Process for increasing enzyme concentration and dry matter in hydroponically grown cellulosic materials using reactive oxygen species

Abstract

An external reactive oxygen species application system and method for increasing enzyme activity and plant dry matter through the use of reactive oxygen species in hydroponically grown cellulosic material. The external reactive oxygen species application system disinfects a plurality of seeds with reactive oxygen species at a first concentration using a liquid applicator during the seedling stage. The reactive oxygen species increases gibberellic acid expression. The liquid applicator applies the reactive oxygen species at a second concentration during the plant development stage to increase plant dry matter.

Description

[Technical field]

[0001] The present disclosure relates to the use of reactive oxygen species to increase enzyme activity and dry matter in hydroponically germinated seeds.Reactive oxygen species such as hydrogen peroxide have been used to increase seedling germination percentage.More specifically, but not exclusively, the present invention relates to a process and composition for increasing enzyme concentration in hydroponically grown cellulosic materials using reactive oxygen species. [Background technology]

[0002] Germination and seedling development in angiosperms are generally controlled by two antagonistic plant hormones, gibberellin and abscisic acid. Gibberellin release supports dormancy interruption, cell division, and hydrolytic enzyme release, while abscisic acid release maintains seed dormancy, inhibits hydrolytic enzyme release, and reduces cell division. The oxidative mode of action of gibberellin is mimicked in vivo when reactive oxygen species are introduced; supporting the release of hydrolytic enzymes from the aleurone layer and other plant cell tissues. In addition, in contrast to lignification reactions in the absence of reactive oxygen species, ligninolytic enzymes such as peroxidases favor the delignification pathway in the presence of reactive oxygen species. Since lignin is generally considered to be the most complex and indigestible fiber complex in higher plants, delignification promoted by ligninolytic enzyme hydrolysis supports improved fiber digestibility. Therefore, application of reactive oxygen species in hydroponic environments to increase dry matter and enzyme activity is required. Summary of the Invention

[0003] Thus, a primary object, feature, or advantage of this disclosure is to improve over the state of the art. A further object, feature, or advantage of the present disclosure is to increase the dry matter of hydroponically grown plants. Yet a further object, feature, or advantage of the present disclosure is to increase enzyme activity in hydroponically grown plants.

[0004] Another object, feature, or advantage is to reduce abscisic acid activity by exogenously introducing reactive oxygen species (ROS) to a plant or seed. Another object, feature, or advantage is to increase gibberellin activity by exogenously introducing ROS to a plant or seed.

[0005] One or more of these and / or other objects, features, or advantages of the present disclosure will become apparent from the following specification and claims. It is not necessary for a single embodiment to provide each and every object, feature, or advantage. Different embodiments may have different objects, features, or advantages. Thus, the present disclosure should not be limited to or by any object, feature, or advantage described herein.

[0006] In one aspect of the present disclosure, a method for external application of ROS to increase dry matter and enzyme activity is disclosed. The method can include disinfecting a plurality of seeds with at least one ROS, the ROS being configured to kill at least one of harmful bacteria, mold, or fungi. The method can also include externally applying at least one ROS to a plurality of seeds on a cultivation surface during the germination stage at a first concentration, the at least one ROS increasing gibberellic acid expression. The method can also include germinating a plurality of seeds and externally applying at least one ROS to the plurality of germinated seeds over a plant development stage until the germinated seeds reach maturity at a second concentration.

[0007] In another aspect of the present disclosure, an external ROS dispersal system is disclosed. The system can include a cultivation surface operably supported by a frame structure and disposed across the length and width of the frame structure. The cultivation surface can be configured to house a plurality of seeds. The system can also include a ROS source operably connected to the frame structure. The ROS source can house at least one type of ROS. The system can further include one or more liquid applicators operably secured to the frame structure adjacent to the cultivation surface for releasing at least one type of ROS from the ROS source to the plurality of seeds housed on the cultivation surface. The one or more liquid applicators can release at least one type of reactive oxygen species at different concentrations. The at least one type of reactive oxygen species promotes germination of the plurality of seeds on the cultivation surface.

[0008] In another aspect of the present disclosure, a method for increasing the dry matter of a plurality of plants by external spraying of ROS is disclosed. The method can include placing a plurality of seeds on a cultivation surface, and the cultivation surface can include a top surface for hydroponically cultivating the plurality of seeds on the cultivation surface. The method can further include introducing at least one ROS onto the cultivation surface at a first concentration for a first time by a liquid sprayer. The method can further include disinfecting the plurality of seeds with a first ROS of the plurality of reactive oxygen species introduced by the liquid sprayer, and germinating the plurality of seeds on the cultivation surface. Finally, the method can include releasing a second reactive oxygen species of the plurality of reactive oxygen species at a second concentration for a second time by the liquid sprayer. Exemplary embodiments of the present disclosure are described in detail below with reference to the accompanying drawings, which are incorporated herein by reference. [Brief description of the drawings]

