Methods of using colloidal silver-based compositions in reducing or preventing microbial contamination of plants or explants in tissue culture processes

Abstract

The present disclosure provides a method for using an environmentally friendly colloidal silver-based composition in preventing and reducing contamination caused by microorganisms in plant tissue culture processes. The composition comprises colloidal silver, a copolymer, a surfactant, a base, and water. The method allows for culturing plants or explants in plant tissue culture medium under non-sterile conditions, thus eliminating the need for cumbersome and labor-intensive procedures for sterilizing culture medium and culture vessels to create sterile conditions for plant or explant culture.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 964,807, filed January 23, 2020. The above-mentioned application is incorporated herein by reference.

[0002] The present disclosure relates generally to methods for reducing or preventing microbial contamination of plants or explants in tissue culture processes, and more particularly to methods of using colloidal silver-based compositions in reducing or preventing microbial contamination of plants or explants in tissue culture processes, including micropropagation, cell culture, suspension culture, callus culture, germplasm preservation, and the like, under sterile or non-sterile conditions. [Background technology]

[0003] Tissue culture is a technique for propagating plant cells by incubating small pieces of plant tissue (also known as explants) on a suitable nutrient medium. This technique allows for the in vitro cultivation of plant cells and organs, which can divide and regenerate into callus or new plant organs. Explants are obtained from any part of the plant and used as a starting point by introducing the explant into a nutrient medium to promote its growth.

[0004] The tissue culture process generally requires a sterile environment to avoid microbial contamination. The nutrient medium used presents a combination of micro- and macronutrients available as a nutrient source for the explants. However, the same nutrients are also available to microorganisms that invade the system, either during the introduction process or by attaching to the explants. Plant cell division occurs at a much slower rate than bacterial and fungal cell division; therefore, the latter can easily outgrow the plant tissue inside the container used for tissue culture. For this reason, all materials used (glassware, containers, tools, and the medium itself) must be sterilized or disinfected to remove all viable microorganisms by methods such as autoclaving and the use of laminar flow hoods to provide sterile materials and aseptic conditions.

[0005] Typically, sterilization of culture media and culture vessels is performed using a high-pressure steam sterilizer (autoclave), while inoculation of plant tissues is performed in a sterilization room on a clean bench equipped with a laminar flow hood. Therefore, after sterilization, the culture media, etc. must be removed from the autoclave and transferred to the sterilization room. This task is tedious because it requires great care to avoid the introduction of microorganisms. Another problem is that the capacity of the autoclave or sterilization room limits the amount of plant tissue that can be cultured, making it impossible to simultaneously culture a large amount of plant tissue.

[0006] Despite the existence of methods for providing sterile conditions, the problem of microbial contamination is ever-present. This poses significant difficulties to the culture of some explants and / or plant species due to the presence of endogenous microorganisms. Commercial tissue culture operations often must assume the loss of a certain percentage of their production due to microbial contamination. Summary of the Invention [Problem to be solved by the invention]

[0007] Thus, there is an urgent need for methods and reagents to prevent and reduce contamination levels in the various variations and stages of plant tissue culture. [Means for solving the problem]

[0008] The present disclosure addresses the above-mentioned needs in multiple aspects. In one aspect, the present disclosure provides a method for reducing or preventing microbial contamination of plants or explants in a tissue culture process. The method includes culturing the plants or explants in a plant tissue culture medium formed from a colloidal silver-based composition. The silver-based composition includes colloidal silver, a copolymer (e.g., methyl vinyl ether copolymer), a surfactant (e.g., polyoxyethylene octylphenyl ether), a base (e.g., sodium hydroxide, potassium hydroxide), and water.

[0009] In some embodiments, the method further comprises pre-treating the plant or explant with the composition. In some embodiments, the method comprises applying the composition to the surface of the plant or explant. In some embodiments, the composition is applied as a spray, mist, or drops. In some embodiments, the method comprises inoculating the plant or explant in plant tissue culture medium, followed by applying the composition onto the plant or explant, thereby forming a barrier (e.g., a microfilm) that protects the plant or explant against microbial contamination.

[0010] In some embodiments, the composition comprises 0.1-20% (w / v) colloidal silver, 1.75-4.38% (w / v) methyl vinyl ether copolymer, 0.02-0.04% (w / v) polyoxyethylene octylphenyl ether, 0.02-0.04% (w / v) sodium hydroxide, and 89-95% (w / v) water. In some embodiments, the composition comprises about 5.00% (w / v) colloidal silver, about 3.50% (w / v) methyl vinyl ether copolymer, about 0.099% (w / v) polyoxyethylene octylphenyl ether, about 0.032% (w / v) sodium hydroxide, and about 91.36% (w / v) water.

[0011] In some embodiments, the water is distilled or deionized. In some embodiments, a base (e.g., sodium hydroxide, potassium hydroxide) is used to neutralize the composition.

