaction (RT-PCR) was performed with the extracted RNA using iScript™ gDNA Clear cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, Calif., USA). Then, real-time PCR was performed using iQ™ SYBR® (Bio-Rad Laboratories) in CFX Connect™ Real-Time PCR
Detection System (Bio-Rad Laboratories) with the primers listed in Table 12.
3. Results
A. Corn
A-3.1 A composition of the present invention increases crop growth.
As shown in
A-3.2 A composition of the present invention improves the efficiency of plants in converting solar energy into chemical energy.
As shown in
A-3.3 A composition of the present invention improves activities of key enzymes in nitrogen assimilation in plants.
As shown in
A-3.4 A composition of the present invention induces more expression of genes related to growth, photosynthesis, and water use efficiency.
As shown in
B. Soybean
B-3.1 A composition of the present invention increases crop growth.
As shown in
B-3.2 A composition of the present invention improves the efficiency of plants in converting solar energy into chemical energy.
As shown in
B-3.3 A composition of the present invention improves activities of key enzymes in nitrogen assimilation in plants.
As shown in
GOGAT of soybean plants than Test group 1.
B-3.4 A composition of the present invention induces more expression of genes related to growth, photosynthesis, and water use efficiency.
As shown in
To sum up, the examples demonstrate that a composition of the present invention enhances plant growth by increasing root growth, increasing photosynthesis, increasing nutrient uptake and assimilation, and increasing water management by crops.
1. Plant Growth and Treatment
Soybean seeds (Roundup Ready 2 Xtend®) were seeded in pots containing culture medium (peat soil:vermiculite=3:1) and placed in a phytotron, which was operated at 25/23° C. day/night temperature and on a 16/8 light/dark cycle. One seed was seeded in one pot. Soybean plants were applied with 0.1% (v/v) Tween® 80 (Control group) or Sample 1 (8 mg/L IBA, 1.8 mg/L Kinetin, 5 mg/L GABA, and 25 mg/L choline chloride) and 0.1% (v/v) Tween® 80 (Test group) at the stage of V1 (one set of unfolded trifoliolate leaves) once at a rate of 10 mL/12 pots using a foliar spray treatment.
2. Analyses
2.1 RNA-seq profiling: Three (3) replicates were performed. Six (6) days after the application of reagents, all trifoloalate leaves were collected for RNA extraction by LabPrep™ RNA plus mini kit (LabTurbo Biotech Co.). Genomic DNA was removed by TURBO DNA-Free™ Kit (Thermo Fisher Scientific, Waltham, Mass., USA). The RNA-seq libraries were prepared using the TruSeq RNA Library Prep Kit v2 (Illumina, Inc., San Diego, Calif., USA) for sequencing on HiSeq 2500 platform (Illumina, Inc.). RNA-Seq reads were assembled into contigs, and then mapped to the Arabidopsis genome and cDNA sequences. To identify differentially expressed genes, RNA-seq reads with the fold change more than 2 were selected.
2.2 Statistics: Methods of statistics are the same as described in Example 1.
3. Results
As shown in Table 13 and
Many changes and modifications in the above described embodiment of the invention can, of course, be carried out without departing from the scope thereof. Accordingly, to promote the progress in science and the useful arts, the invention is disclosed and is intended to be limited only by the scope of the appended claims.
This application claims priority to U.S. Provisional Application 63/209,581, filed on Jun. 11, 2021, which is herein incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63209581 | Jun 2021 | US |