[0009] [Figure 1] 1 is a diagrammatic representation of a ROS dispersal system according to an exemplary embodiment of the present disclosure. [Diagram 2]1 is a flow chart illustrating ROS interactions with plant hormones according to an exemplary embodiment of the present disclosure. [Diagram 3] 1 is a diagram of interactions between plant hormones according to an exemplary embodiment of the present disclosure. [Figure 4] 1 is a diagram of a chemical reaction between lignin and ROS according to an exemplary embodiment of the present disclosure. [Diagram 5] FIG. 1 illustrates the germination percentage of barley for different hydrogen peroxide concentrations and salinity treatments, according to an exemplary embodiment of the present disclosure. [Figure 6] 1 is a diagrammatic representation of interactions between plant hormones and dry matter according to an exemplary embodiment of the present disclosure. [Figure 7] FIG. 2 is a perspective view of a seed cleaner of a ROS application system according to an exemplary embodiment of the present disclosure. [Figure 8] FIG. 2 is a side perspective view of a portion of a holding vessel of a ROS dispersal system according to an exemplary embodiment of the present disclosure. [Figure 9] FIG. 2 is another side perspective view of a portion of the ROS distribution system illustrating the ROS irrigation system. [Figure 10] FIG. 2 is a side perspective view of a portion of a hydroponic cultivator showing the alternative ROS irrigation system. [Figure 11] FIG. 11 is a perspective end view of a portion of the hydroponic cultivator further illustrating the ROS irrigation system of FIG. [Figure 12] FIG. 2 is a side perspective view of a portion of a hydroponic cultivator showing the alternative ROS irrigation system. [Figure 13] 1 is a flowchart illustrating a method of ROS dispersal by a ROS dispersal system according to an exemplary embodiment of the present disclosure. [Figure 14] 1 is another flowchart illustrating a method of ROS dispersal by a ROS dispersal system according to an exemplary embodiment of the present disclosure. [Figure 15] 1 is another flowchart illustrating a method of ROS dispersal by a ROS dispersal system according to an exemplary embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] In a hydroponic environment, seeds can be grown hydroponically to a fully grown state in approximately 5 to 7 days depending on height, additional nutrients, irrigation, lighting, etc. External application of ROS can affect the phytohormonal balance within higher plants, favoring gibberellin synthesis. Through affecting the phytohormonal balance during germination, enzyme activity, mobilization of stored nutrients, and dry matter accumulation are favorably increased.

[0011] The external ROS application system 10 shown in FIG. 1 and FIGS. 8-12 can include a plurality of vertical members 12 and a plurality of horizontal members 14 that are removably interconnected to form an upright seed growing table 16 having one or more holding containers 58. In some embodiments of the present disclosure, the ROS application system 10 can have one or more seed growing tables and one or more holding containers 58. Each vertical member 12 can be configured to terminate at a lower portion in a height-adjustable leg 20. Each leg 20 can be adjusted to change the relative vertical position or height of one vertical member 12 of the seed growing table 16 relative to another vertical member (number) 12. The horizontal member 14 can be configured to include one or more lateral members removably interconnected to one or more longitudinal members 24. A pair of the vertical members 12 can be laterally separated by a lateral member 22, thereby defining a width or depth of the seed growing table 16. The longitudinal members 24 may be removably interconnected to the transverse members 22 by one or more connectors 26. The ROS distribution system may be configured to distribute at least one ROS to a plurality of seeds contained within a holding container 58.

[0012] Through the application of ROS in a hydroponic environment, the oxidative mode of action of gibberellins is mimicked in vivo, supporting the release of hydrolytic enzymes from the aleurone layer and other plant cell tissues. In addition, in contrast to lignification reactions in the absence of ROS, in the presence of ROS, ligninolytic enzymes such as peroxidases favor the delignification pathway. Since lignin is generally considered to be the most complex and indigestible fiber complex in higher plants, delignification promoted by ligninolytic enzyme hydrolysis supports improved fiber digestibility. External ROS increases the amount of dry matter in hydroponically grown cellulosic materials and maximizes enzyme activity. In higher plants, enzyme release during the germination process is usually controlled by the release of gibberellic acid (GA) from the embryo. Prior to photosynthesis, the rate of GA release is positively correlated with the metabolic demand of the seedling. Greater metabolic demand conveys an increased release rate of gibberellic acid. By using systems and methods that provide an external application of ROS to plants or seeds, hypoxic conditions are limited and the efficiency of cellular respiration is maximized. Lignin peroxidase is energetically favorable for the ligninolytic pathway rather than lignification.

[0013] The term "dry matter" means all plant material excluding water. Nutrient or mineral content in animal feed or plant tissues can be expressed on a dry matter basis or as a percentage of the total dry matter in the material. Plants or seeds can mean any plant from the kingdom Plantae or angiosperms, including flowering plants, cereals, legumes, grasses, root and tuber crops, vegetable crops, fruit plants, pulses, medicinal crops, flavor crops, beverage plants, sugar and starch, spices, oil plants, fiber crops, latex crops, food crops, fodder crops, plantation crops or pasture crops.

[0014] Grains include rice (Oryza sativa), wheat (Triticum), maize (Zea mays), rye (Secale cereale), oats (Avena sativa), barley (Hordeum vulgare), sorghum (Sorghum bicolor), pearl millet (Pennisetum glacucum), finger millet (Eleusine coracana), barnyard millet (Echinochloa frumentacea), foxtail millet (Setaria italica), millet (Paspalum scrobiculatum), and proso millet (Panicum millaceum).

[0015] Pulses include black gram (kalai or urd) (Vigna mungo var, radiatus), grass pea (Lathyrus sativus), chickpea (Cicer arietinum), cowpea (Vigna sinensis), mungbean (Vigna radiatus), horse gram (Macrotyloma uniflorum), lentil (Lens esculenta), moth bean (Phaseolus aconitifolia), pea (Pisum sativum), pigeon pea (Cajanas cajan, Cajanus indicus), philipesara (Phaseolus trilobus), and soybean (Glycine max).

[0016] Oilseeds include black mustard (Brassica nigra), castor bean (Ricinus communis), coconut (Cocus nucifera), groundnut (Arachis hypgaea), mustard (Brassica juncea), toria (Napus), niger (Guizotia abyssinica), linseed (Linum usitatissumun), safflower (Carthamus tinctorious), sesame (Seasmum indicum), sunflower (Helianthus annus), white mustard (Brassica alba), and oil palm (Elaeis guniensis). Fiber crops include Crotalaria juncea, jute (Corchorus), cotton (Gossypium), mesta (Hibiscus), or tobacco (Nicotiana).

[0017] Sugar and starch crops include potato (Solanum tberosum), sweet potato (Ipomea batatus), cassava (Manihunt esculenta), sugarcane (Saccharum officinarum), and beet (Beta vulgaris).Spices include pepper (Piper nigrum), betel nut (Piper betle), cardamomum (Elettaria cardamomum), garlic (Allium sativum), ginger (Zingiber officinale), onion (Allium cepa), capsicum or chilli (Capsicum annum), or turmeric (Curcuma longa). Pasture grasses include buffel grass or anjan (Cenchrus ciliaris), striped barnyard grass (Paspalum dilatatum), dinanath grass (Pennisetum), guniea grass (Panicum maximum), marbel grass (Dicanthium annulatum), napier grass or elephant grass (Pennisetum purpureum), pangograss (Digitaria decumbens), paragrass (Brachiaria mutica), sudan grass (Sorghum sudanense), teosinte (Echlaena mexicana), or blue panic grass (Panicum antidotale).Pasture legume crops include berseem or Egyptian clover (Trifolium alexandrinum), centrosema (Centrosema pubescens), guar bean or cluster bean (Cyamopsis tetragonoloba), alfalfa or lucerne (Medicago sativa), sirato (Macroptlium atropurpureum), and velvet bean (Mucuna cochinchinensis).