[0012] In some embodiments, the colloidal silver has the following characteristics: (a) silver suspended in distilled water and produced by dispersion according to published guidelines (NIST, 2012) or by electrical methods at a silver electrode; (b) has an atomic mass of 107,868 g / mol; (c) has a melting point of 960.5°C; (d) has a boiling point of 2000°C; (e) has a density of 10.49 g / mL at 15°C; (f) is not attacked by water or atmospheric oxygen; (g) becomes tarnished by ozone and hydrogen sulfide; (h) is inert to many acids and reacts readily with dilute nitric acid and hot sulfuric acid; and (i) is not sensitive to light in its metallic form.

[0013] In some embodiments, the colloidal silver particles have an average particle size between about 60 nm and about 140 nm. In some embodiments, at least 50% of the colloidal silver particles have a particle size between about 60 nm and about 140 nm. In some embodiments, at least 90% of the colloidal silver particles have a particle size between about 60 nm and about 140 nm.

[0014] In some embodiments, the composition further comprises a carrier that is acceptable for tissue culture processes. In some embodiments, the composition further comprises at least one of a second fungicide and a second bactericide.

[0015] In some embodiments, the composition is provided in a concentrated form, in a powder form, or in a hydrogel form (or concentrated gel form).

[0016] In some embodiments, the composition is prepared by the following steps: (a) placing 89.2 to 95.7 kg of water into a 100 L stainless steel vessel; (b) slowly adding 2.50 to 6.25 kg of colloidal silver concentrate with stirring; (c) adding 50 to 120 grams of polyoxyethylene octylphenyl ether; (d) adding 1.75 to 4.38 kg of methyl vinyl ether copolymer, which has been prepolymerized in a stainless steel vessel containing 1.75 to 4.38 L of deionized water at 40° C.; (e) adding 1 L of 20 to 40 g / L sodium hydroxide solution; and (f) continuously stirring until the resulting mixture is clear.

[0017] In some embodiments, the composition is prepared by the following steps: (a) placing 91.4 kg of water into a 100 L stainless steel vessel; (b) slowly adding 5 kg of colloidal silver concentrate with stirring; (c) adding 99 grams of polyoxyethylene octylphenyl ether; (d) adding 3.5 kg of methyl vinyl ether copolymer, which has been pre-polymerized in a stainless steel vessel containing 3.5 L of deionized water at 40° C.; (e) adding 1 L of 35 g / L sodium hydroxide solution; and (f) continuously stirring until the resulting mixture is clear.

[0018] In some embodiments, the microbial contamination is caused by a fungus or a bacterium. In some embodiments, the fungus is a fungus selected from the genera Blumeria, Sphaerotheca, Phytophthora, Rhizoctonia, Fusarium, Penicillium, Aspergillus, Colletotrichum, Botrytis, Magnaporthe, Pythium, The species is selected from the group consisting of Pythium, Puccinia, Erysiphe, Alternaria, Pseudoperonospora, Plasmodiophora, Sclerotinia, Fulvia, Peronospora, Ustilago, and Rhizopus. In some embodiments, the bacterium is selected from the group consisting of Corynebacterium, Bacillus, Staphylococcus, Escherichia, Pseudomonas, Xanthomonas, Erwinia, Clavibacter, Ralstonia, Burkholderia, and Agrobacterium.

[0019] The above summary is not intended to define all aspects of the disclosure, and additional aspects are described in other sections, such as the following detailed description. It should be understood that the entire document is intended to be related as an integrated disclosure, and that all combinations of features described herein are contemplated, even if the combinations of features are not found together in the same sentence, paragraph, or section of the document. Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the disclosure, are given for purposes of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present disclosure provides a method for reducing or preventing microbial contamination of plants or explants in tissue culture processes. The method comprises culturing the plants or explants in a plant tissue culture medium formed from a colloidal silver-based composition. The silver-based composition comprises colloidal silver, a copolymer (e.g., methyl vinyl ether copolymer), a surfactant (e.g., polyoxyethylene octylphenyl ether), a pH adjuster (e.g., a base), and water.

[0021] The method allows for culturing plants or explants in plant tissue culture medium under non-sterile conditions, i.e., the method obviates the need for cumbersome and laborious procedures to sterilize culture medium and culture vessels to create sterile conditions for plant or explant culture.

[0022] The antimicrobial properties of silver are widely recognized, and its effectiveness as an antimicrobial agent has been demonstrated. To meet the need for the development of an effective antimicrobial agent to address the ubiquitous and undesirable contamination caused by bacteria and fungi in tissue culture processes, the present disclosure provides methods based on colloidal silver-based compositions that have the ability to prevent and control infections caused by microorganisms in tissue culture processes, for example, by using a contact mode of action (e.g., non-systemic).

[0023] The composition contains colloidal silver as an active ingredient, mixed with a polymer. The composition can be added to a tissue culture nutrient medium, applied directly to explants, or used as part of the sterilization process of explants prior to their introduction into a nutrient medium or as part of a tissue culture process, either by being present in the nutrient medium, forming a microfilm on the surface of the explant, or by assisting in the sterilization process of the explant. The composition prevents the development of fungal and bacterial contamination without any adverse effects on the explants and their development. Due to the nature of the composition and its non-systemic application, the composition is non-toxic to the host explants and plants, as well as humans, and does not contaminate ecosystems. The composition can be used as part of the sterilization process of explants prior to their introduction. The composition can be used to introduce plant tissue into non-sterile conditions and / or to grow plant tissue in non-sterile conditions. The compositions include preparations containing colloidal silver that function as fungicides and bactericides to prevent and control many of the contaminations caused by fungi and bacteria in tissue culture processes.