[0018] Plantation crops include bananas (Musa paradisiaca), areca palm (Areca catechu), arrowroot (Maranta arundinacea), cocoa (Theobroma cacao), coconut palm (cocos nucifera), coffee (Coffea arabica), and tea (Camellia theasinesis). Vegetable crops include: wax gourd (Beniacasa cerifera), bitter melon (Momordica charantia), bottle gourd (Lagenaria leucantha), eggplant (Solanum melongena), broad beans (Vicia faba), cabbage (Brassica), carrot (Daucus carota), cauliflower (Brassica), taro (Colocasia esulenta), cucumber (Cucumis sativus), lima bean (Phaseolus lunatus), taro or edible arum (Colocasia antiquorum), elephant yam or yam (Amorphophallus campanulatus), green beans (Phaseolus vlugaris), kohlrabi (Brassica spp.), yam (Dioscorea spp.), lettuce (Lactuca sativa), muskmelon (Cucumis melo), pointed gourd or parwal (Trchosanthes diora), pumpkin (Cucrbita spp.), radish (Raphanus sativus), okra (bhendi) (Abelmoschus esculentus), Luffa acutangular, spinach (Spinacia oleracea), snake gourd (Trichosanthes anguina), tomato (Lycoperscium esculentus), turnip (Brassica spp.), or watermelon (Citrullus vulgaris).

[0019] Medicinal crops include aloe vera, ashwagnatha (Withania somnifera), belladonna (Atropa belladonna), bishop's laurel (Ammi visnaga), bhringaraj (Eclipta alba), cinchona (Cinchona sp.), coleus (Coleus forskholli), yam (Dioscorea spp.), gloriosa (Gloriosa superba), ipecae (Cephaelis ipecauanha), long pepper (Poper longum), primrose (Oenothera lamarekiana), roselle (Hibiscus sabdariffa), sarpagan (Rauvalfia serpentine, senna (Cassia angustifolia), calamus (Acorus calamus), or valeriana (Valeriana wallaichii).

[0020] Fragrance crops include ambrette (Abelmoschus moschatus), celery (Apium graveolens), citronella (Cymbopogon winterianus), geranium (Pelargonium graveolens), jasmine (Jasminum grantiflorum), vetiver (khus) (Vetiveria zizanoids), lavender (Lavendula sp.), lemongrass (Cymbopogon flexuosus), mint, palmarosa (cymbopogon martini), patchouli (Pogostemon cablin), sandalwood (Santalum album), holy basil (Ocimum sanctum), or tuberose (Polianthus tuberosa). Food crops are harvested for human consumption and fodder crops are harvested for livestock consumption. Pasture crops include crops that animals can feed directly or that can be cut and fed to livestock.

[0021] ROS are a type of unstable molecule that contains oxygen, which can easily react with other molecules, such as seed coats, pathogens, or molecules within the cell. ROS can be formed due to the electron-accepting nature of O2. ROS play an important role in functions such as important signaling molecules that regulate normal plant growth and responses to stress. ROS are involved in photoprotection and resistance of plants or seeds to different types of stress. However, too much ROS can cause damage to DNA, RNA, or other molecules when it oxidizes, and in some cases interfere with cellular function. Oxygen toxicity can result from both uncontrolled production and inefficient removal of ROS by antioxidants. During periods of environmental stress, such as UV exposure, heat exposure, drought, salinity, low temperature, defense against pathogens, nutrient deficiency, or other types of environmental stressors, ROS levels can naturally increase. ROS include hydrogen peroxide (HO), hydroxyl radical (OH), hypochlorous acid (HOCL), nitric acid (NO), peroxyl radicals (ROO, where R can be H), including both alkylperoxyls and hydroperoxyls, peroxynitrite anion (ONOO), and nitrite radicals (NO). - ), oxygen (O2), superoxide anion (O2 - ), peroxide (O2 -2 ). H2O2 is somewhat reactive and has a relatively long half-life, which allows it to diffuse some distance from the original site of release or production.

[0022] Internal ROS generation in plants is mainly found in chloroplasts, mitochondria and peroxisomes, but can also be found in the endoplasmic reticulum, plasma membrane, cell wall and apoplast. The chloroplast photosystems PSI and PSII are the major source of internal ROS generation. Abiotic stress factors trigger the formation of ROS through the Mehler or Fenton reaction, followed by O ·-2 to H2O2. In mitochondria, ROS are generated under normal conditions, but production is greatly increased by abiotic stress conditions. Peroxisomes are the major site of ROS generation due to their oxidative metabolism. In stressful conditions, when water availability is low and stomata remain closed, increased photorespiration leads to glycolate formation. Glycolic acid is oxidized by glycolate oxidase in peroxisomes to release H2O2, which is the major producer of H2O2 during photorespiration. During periods of adverse environmental conditions, stress signals combined with abscisic acid (ABA) make the apoplast a prominent site for H2O2 production and induce stomatal closure. The plasma membrane provides the information necessary for plant cell survival. The electron transport chain of the endoplasmic reticulum generates local ROS.

[0023] External ROS are ROS that are not produced internally by the plant or seed. External ROS can be sprayed externally to the plant or plant seed by a sprayer or introduced through the plant growing surface or soil. External ROS can be a single type of ROS, such as H2O2, or a combination of H2O2 and O2. -2 The ROS may include multiple types of ROS such as

[0024] Accumulation of internal and external ROS in plants, seeds, or cells leads to various cellular responses. Plant responses can be ROS dose-dependent. ROS allow for the hormone balance necessary for life. ROS can function as plant signaling factors, can cross biological membranes, and may inactivate or activate enzymes. Therefore, control of the amount of ROS introduced into plants may be necessary. ROS can oxidize ABA, rendering it inactive. ROS can do so by activating GA, as shown in Figure 2.

[0025] ROS can interact with the external chemistry of seeds or cell walls. Some seeds have waxy outer membranes, which may contain chemicals or physical barriers that prevent germination or water from entering the seed. Seeds with waxy outer layers include cereals such as wheat, barley, and rye. In some embodiments, the O2 component of ROS reacts with the outer layer of the cell wall or seed coat to weaken, loosen, or create bubbles in the cell wall or waxy outer layer, thereby softening the cell wall. ROS may even break open the seed coat or cell wall. ROS can also destroy cell walls by mediating polysaccharide degradation and activating calcium channels and mitogen-activated protein kinases, expanding and loosening the cell wall, causing weaknesses in the cell wall. External ROS can be introduced into the seed environment to create weak spots in the seed coat, allowing water to enter the seed more quickly. This may help the seed begin to germinate. The creation of weak spots by external ROS causes the seed to release additional internal ROS within the seed, which interact with the interior of the cell wall or seed coat, further weakening the wall. In the absence of ROS, the cell wall may be strengthened and dormancy may continue.

[0026] External introduction of ROS can activate seed germination and end dormancy. As shown in Figure 2, ROS action during seed germination is based on the interaction between plant hormones that regulate seed dormancy or seed germination, such as ABA, GA, and ethylene (ET). ABA inhibits ROS-mediated action on seed germination by promoting ROS-scavenging enzyme activity. As shown in Figure 3, the ratio of ABA to GA regulates seed dormancy. A high ABA / GA ratio favors dormancy, while a low ABA / GA ratio leads to seed germination. A high ABA / GA ratio can be countered by the controlled introduction of additional external ROS into the soil or cultivation surface, or directly onto the seed or plant. ROS are absorbed by the seed or plant. GA can also counter ROS-scavenging enzymes by down-regulating the enzymes. ROS can also oxidize ABA in a similar manner, thereby decreasing the amount of ABA relative to GA. In some cases, ROS can release seed dormancy by activating GA signaling and synthesis, rather than ABA signaling or suppressing ABA catabolism. GA synthesis can induce additional ROS production in seeds. ROS then subsequently functions as a signal molecule and antagonizes ABA signaling. External ROS can increase the internal ROS content of seeds, synthesizing or activating additional GA or suppressing more ABA signaling. External application of ROS reduces ABA levels and increases GA concentration, which leads to seed germination. However, the amount or concentration of ROS may need to be monitored. Beyond a certain limit, ROS is either too low to allow germination or too high to affect embryo viability and thus prevent or delay germination. This creates an "oxidative window" for germination, which limits successful seedling development within certain boundaries of increased ROS levels.

[0027] During germination, GAs translocate to the aleurone layer. When GAs interact with the aleurone layer of the seed, α-amylase synthesis is triggered, which results in the release or synthesis of hydrolytic enzymes, including α-amylase. Aleurone is localized around the starchy endosperm. Hydrolytic enzymes such as 1,3;1,4-β-glucanase (β-glucanase), α-amylase, and β-amylase are released to catalyze the breakdown of cell wall polysaccharides, proteases, storage proteins, and starchy energy stores that are essential for germination and provide sugars to drive root growth. β-glucanases can hydrolyze 1,3;1,4-β-glucans, which are the predominant cell wall polysaccharides. α-amylase cleaves internal amylose and amylopectin residues. β-amylase exohydrolase liberates maltose and glucose from starch molecules. In the absence of ABA repression by the introduction of external ROS, ABA inhibits the transcription of hydrolases and prevents germination.

[0028] In aerobic moist seeds, ROS production and external ROS dispersal correlate with increased metabolism in chloroplasts, mitochondria, glyoxysomes, peroxisomes, and plasma membranes. During seed imbibition, compartmentalization of ROS and their target molecules in different subcellular structures regulates the expression of various genes. Water allows ROS to be transported or travel longer distances, whereas in dry seeds, ROS production must be near the target during seed imbibition. As shown in Figure 6, when the seed or plant is moistened, external ROS can easily translocate from outside the cell, seed, or plant to inside the cell, seed, or plant, thereby increasing enzyme activity and dry matter. Figure 5 illustrates the germination percentage of barley for different H2O2 concentrations and salinity treatments. Salinity treatments expressed as salinity in parts per thousand. Values ​​shown in Figure 5 are expressed as fixed effects linear model estimates with 95 percent confidence intervals illustrating surrounding estimates. Through the application of ROS, the inhibitory effects of ABA, including reduced stem elongation and germination, are reduced.

[0029] During stress, lipoxygenases localized in the cell wall cause hydroperoxidation of polyunsaturated fatty acids (PUFAs), which are an active source of ROS. During pathogen attack, lignin precursors undergo excessive cross-linking through ROS-mediated pathways to reinforce cell walls with lignin. Lignin fills the spaces in cell walls between cellulose materials, hemicellulose, and pectin components, especially in vascular and supporting tissues: xylem tracheids, vessel elements, and sclerenchyma cells. When external ROS are distributed to seeds or plants, they can sterilize the seeds or plants or kill some or all of the pathogens. This stops the cross-linking of lignin precursors and strengthening of cell walls, which prevents plant germination or growth.

[0030] The term "cellulosic material" refers to any material that contains cellulose. The predominant polysaccharide in the primary cell walls of biomass is cellulose, the second most abundant is hemicellulose, and the third is pectin. Secondary cell walls, which are produced after the cells stop growing, also contain polysaccharides and are strengthened by polymeric lignin covalently cross-linked to hemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thus a linear β-(1-4)-D-glucan, while hemicellulose contains a variety of compounds such as xylans, xyloglucans, arabinoxylans, and mannans in complex branched structures containing a spectrum of substituents. Although generally polymorphic, cellulose is found primarily as an insoluble crystalline matrix of parallel glucan chains in plant tissues. Hemicellulose usually hydrogen bonds to cellulose as well as other hemicelluloses, helping to stabilize the cell wall matrix.