[0024] The mechanisms involved in the antimicrobial activity of colloidal silver may include: (1) altering and damaging the membrane structure of microorganisms, thereby increasing their permeability and disrupting their transport functions, resulting in cell death; (2) microbial penetration and interaction with phosphorus- and sulfur-containing compounds such as DNA and proteins; (3) disrupting DNA replication; (4) inactivating certain enzymes; (5) attacking the respiratory chain; and (6) generating hydrogen peroxide and free radicals. The antimicrobial activity exerted by the composition is diverse and relies on the principles of microfilm technology and surface chemistry: (1) when applied to explants, the composition forms a microfilm that functions as an effective physical barrier against microorganisms (e.g., bacteria, fungi). The composition is an aqueous suspension of colloidal silver in a matrix of protective methyl vinyl ether copolymer, in which the colloidal silver associated with the methyl vinyl ether copolymer forms an effective protective microfilm. The microfilm is produced by a polymer, and after the microfilm is formed, the embedded colloidal silver comes into direct contact with microorganisms (e.g., bacteria, fungi) on the surface of the explant; (3) when applied near fungi and bacteria on the surface of the explant or nutrient medium or solution used to sterilize the explant, the composition neutralizes enzymes used by microorganisms to metabolize oxygen, and (4) simultaneously changes the permeability of membranes of unicellular organisms, thereby inducing effective asphyxiation of the microorganisms.

[0025] One of the advantages of the disclosed composition is that the methyl vinyl ether copolymer used in the formulation creates an invisible mesh that holds the components together without interfering with the normal functionality of the explant tissue and without allowing cell regeneration and proliferation.

[0026] In some embodiments, the method can include obtaining an explant, which can originate from another in vitro cultured plant, a living plant, a fragment of a living plant, a seed, or another form of plant tissue, which can be selected from plant stocks maintained for culture initiation. The plant tissue can be sterilized using a substance that helps remove microbial contamination already present on or in the explant. This process often begins with scrubbing with soap and water, followed by treatment with a disinfectant. In some embodiments, the composition can be used in addition to or instead of a commonly used disinfectant. The sterilized explant can be prepared for inoculation in culture medium. This preparation consists of chopping the explant to expose the desired tissue and removing undesired tissue. The preparation process can be performed in a sterile environment, such as in a laminar flow hood. However, when using the compositions of the present invention, the process can be performed without a laminar flow hood. In some embodiments, the method can further include inoculating the processed plant explant into culture medium using a sterile container. This step can be carried out in a sterile environment, such as in a laminar flow hood. However, when using the compositions of the present invention, the process can be carried out without a laminar flow hood. The container containing the plant explants therein can be transferred to an environment with suitable growth conditions, such as an incubation chamber that provides appropriate temperature and light conditions. Additionally and / or optionally, after the explants are inoculated into the culture medium, a solution of the composition described herein can be added onto the explants, thereby forming a barrier (e.g., a microfilm) that will protect the explants against microbial contamination.

[0027] In some embodiments, the method includes pretreating a plant or explant with the composition. Surface sterilization of plant material prior to introduction into sterile culture medium is a critical step in establishing plant tissue culture protocols. Heavy microbial contamination present on plant material and explants makes in vitro culture establishment problematic. This contamination is usually overcome through effective surface sterilization of the plant material, among other aseptic techniques. Explants are treated for surface sterilization through a process involving immersion of the explant in an appropriate concentration of a chemical sterilant or fungicide for a specified period of time. This treatment results in the establishment of cultures with little or reduced levels of contamination. Various types of fungicides can be used, including, but not limited to, ethanol, isopropyl alcohol, sodium hypochlorite (NaOCl) / calcium (Ca(ClO)), hydrogen peroxide (H2O2), mercuric chloride (HgCl2), silver nitrate (AgNO3), and bromine water. The type, concentration, and exposure time of the fungicide required will vary for different plant species and plant parts. Ethanol is a very powerful sterilant, but it is also phytotoxic, causing damage to the explant and reducing the chances of successful introduction. Follow-up treatment with another disinfectant is generally required. Hypochlorite and related compounds are also very effective in significantly reducing microbial populations on explants, even at micromolar concentrations, but are also aggressive to plant tissue.

[0028] Effective surface pretreatment of plant tissue introduced into the culture medium significantly increases the chances of success. Reducing the microbial load in the explant will reduce the occurrence of microbial contamination in subsequent steps of the process.

[0029] In some embodiments, the method comprises applying the composition to a surface of the plant or explant, hi some embodiments, the composition is applied as a spray, mist, or drops.