[0031] As shown in Figure 4, lignin depolymerization can be achieved mainly by one-electron oxidation reactions catalyzed by extracellular oxidases and peroxidases in the presence of extracellular or external ROS. Hydroxyl radicals attack the lignin structure, creating access points for hydrolysis by whole cells, enzymes, or other chemicals. External sparging of ROS allows additional ROS to attack the lignin structure, thus creating additional access points for hydrolysis of lignin, cellulose, and hemicellulose by ligninolytic enzymes, hemicellulolytic enzymes or hemicellulose, cellulolytic enzymes or cellulases, and endoglucanases.

[0032] Lignin degrading enzymes are enzymes that hydrolyze the structure of lignin polymers. Enzymes that can degrade lignin include lignin peroxidase, manganese peroxidase, laccase and feruloyl esterase, as well as other enzymes described in the art that are known to depolymerize or otherwise degrade lignin polymers. Enzymes that can hydrolyze the bonds formed between hemicellulose sugars (especially arabinose) and lignin are also included. Hemicellulolytic enzymes or hemicellulases are one or more enzymes that hydrolyze hemicellulose materials. Endoglucanases are endo-1,4-(1,3:1,4)-β-D-glucan 4-glucanohydrolases (EC 3.2.1.4) that catalyze the endo-hydrolysis of β-1,4-linkages in cellulose, cellulose derivatives (such as carboxymethylcellulose and hydroxyethylcellulose), lichenin, mixed β-1,3-glucans such as cereal β-D-glucans or xyloglucans, and other plant materials that contain cellulosic components. Cellulolytic enzymes or cellulases refer to one or more enzymes that hydrolyze cellulosic materials. Such enzymes include endoglucanases, cellobiohydrolases, β-glucosidases, or combinations thereof.

[0033] ROS can oxidize the pericarp of the plant ovary. The pericarp is the ripened and variously modified wall of the plant ovary. The pericarp has an outer exocarp, a central mesocarp, and an inner endocarp, which is the wall of the plant fruit that develops from the ovary wall. External ROS can initiate redox signaling during plant organ development, including fruit ripening and flower development. Oxidative stress, an imbalance between ROS production and ROS removal, occurs in mitochondria due to increased respiration rates during ripening, when sugars become the rate-limiting factor and initiate ripening, affecting the redox state. External ROS can increase the imbalance and allow the plant to ripen. Oxidative stress also occurs in plastids during the chloroplast to chromoplast transition at the onset of fruit ripening.

[0034] As shown in Figures 8-12, at the beginning of the germination stage, ROS can be introduced into the environment of the seeds to sanitize the seeds, the growing material in which the seeds will be planted, the growing surface 18 for housing the growing material, such as a seed belt, a hydroponic machine, or a tray system. The growing surface 18 can include a top surface for hydroponically growing a plurality of seeds on the growing surface. The ROS kill harmful bacteria, mold, or fungi growing on the seeds, growing material, or growing surface while preventing good bacteria present in the seeds from being destroyed during the sanitization process. By killing harmful bacteria, mold, or fungi with ROS, beneficial or useful bacteria can grow in the growing material or on the seeds. The material of the growing surface 18 can be designed to help maintain an oxidative environment. For example, the bottom or sides of the growing surface 18 can be hydrophobic, permeable, or semi-permeable. In some embodiments of the present disclosure, the growing surface 18 can be sanitized with ROS prior to the germination stage. The seeds can also be washed or exposed to ROS for disinfection in a seed washer 30 before or as they are placed on the growing surface, as shown in FIG.

[0035] Fresh or bulk seeds often contain debris and other contaminants, and the seed cleaner 30 can remove the debris and contaminants from the seeds before conveying the seeds to the growing surface 18 or frame structure 12. The seed cleaner 30 can include a separator housing 32 having a seed inlet 34, a seed discharge 36 and disposed between the seed inlet 34 and the seed discharge 36, and can include one or more augers 38 disposed vertically in a conduit 40 having a diameter of the vane 42. The vanes 42 are preferably discontinuous, thereby leaving gaps between intermittently spaced vane sections for seeds, debris, and contaminants to fall freely from one vane level to the next vane level. A vacuum line 44 can be operably connected to or near the seed inlet 34 and connected to a vacuum source at an opposite end. Seeds are introduced into the seed cleaner 30 through the seed inlet 34 and can fall freely down the vanes 42 between the gaps in the vanes 42 on each auger 38. Debris and contaminants can be sucked into the vacuum line 44, and the seeds can drop through the seed outlet free of debris and contaminants, which are sucked into a trash bin. The cleaned seeds can exit the seed outlet 36 and be transported or placed on the growing surface 18. In some cases, the seeds may contain debris that is too large to be sucked in without also sucking the seeds. A screen (not shown) can be operatively positioned at the outlet of the seed outlet 36 to screen out debris that is too large to be sucked in as it passes through the seed cleaner 30. One or more piping elements can be operatively configured to disinfect the seeds in the seed cleaner using a ROS applicator. One or more UV lighting elements can be operatively configured to irradiate the seeds in the seed cleaner 30 with UV light to kill bacteria on the seeds. The process of cleaning the seeds and irradiating the seeds with UV light by the seed cleaner 30, and the general operation of the seed cleaner 30, can be automated by a controller, a graphical user interface, and / or a remote control device.