[0030] In some embodiments, the composition comprises 0.1-20% (w / v) colloidal silver, 1.75-4.38% (w / v) methyl vinyl ether copolymer, 0.02-0.04% (w / v) polyoxyethylene octylphenyl ether, 0.02-0.04% (w / v) sodium hydroxide, and 89-95% (w / v) water. In some embodiments, the composition comprises about 5.00% (w / v) colloidal silver, about 3.50% (w / v) methyl vinyl ether copolymer, about 0.099% (w / v) polyoxyethylene octylphenyl ether, about 0.032% (w / v) sodium hydroxide, and about 91.36% (w / v) water.

[0031] In some embodiments, the water is distilled or deionized. The colloidal silver contained in the composition has high shelf life and can therefore be used diluted in tap water or distilled water. As a result, the composition is easier to handle and has reduced costs.

[0032] In some embodiments, the copolymer may be any one of methyl vinyl ether / maleic anhydride, poly(methyl methacrylate) (PMMA), polyether-silicone, iso-dimethicone, methylene diphenyl diisocyanate, phenylenediamine, polyvinyl acetate, ethylene vinyl acetate (EVA), polyvinyl alcohol, polyvinylpyrrolidone, vinyl chloride, vinylidene chloride copolymer, calcium lignosulfonate, acrylic acid copolymer, polyvinyl acrylate, polyethylene oxide, acylamide, polyhydroxyethyl acrylate, and the like.

[0033] In some embodiments, the surfactant may be any one of polyoxyethylene glycol octylphenol ether, polyoxyethylene glycol alkylphenol ether, polysorbate, sorbitan alkyl ester, polyethylene glycol, polypropylene glycol, Prefer 28 (CENEX), SurfN (US), Inhance (BRANDT), P-28 (WILFARM), and Patrol (HELENA); esterified seed oils include SunIt II (AMCY), MSO (UAP), Scoil (AGSCO), Hasten (WILFARM), and Mes-100 (DREXEL); and organosilicone surfactants include Silwet L77 (UAP), Silikin (TERRA), Dyne-Amic (HELENA), Kinetic (HELENA), Sylgard 309 (WILBUR-ELLIS), and Century (PRECISION).

[0034] In some embodiments, a pH adjuster such as sodium hydroxide may be used to neutralize the composition. It will be appreciated that other bases (e.g., strong or weak bases) may be used to neutralize the composition, including, but not limited to, lithium hydroxide, potassium hydroxide, cesium hydroxide, calcium hydroxide, zinc hydroxide, and ammonium hydroxide.

[0035] In some embodiments, the colloidal silver has the following characteristics: (a) silver suspended in distilled water and produced by dispersion according to published guidelines (NIST, 2012) or by electrical methods at a silver electrode; (b) has an atomic mass of 107,868 g / mol; (c) has a melting point of 960.5°C; (d) has a boiling point of 2000°C; (e) has a density of 10.49 g / mL at 15°C; (f) is not attacked by water or atmospheric oxygen; (g) becomes tarnished by ozone and hydrogen sulfide; (h) is inert to many acids and reacts readily with dilute nitric acid and hot sulfuric acid; and (i) is not sensitive to light in its metallic form.

[0036] In some embodiments, the compositions are provided in concentrated form (e.g., stock solution), powder form, or hydrogel form (or concentrated gel form) and can be further formulated into plant tissue culture medium, for example, by dilution in a liquid, which can be water, such as distilled or deionized water.

[0037] In some embodiments, the compositions used in the methods disclosed above can be prepared by a multi-step process. The process begins with the preparation of a colloidal silver concentrate. 100 liters of distilled or deionized water are poured into a stainless steel container and a polyethylene hose connected to a water pump is inserted. The water pump drives the water into a colloidal silver generator connected to a 110V voltage source. The generator consists of a pump to control the amount of water entering the electrodes and another pump to regulate the amount of water leaving the system. A second water pump connected to a 110V outlet recirculates the colloidal silver-containing water in the system for 1 minute to increase the silver concentration, thus ensuring that the final product contains the required silver concentration. The controlled size of the colloidal silver is obtained by filtration. A solution is then prepared by placing 89.2 to 95.7 kg of water in a 100 L stainless steel vessel and slowly adding 2.50 to 6.25 kg of colloidal silver concentrate with stirring; followed by adding 50 to 120 grams of polyoxyethylene octylphenyl ether and 1.75 to 4.38 kg of methyl vinyl ether copolymer, where the methyl vinyl ether copolymer was prepolymerized in a stainless steel vessel containing 1.75 to 4.38 L of deionized water at 40° C.; followed by adding 1 L of 20 to 40 g / L sodium hydroxide solution and continuously stirring until the resulting mixture is clear.

[0038] In some embodiments, the composition is prepared by the following steps: (a) placing 91.4 kg of water into a 100 L stainless steel vessel; (b) slowly adding 5 kg of colloidal silver concentrate with stirring; (c) adding 99 grams of polyoxyethylene octylphenyl ether; (d) adding 3.5 kg of methyl vinyl ether copolymer, which has been pre-polymerized in a stainless steel vessel containing 3.5 L of deionized water at 40° C.; (e) adding 1 L of 35 g / L sodium hydroxide solution; and (f) continuously stirring until the resulting mixture is clear.

[0039] The process for preparing the composition can utilize a generator such as a Robey device. The generator uses a 110V voltage source, a current controller, two silver electrodes, and a cellulose filter. The cellulose filter restricts the passage of silver colloids with an average particle size of less than about 60 nm and more than about 600 nm.

[0040] In some embodiments, the colloidal silver particles have an average particle size between about 60 nm and about 600 nm. In some embodiments, at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, 100%) of the colloidal silver particles have a particle size between about 60 nm and about 600 nm. In some embodiments, at least 90% of the colloidal silver particles have a particle size between about 60 nm and about 600 nm. In some embodiments, at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, 100%) of the colloidal silver particles in the composition are present as nanoparticles having a particle size between about 60 nm and about 100 nm.

[0041] In some embodiments, the colloidal silver particles have an average particle size between about 60 nm and about 140 nm. In some embodiments, at least 50% of the colloidal silver particles have a particle size between about 60 nm and about 140 nm. In some embodiments, at least 90% of the colloidal silver particles have a particle size between about 60 nm and about 140 nm.

[0042] In some embodiments, the colloidal silver particles may have a maximum dimension (e.g., diameter) between about 60 nm and about 600 nm (e.g., between about 60 nm and about 500 nm, between about 60 nm and about 400 nm, between about 60 nm and about 300 nm, between about 60 nm and about 200 nm, between about 60 nm and about 140 nm, between about 60 nm and about 100 nm).

[0043] Colloidal silver particles can have a variety of different shapes, including spheres, oblate spheroids, cylinders, ovals, ellipsoids, shells, cubes, cuboids, cones, pyramids, rods (e.g., cylinders or elongated structures having a square or rectangular cross section), tetrapods (particles with four leg-like appendages), triangles, prisms, etc.

[0044] In some embodiments, one or more substantially uniform populations of colloidal silver particles are used, for example, two, three, four, five or more substantially uniform populations having distinguishable characteristics (e.g., size, optical properties). It will be understood that a combination of two or more populations having distinguishable characteristics may be considered a single population.

[0045] As used herein, "tissue culture process," "tissue culturing process," or "culturing plant tissue" refers to any process for germinating seeds or otherwise propagating or differentiating plants, plant organs, plant tissues, or plant cells. In some embodiments, plant tissue cultures in culture media (e.g., solid or liquid media) can be further subcultured. Plant tissue cultures can be maintained in defined or undefined media, typically under sterile (aseptic) conditions. However, the methods disclosed above advantageously allow for maintaining tissue cultures under non-aseptic conditions.

[0046] Plant tissue culture media can contain suitable concentrations of plant hormones or a suitable mixture of mineral salts, such as auxins, cytokinins, or gibberellins, one or more vitamins, such as vitamin B, one or more carbon sources, including, for example, sucrose or glucose, and one or more growth promoters of unknown composition, such as coconut milk.

[0047] The components of the mineral salt mixture can be selected according to the requirements of the specific plant species being propagated and prepared. The appropriate composition of the mineral salts can be determined experimentally or selected from mineral salts already known in the plant tissue culture field. Alternatively, the mineral salts can be selected from commercially available mixtures (e.g., from Sigma Chemical Co., St. Louis, MO, USA). In addition, other macronutrient and vitamin components can be combined in various ways to produce a medium suitable for the type of plant being propagated.

[0048] According to the methods disclosed herein, colloidal silver-based compositions are added to plant tissue culture media at a concentration that reduces or prevents bacterial or fungal growth, or both, and allows for normal germination of the seed or propagation of the plant, plant organ, plant tissue, or plant growing on the seed, and promotes normal cell growth and development (e.g., substantially normal seed germination).

[0049] In some embodiments, plant tissue culture media may be prepared by combining its components according to established protocols for the plant species being used. The media should generally contain one or more of the following components: macronutrients (such as N, P, K, S, Mg, and Ca), micronutrients (such as Cu, Fe, Zn, Mn, B, and Mo), vitamins, amino acids or nitrogen supplements, carbon sources, organic additives, growth regulators, and solidifying agents.

[0050] There are several commonly used basal media of known composition, including Murashige-Skoog (MS) medium, Linsmeyer-Skoog (LS) medium, Gamborg (B5) medium, and Nitsch-Nitsch (NN) medium. The culture medium used for a particular process may be one of these basal medium recipes with specific additions of one or more of the following components: macronutrients (such as N, P, K, S, Mg, and Ca), micronutrients (such as Cu, Fe, Zn, Mn, B, and Mo), vitamins, amino acids or nitrogen supplements, carbon sources, organic additives, growth regulators, and coagulants.

[0051] The amount (or final concentration) of the compositions described herein used in culture media, disinfectant solutions, or other applications can vary for each use case. For example, it can depend on the plant species used (some are more susceptible to contamination), the source of the explants (some are dirtier than others), and the technique used, among other variables. In some embodiments, the culture media can contain between about 0.01% (w / v) and about 25% (w / v) of the composition. Similarly, the compositions can be used at concentrations between about 0.01% (w / v) and about 25% (w / v) when applied using a dropper or used as a disinfectant solution or other application.