[0036] Each growing surface 18 may include a liquid applicator 46A, 46B, and / or 46C operably configured above each growing surface 18 to irrigate the seeds disposed thereon. The dimensions of the growing surface 18 may be configured to accommodate the desired plant production required or for ROS distribution coverage. The liquid applicator 46A may be configured adjacent at least one longitudinal edge of the growing surface 18. The liquid applicator 46A may also be configured operably adjacent at least one lateral edge of the growing surface 18. Preferably, the liquid applicator 46A may be configured adjacent a longitudinal edge of the growing surface 18 to provide drip flood irrigation to the growing surface 18 and the seeds disposed thereon. The liquid spreader 46A can include a ROS guide 48 and a ROS distributor 50A, 50B, 50C having a ROS outlet 52, the ROS outlet 52 generally having an undulating profile, such as a sawtooth or wavy profile providing peaks (higher) and valleys (lower) portions. The liquid spreader 46A can include a ROS conduit 54 configured to carry ROS from a ROS source 56, such as a ROS collector 58 or a piped ROS source 56. The ROS can exit the ROS conduit 54 through one or more openings and can be captured by the ROS guide 48 and the ROS distributor 50A as it exits the ROS conduit 54. One or more openings in the ROS conduit 54 can be configured as a ROS dripper that intermittently drips a known or quantifiable amount of ROS into the ROS guide 48 over a set time frame. The one or more openings can be configured intermittently along the length of the ROS conduit 54 or can be distributed in groups along the length of the ROS conduit 54. The one or more openings in the ROS conduit 54 can be operatively configured to distribute the ROS evenly over the growing surface 18 and slowly drip the liquid onto the growing surface 18. Drip or flood irrigation of the growing surface can provide a layer of ROS 62 for soaking the seeds and provide the liquid to the seeds on the growing surface 18 in a controlled, evenly distributed flow.The ROS distributor 50A can be configured with a ROS guide 48 adapted to collect the liquid as it exits the ROS conduit 54. The collected ROS can be evenly dispersed by the ROS distributor 50A and exit the ROS distributor 50A via the ROS outlet 52 and fall onto the growing surface 18.

[0037] According to at least one embodiment, the ROS exiting the ROS distributor 50A can travel on the growing surface 18, under and / or between the seed masses on the growing surface 18. Other configurations of the liquid spreader 46 are also contemplated herein. For example, in one embodiment, the ROS can enter the liquid spreader 46 through the ROS line 54 and exit the ROS line 54 through multiple openings. The ROS from the ROS line 54 can combine to form a small puddle, resulting in an even distribution of liquid over the length of the ROS distributor 50A. When this small puddle is full, the ROS can spill out of the ROS outlet 52, such as between the teeth of the ROS outlet 52. In this manner, the ROS can be distributed evenly over the entire length and width of the growing surface. The ROS exiting the ROS outlet 52 can drip onto the growing surface 18 and flow under the seed masses on the growing surface 18 to soak or contact the seeds. The root systems of the seeds on the growing surface 18, along with capillary action, can lift the ROS up through the seeds to water all of the seeds and / or plants.

[0038] A liquid applicator 46B can be disposed above each growing surface 18. The liquid applicator 46B can include a plurality of ROS distributors 50B operably configured within ROS lines 54 operably plumbed to a ROS source 56. The ROS distributors 50B can include a spray head, such as a single or dual band spray head / tip, for spray irrigating the seeds disposed on each growing surface 18. In one embodiment, a plurality of ROS lines 54 can be disposed in a spaced apart array above each growing surface 18. Each ROS line 54 can traverse the length of the holding vessel and can be plumbed to connect to a ROS source 56. The other ROS line 54 can be configured to traverse the width of the growing surface 18. ROS can be discharged from each ROS distributor 50B for spray irrigating the seeds on each growing surface 18. In another embodiment, each ROS line 54 can be oscillated back and forth over a radius of travel of 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, or more to cover the entire surface area of ​​the seeds on each growing surface 18. If a dual angle spray head is used in the ROS distributor 50B, the oscillation stroke of each ROS line 54 can be reduced, thereby reducing friction and wear and tear of the liquid applicator 46B. The process of applying ROS to the seeds or plants can be automated by a controller, a graphical user interface, and / or a remote control device. A drive mechanism 66 can be operably connected to each ROS line 54 to oscillate or rotate each line over a radius of travel. The liquid applicator 46 can be operated manually or automatically using one or more controllers operated by a control system.

[0039] The liquid applicator 46 can be configured to remove debris, contaminants, mold, fungus, bacteria, and other foreign / unwanted material from the growing surface 18. The liquid applicator 46 can also be used to irrigate the seeds with disinfectant or ROS as the seeds are released from the seed dispenser onto the growing surface 18. A time delay can be used to allow the ROS to remain on the seeds for a desired amount of time before applying a fresh water spray or irrigation. The process of cleaning, descaling, and disinfecting the growing surface 18 using the liquid applicator 46D can be automated by a controller, a graphical user interface, and / or a remote control device.

[0040] The liquid applicator 46 can be operated to wet the seeds with ROS immediately after sowing on the growing surface 18. Seeds in early, middle and later stages of growth can be irrigated with ROS using the liquid applicator 46. The liquid applicators 46A-D can be operated simultaneously, intermittently, alternately and independently of each other. During the early stages of seed growth, both liquid applicators 46A-B are operated to best wet the seeds to promote seedling and germination. During the later stages of growth, the liquid applicator 46A can be used to irrigate more than the liquid applicator 46B. Alternatively, the liquid applicator 46B can be used to irrigate more than the liquid applicator 46A depending on the degree of soaking of the seed growing. The liquid applicator 46C can be operated to spray the seeds dispensed onto the growing surface 18 during sowing onto the growing surface 18 and during movement of the growing surface 18 in the second direction to wet the seeds with ROS. The ROS provided to the liquid applicators 46A-D can include additives such as disinfectants and / or nutrients. Nutrients, such as commonly known plant nutrients, can be added to the ROS dispensed from the liquid applicators 46A-D to promote healthy plant growth and / or increase the presence of desired nutrients in the harvested seeds. The liquid applicators 46C-D can also be used to sanitize the growing surface 18 before and / or after winding or unwinding onto the seed belt, growing surface 18, or growing surface 68.

[0041] By including one or more disinfectants, such as ROS, used by each ROS distributor 50A-D, the liquid distributors 46A-D and their various components can be sanitized along with other components of the external ROS distribution system 10. For example, the ROS guide 48, the ROS conduit 54, the ROS outlet 52, the drain 60, the ROS collector 58, the growing surface 18, the ROS distributors 50A-C, and other components of the growing system. In another embodiment, the liquid distributors 46A-D can be used to clean and sanitize the growing surface 18 before, during, or after sowing and harvesting. Separate liquid distributors or manifolds can be configured to disinfect or sanitize any components of the growing system that carry ROS for irrigation and cutting or receive irrigation or cutting runoff from one or more holding vessels.