[0052] In some embodiments, methods of using colloidal silver-based compositions in plant tissue culture processes reduce the rate of bacterial or fungal contamination by at least 20% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%) compared to control medium lacking the composition.

[0053] As used herein, substantially normal seed germination is defined as a percent germination that is at least 50% of a control that does not contain the composition in the medium.

[0054] In some embodiments, methods of using colloidal silver-based compositions in plant tissue culture processes achieve an increased growth rate or increased cell division rate of plant cells, plants, plant organs, or plant tissues that is at least 20% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%) greater than a corresponding control that does not contain the composition.

[0055] In some embodiments, the effectiveness of the method may be studied using morphological, anatomical, physiological, and biochemical assays known in the art. For example, morphological analysis may include comparing the shape, size, or number of roots, shoots, leaves, or reproductive organs, or portions thereof. Anatomical analysis may include comparative analysis of the size, shape, pattern, or cellular differentiation, such as the amount, location, or maturation of vascular tissue, trichomes, or stomatal pores, or the presence or absence of actively dividing meristems. Physiological analysis may involve comparative analysis of respiration, biosynthesis, stomatal resistance, or ethylene production rates. Biochemical analysis may include comparative analysis of protein or DNA synthesis, chlorophyll degradation, or the presence, absence, or amount of other pigments. These analyses may also be used to determine or select optimal concentrations of compositions in tissue culture media and, therefore, implement the method for a particular plant species.

[0056] In some embodiments, microbial contamination may be caused by any undesirable microorganism, such as a fungus or a bacterium.

[0057] The fungus may be one of the fungi that cause contamination in tissue culture processes, including, but not limited to, Blumeria, Sphaerotheca, Phytophthora, Rhizoctonia, Fusarium, Penicillium, Aspergillus, Colletotrichum, Botrytis, Magnaporthe, Pythium, Puccinia, Erysiphe, Alternaria, Pseudoperonospora, Plasmodiophora, Sclerotinia, Fulvia, Peronospora, Ustilago, and Rhizopus. The composition inhibits the growth and development of both gram-positive and gram-negative bacteria.

[0058] In some embodiments, exemplary bacteria include, but are not limited to, Corynebacterium, Bacillus, Staphylococcus, Escherichia, Pseudomonas, Xanthomonas, Erwinia, Clavibacter, Ralstonia, Burkholderia, and Agrobacterium.

[0059] In some embodiments, the compositions further comprise a carrier that is compatible or acceptable for tissue culture processes. The compatible or acceptable carrier may be a nutrient or a surfactant. Also within the scope of the present disclosure is a tissue culture nutrient medium formulated from the compositions as described for controlling microbial contamination. In embodiments, the nutrient medium can further comprise a carrier that is acceptable for tissue culture processes. Also within the scope of the present disclosure is a disinfectant solution formulated from the compositions as described for controlling microbial contamination in explants prior to their introduction into the nutrient medium.

[0060] "Carrier acceptable for tissue culture processes" or "carrier compatible or acceptable for tissue culture processes" means any material, other than water, that can be added to a composition without causing or having an adverse effect on plants, explants, etc. In some embodiments, the carrier may be in a variety of forms, including solid or liquid carriers, microspheres, powders, emulsions, etc. The carrier may be any one or more of a number of carriers that impart various properties, such as increased stability, wettability, or dispersibility. In some embodiments, the carrier includes solidifying agents such as glycerol, cellulose, PEG, natural substances or extracts (protein hydrolysates, coconut milk, yeast extract, malt extract, etc.), activated charcoal, agar, agarose, and gellan gum.

[0061] In some embodiments, exemplary carriers include, but are not limited to, alginates, gums, starches, lecithin, formononetin, polyvinyl alcohol, alkali formononetinate, hesperetin, polyvinyl acetate, cephalin, gum arabic, xanthan gum, mineral oil, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), 1-arabinogalactan, methylcellulose, PEG 400, chitosan, polyacrylamide, polyacrylate, polyacrylonitrile, glycerol, triethylene glycol, vinyl acetate, gellan gum, polystyrene, polyvinyl, carboxymethylcellulose, gum ghatti, and polyoxyethylene-polyoxybutylene block copolymers. Carriers may also be non-naturally occurring compounds, such as polymers and copolymers. For example, non-limiting examples of polymers that can be used as adhesives include: polyvinyl acetate, polyvinyl acetate copolymers, ethylene vinyl acetate (EVA) copolymers, polyvinyl alcohol, polyvinyl alcohol copolymers, cellulose (e.g., ethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, and carboxymethyl cellulose), polyvinylpyrrolidone, vinyl chloride, vinylidene chloride copolymers, calcium lignosulfonate, acrylic acid copolymers, polyvinyl acrylate, polyethylene oxide, acylamide polymers and copolymers, polyhydroxyethyl acrylate, methylacrylamide monomer, and polychloroprene. In some embodiments, the composition may also contain a surfactant.Non-limiting examples of surfactants include nitrogen surfactant blends such as Prefer 28 (CENEX), SurfN (US), Inhance (BRANDT), P-28 (WILFARM), and Patrol (HELENA); esterified seed oils include SunIt II (AMCY), MSO (UAP), Scoil (AGSCO), Hasten (WILFARM), and Mes-100 (DREXEL); and organosilicone surfactants include Silwet L77 (UAP), Silikin (TERRA), Dyne-Amic (HELENA), Kinetic (HELENA), Sylgard 309 (WILBUR-ELLIS), and Century (PRECISION). In some embodiments, the surfactant is present at a concentration of between 0.001% v / v and 10% v / v (e.g., between 0.001% v / v and 1% v / v). In some embodiments, the composition may include a stabilizer. Such agents include one or more of trehalose, sucrose, glycerol, and methylene glycol. Other suitable stabilizers include, but are not limited to, non-reducing sugars and sugar alcohols (e.g., mannitol or sorbitol).