[0042] As shown in FIG. 13, in one embodiment of ROS application, ROS can be applied to disinfect a plurality of seeds (step 200). Next, the plurality of seeds can be placed on a cultivation surface during the germination stage (step 202). In some embodiments of the present disclosure, the seeds can be disinfected after being placed on the cultivation surface. Next, at least one type of ROS can be applied to the plurality of seeds on the cultivation surface during the germination stage (step 204). The external ROS can be applied all the time, or the applicator can apply the ROS in a set time frame or quantifiable amount. For example, the liquid applicator can apply the ROS for a first period of time, such as 1 minute, followed by the liquid applicator stopping the application of the ROS for a second period of time, such as 4 minutes, or 1 minute of ROS application every 5 minutes. Next, the plurality of seeds can be germinated (step 206). Next, the ROS can be applied to the plurality of germinated seeds during the plant development stage (step 208). Finally, the germinated seeds can be allowed to mature, step 210. ROS can be applied at specific concentrations during both the seedling and plant development stages.

[0043] As shown in FIG. 14, in another embodiment of the application of ROS, ROS can be applied to disinfect a plurality of seeds (step 300). The plurality of seeds can then be placed on a growing surface during the germination stage (step 302). In some embodiments of the present disclosure, the seeds can be disinfected after being placed on the growing surface. At germination, ROS can be applied at a first concentration, such as a concentration in the range of 1000-3000 ppm (step 304). The germination stage can then be terminated (step 306). The germination stage can last for 18-24 hours. However, the germination stage can be shorter or longer than 18-24 hours. The moisture level during germination can range from 10-60 percent, however, the moisture level can be higher or lower. After the germination stage is terminated, ROS can be applied at a second concentration rate, such as 50-200 ppm, throughout the entire plant development until plant maturity (step 308). A higher rate during the germination stage may promote the inactivation of biological contaminants present on the seed coat, including bacterial contamination and fungal spores, pericarp oxidation, and early hydrogen peroxide uptake. A higher rate of ROS application creates weak spots in the seed coat, allowing the seed to increase water uptake, and the uptake of external ROS by the seed increases the production of GA and decreases the amount of ABA or ABA signaling. The reduction in the seed's environmental stress and the increase in GA allow the seed to begin germination. Finally, the seed or seeds are mature and ready to be harvested (step 310).

[0044] A lower rate may be required after the onset of metabolic activity. The reduced rate throughout development supports the continuation of abscisic acid oxidation, thereby increasing the release of gibberellins throughout plant development. The lower rate over a long period of time is believed to mimic the value of a high rate over a reduced duration. As the plant grows, the root system provides capillary action and helps distribute external ROS. The absence of hydrogen peroxide may result in lower yield, lower feed value, and lower quality of plant product over the entire development. In some embodiments of the present disclosure, ROS can be sprayed at a first concentration during the soaking phase. The soaking phase may last for 10 minutes or 15 minutes. The lower rate over the extended soaking phase may have the value of a high rate over a reduced duration. For example, seeds can be exposed to 200 / 300 ppm ROS for a duration of 10 minutes during the soaking phase. After the soaking period, the ROS application can be switched to a maintenance rate that can be 50-200 ppm throughout development.

[0045] During the application process, the ROS can be applied by the liquid applicator 46 as a liquid concentrate. Application can include flood irrigation, drip irrigation, spray irrigation, subirrigation, or any other irrigation method that allows the ROS to reach the seeds or plants. The liquid applicator 46 can apply the ROS in a set time frame or in a quantifiable amount. For example, the liquid applicator 46 can apply the ROS for a first period of time, such as one minute, followed by the liquid applicator 46 stopping the application of the ROS for a second period of time, such as four minutes, or one minute of ROS application every five minutes. The cycle can continue until the development or seedling stage is over. In another example, the ROS can be applied for one minute every two hours. The liquid applicator 46 can provide a flow with a controlled and even distribution, allowing the ROS to reach a maximum number of seeds. The excess ROS can be captured, recirculated, and reused by the external ROS application system 10. If the growing surface 18 has an outlet or slope, the slope can aid in the even distribution of the ROS as it exits through the holding vessel. In some embodiments, the liquid applicator 46 can direct the distribution of the ROS to an area in the holding vessel, a portion of a seed, or a portion of a plant that requires further application. The liquid applicator 46 can also vibrate to cover a larger area of ​​the growing surface 18 or the entire length and width of the growing surface 18 or seedbed.

[0046] The ROS can be applied separately from other liquids being provided to the growing system. The ROS can be mixed with another liquid, such as water, an alkaline solution, or fertilizer, prior to distribution by the liquid applicator 46. The liquid applicator can have multiple nozzles for liquid distribution, each responsible for dispensing one or more types of liquid. For example, the ROS nozzle and the water nozzle can be converging nozzles, and the ROS and water can be released simultaneously onto the substrate.

[0047] In another embodiment of the application of ROS to increase dry matter, a plurality of seeds can be placed on a growing surface (step 400). A first reactive oxygen species of the plurality of reactive oxygen species can then be introduced by a liquid applicator onto the growing surface at a first concentration for a first time (step 402). The plurality of seeds can then be disinfected by the first reactive oxygen species of the plurality of reactive oxygen species introduced by the liquid applicator (step 404). In some embodiments of the present disclosure, the seeds can be disinfected after being placed on the growing surface. The plurality of seeds can then be germinated on the growing surface (step 406). A second reactive oxygen species of the plurality of reactive oxygen species can then be released by a liquid applicator at a second concentration for a second time (step 408). In some embodiments, different concentrations of ROS can be used during different stages of plant development or when it is determined that the plant or seed requires additional ROS to increase dry matter. Finally, the plant can be allowed to mature and be harvested (step 410).

[0048] The present disclosure is not limited to the specific embodiments described herein. In particular, the present disclosure contemplates numerous variations of increasing enzyme activity and dry matter using ROS in hydroponic systems. The above description is presented for illustrative and explanatory purposes. It is not intended to be an exhaustive list or to limit any of the present disclosure to the exact form disclosed. It is contemplated that other alternatives or exemplary embodiments are considered to be encompassed by the present disclosure. The present description is merely an example of the embodiments, processes, or methods of the present disclosure. It is understood that any other modifications, substitutions, and / or additions can be made and are within the intended spirit and scope of the present disclosure. [Explanation of symbols]

[0049] 10 External ROS Spray System 12 Vertical Members 14 Horizontal Members 16 Seed cultivation stand 18 Cultivation surface 20 Legs 22 Transverse Members 24 Longitudinal members 26 Connectors 30 Seed Cleaning Machine 32 Separator housing 34 Seed inlet 36 Seed outlet 38 Ogre 40 Conduit 42 Feather 44 Vacuum line 46A~D Liquid sprayer 48 ROS Guide 50A~C ROS distributor 52 ROS exit 54 ROS conduit 56 ROS sources 58 Holding vessel, ROS collector 60 Drain 62 RO 66 Drive Mechanism 68 Cultivation surface 74 seeds

Claims

1. 1. A method for the external application of reactive oxygen species to increase dry matter and enzyme activity, comprising: disinfecting the plurality of seeds with at least one reactive oxygen species, the reactive oxygen species configured to kill at least one of harmful bacteria, mold, or fungi; exogenously applying the at least one reactive oxygen species to the plurality of seeds on a cultivation surface at a first concentration during seedling stage, wherein the at least one reactive oxygen species increases gibberellic acid expression; germinating the plurality of seeds; and exogenously applying the at least one reactive oxygen species to the germinated seeds at a second concentration over a plant developmental period until the germinated seeds reach maturity. The above method.

2. 10. The method of claim 1, wherein the at least one reactive oxygen species comprises hydrogen peroxide.

3. 2. The method of claim 1, wherein the first concentration is between 1000 ppm and 3000 ppm.

4. weakening a seed coat of at least one of said plurality of seeds with said at least one reactive oxygen species during said germination stage, wherein weakening said seed coat allows water to penetrate said seed coat at a faster rate. The method of claim 1, further comprising:

5. Reducing abscisic acid expression by exogenous application of said at least one reactive oxygen species. The method of claim 1, further comprising:

6. 2. The method of claim 1, wherein the step of externally dispersing at least one reactive oxygen species during the germination stage supports the release of hydrolytic enzymes from a plurality of cellular tissues of the plurality of seeds.

7. The method of claim 1, wherein the step of external application of at least one reactive oxygen species during the seedling stage promotes a delignification pathway.

8. 1. An external reactive oxygen species application system for increasing plant dry matter, comprising: a growing surface operatively supported by and disposed across a length and width of the frame structure, the growing surface configured to contain a plurality of seeds; a source of reactive oxygen species operably connected to the framework and configured to contain at least one reactive oxygen species; and one or more liquid applicators operably secured to the frame structure adjacent to the cultivation surface for releasing the at least one reactive oxygen species from the reactive oxygen species source to the plurality of seeds contained on the cultivation surface, the one or more liquid applicators being configured to release the at least one reactive oxygen species at different concentrations. Including, The external reactive oxygen species distribution system, wherein the at least one reactive oxygen species promotes germination of the plurality of seeds on the cultivation surface.

9. 9. The external reactive oxygen species distribution system of claim 8, wherein the growing surface comprises an upper surface for hydroponic cultivation of the plurality of seeds on the growing surface.

10. 9. The external reactive oxygen species distribution system of claim 8, wherein the liquid dispenser comprises a plurality of liquid distributors configured to distribute the at least one reactive oxygen species evenly along the width of the growing surface and evenly along the length of the growing surface.

11. 10. The external reactive oxygen species sparging system of claim 8, wherein the at least one reactive oxygen species is combined with water prior to being released from the liquid sparger.

12. 10. The external reactive oxygen species sparging system of claim 8, wherein the liquid sparger releases the at least one reactive oxygen species at a first concentration for a first time period and at a second concentration for a second time period.

13. 9. The external reactive oxygen species sparging system of claim 8, wherein the liquid sparger is configured to release the at least one reactive oxygen species for a first period of time, and the liquid sparger is configured to stop releasing the at least one reactive species for a second period of time.

14. a seed cleaner operably connected to the frame structure and configured to remove debris from the plurality of seeds prior to placing the plurality of seeds on the growing surface.

9. The external reactive oxygen species sparging system of claim 8, further comprising:

15. 1. A method for increasing dry matter in a plurality of plants by exogenous application of reactive oxygen species, comprising: placing a plurality of seeds on a growing surface, the growing surface including a top surface for hydroponically growing the plurality of seeds on the growing surface; introducing at least one reactive oxygen species at a first concentration for a first period of time onto the growing surface with a liquid applicator; disinfecting the plurality of seeds with a first reactive oxygen species of the plurality of reactive oxygen species introduced by the liquid applicator; germinating the plurality of seeds on the cultivation surface; and Releasing a second one of the plurality of reactive oxygen species with the liquid dispenser at a second concentration for a second time period. The above method.

16. 16. The method of claim 15, wherein the first reactive oxygen species comprises at least one of hydrogen peroxide, hydroxyl radical, hypochlorous acid, nitric acid, peroxyl radical, alkylperoxyl, hydroperoxyl, peroxynitrite anion, oxygen, superoxide anion, or peroxide.

17. 16. The method of claim 15, wherein the second reactive oxygen species comprises at least one of hydrogen peroxide, hydroxyl radical, hypochlorous acid, nitric acid, peroxyl radical, alkylperoxyl, hydroperoxyl, peroxynitrite anion, oxygen, superoxide anion, or peroxide.

18. 16. The method of claim 15, wherein the first concentration is in the range of 200 ppm to 3000 ppm.

19. 16. The method of claim 15, wherein the second concentration is in the range of 50 ppm to 200 ppm.

20. 16. The method of claim 15, wherein the first period of time ranges from 18 hours to 24 hours.

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