[0062] In some embodiments, the composition further comprises a second fungicide and / or a second bactericide. In some embodiments, it may be advantageous for the composition to include an agent such as a fungicide, antibacterial agent, or nutrient. The agent ideally does not raise safety concerns for human, animal, or industrial use (e.g., there are no safety issues or the compound is sufficiently unstable so that commercial plant products derived from tissue culture processes contain negligible amounts of the compound). As used herein, "fungicide" includes commercially available synthetic compounds designed to protect plants and explants from pathogenic fungi. Examples of fungicides include, but are not limited to, the following: 2-(thiocyanatomethylthio)-benzothiazole, 2-phenylphenol, 8-hydroxyquinoline sulfate, ametoctrazin, amisulbrom, antimycin, Ampelomyces quisqualis, azaconazole, azoxystrobin, Bacillus subtilis), benalaxyl, benomyl, benthiavalicarb-isopropyl, benzylaminobenzenesulfonic acid (BABS) salt, bicarbonate, biphenyl, bismerthiazole, bitertanol, bixafen, blasticidin S, borax, Bordeaux mixture, boscalid, bromuconazole, bupirimate, calcium polysulfide, captafol, captan, carbendazim, carboxin, carpropamid, carvone, chloroneb, chlorothalonil, chlozolinate, Coniothyrium minitans minitans), copper hydroxide, copper octanoate, copper oxychloride, copper sulfate, copper sulfate (tribasic), cuprous oxide, cyazofamid, cyflufenamid, cymoxanil, cyproconazole, cyprodinil, dazomet, debacarb, diammonium ethylenebis-(dithiocarbamate), dichlofluanid, dichlorophen, diclocymet, diclomedine, dicloran, diethofencarb, difenoconazole, difenzoquat ion, diflumetrim, dimethomorph, dimoxystrobin, diniconazole, diniconazole-M, dinobuton, dinocap, diphenylamine, dithianon, dodemorph, dodemorph acetate, dodine, dodine free base, edifenphos, enestrobin, epoxyConazole, ethaboxam, ethoxyquin, etridiazole, famoxadone, fenamidone, fenarimol, fenbuconazole, fenfuram, fenhexamid, fenoxanil, fenpiclonil, fenpropidin, fenpropimorph, fentin, fentin acetate, fentin hydroxide, ferbam, ferimzone, fluazinam, fludioxonil, flumorph, fluopicolide, fluopyram, fluoroimide, fluoxastrobin, fluquinconazole, flusilazole, flusulfamide, flutianil, flutolanil, flutri Aphor, fluxapyroxad, folpet, formaldehyde, fosetyl, fosetyl-aluminum, fuberidazole, furalaxyl, furametpyr, guazatine, guazatine acetate, GY-81, hexachlorobenzene, hexaconazole, hymexazole, imazalil, imazalil sulfate, imibenconazole, iminoctadine, iminoctadine triacetate, iminoctadine albesilate, ipconazole, iprobenfos, iprodione, iprovalicarb, isoprothiolane, isopyrazam, isotianil, kasugamycin, kasugamycin hydrochloride Hydrate, kresoxim-methyl, mancopper, mancozeb, mandipropamid, maneb, mepanipyrim, mepronil, mercuric chloride, mercuric oxide, mercurous chloride, metalaxyl, mefenoxam, metalaxyl-M, metam, metam-ammonium, metam-potassium, metam-sodium, metconazole, metasulfocarb, methyl iodide, methyl isothiocyanate, metiram, metominostrobin, metrafenone, mildiomycin, myclobutanil, nabam, nitrothar-isopropyl, nuarimol, octilinone, Ophuras, oleic acid (fatty acid), orysastrobin, oxadixyl, oxine copper, oxpoconazole fumarate, oxycarboxin, pefurazoate, penconazole, pencycuron, penflufen, pentachlorophenol, pentachlorophenyl laurate, penthiopyrad, phenylmercuric acetate, phosphonic acid, phthalide, picoxystrobin, polyoxin B, polyoxin, polyoxorim, potassium bicarbonate, potassium hydroxyquinoline sulfate, probenazole, prochloraz, procymidone, propamocarb, propamocarb hydrochloridePropiconazole, propineb, proquinazid, prothioconazole, pyraclostrobin, pyrametostrobin, pyraoxystrobin, pyrazophos, pyribencarb, pyributicarb, pyrifenox, pyrimethanil, pyroquilon, quinoclamine, quinoxyfen, quintozene, Reynoutria saccharinensis (Reynoutria sachalinensis extract, sedaxane, silthiofam, simeconazole, sodium 2-phenylphenoxide, sodium bicarbonate, sodium pentachlorophenoxide, spiroxamine, sulfur, SYP-Z071, SYP-Z048, tar oil, tebuconazole, tebufloquine, tecnazene, tetraconazole, thiabendazole, thifluzamide, thiophanate-methyl, thiram, tiadinil, tolclofos-methyl, tolylfluanid, triadimefon, triadimenol, triazoxide, tricyclazole, tridemorph, trifloxystrobin, triflumizole, triforine, triticonazole, validamycin, valifenalate, valifenal, vinclozolin, zineb, ziram, zoxamide, Candida oleophila, Fusarium oxysporum oxysporum, Gliocladium spp., Phlebiopsis gigantea, Streptomyces griseoviridis, Trichoderma spp. spp.), (RS)-N-(3,5-dichlorophenyl)-2-(methoxymethyl)-succinimide, 1,2-dichloropropane, l,3-dichloro-l,l,3,3-tetrafluoroacetone hydrate, 1-chloro-2,4-dinitronaphthalene, 1-chloro-2-nitropropane, 2-(2-heptadecyl-2-imidazolin-1-yl)ethanol, 2,3-dihydro-5-phenyl-l,4-dithi-ine 1,1,4,4-tetraoxide, 2-methoxyethylmercury acetate, 2-methoxyethylmercury chloride, 2-methoxyethylmercury silicate, 3-(4-chlorophenyl)-5-methylrhodanine,4-(2-nitroprop-1-enyl)phenyl thiocyanateme, ampropylphos, anilazine, azithiram, barium polysulfide, Bayer 32394, benodanil, benquinox, bentalon, benzamacryl; benzamacryl-isobutyl, benzamform, binapacryl, bis(methylmercury) sulfate, bis(tributyltin) oxide, buthiobate, cadmium calcium copper zinc chromate sulfate), carbamorph, CECA, clobentiazone, chloraniformethane, chlorphenazole, chlorquinox, climbazole, cyclafuramid, cypendazole, cyproflam, decaphentin, dicloron, diclozolin, diclobutrazol, dimethirimol, dinoctone, dinosulfone, dinotervone, dipyrithione, ditalimfos, dodisin, drazoxolone, EBP, ESBP, etaconazole, ethem, ethilim, fenaminosulf, fenapanil, fenitropan, 5-fluorocytosine and its profungicides, fluotrimazole, flucarbanil, fluconazole, fluconazole-cis, flumecyclox, furofanate, gliodin, griseofulvin, halacrinate, Hercules 3944, hexylthiophos, ICIA0858, isopamphos, isovaledion, mebenil, mecarbinzide, metazoxolone, metofloxam, methylmercuric dicyandiamide, metsulfovax, milneb, mucochloroic anhydride, myclozolin, N-3,5-dichlorophenyl-succinimide, N-3-nitrophenylitaconimide, natamycin, N-ethylmercurio-4-toluenesulfonanilide, bis(dimethicone) Nickel ethyldithiocarbamate, OCH, phenylmercuric dimethyldithiocarbamate, phenylmercuric nitrate, phosdifen, picolinamide UK-2A and its derivatives, prothiocarb; prothiocarb hydrochloride, pyracarbollide, pyridinnitrile, pyroxychlor, pyroxyflur, quinacetol; quinacetol sulfate, quinazamide, quinconazole, labenzazole, salicylanilide, SSF-109, sultropene, tecorum,Thiadifluor, thithiofen, thiochlorfenfim, thiophanate, thioquinox, tioximide, triamiphos, triarimol, triazbutyl, triclamide, urvacid, XRD-563, and zaliramide, IK-1140. Examples of bactericides include, but are not limited to, amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, spectinomycin, geldanamycin, herbimycin, rifaximin, streptomycin, loracarbef, ertapenem, doripenem, imipenem / cilastatin, meropenem, cefadroxil, cefazolin, cephalotin (Cefalotin or Cefalothin), cephalexin, cefazolin, cephalosporin ... Cefazlor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftaroline, fosamil, ceftobiprole, teicoplanin, vancomycin, telavancin, clidamycin, lincomycin, daptomycin, azithromycin, clarithromycin, dirithromycin, erythromycin Syn, roxithromycin, troleandomycin, telithromycin, spiramycin, aztreonam, furazolidone, nitrofurantoin, linezolid, pocizolid, radezolid, trezolid, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin 2, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, penicillin G, temocillin, ticarcillin, amoxicillin Cialis / clavulanate, ampicillin / sulbactam, piperacillin / tazobactam, ticarcillin / clavulanate, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin, 2-mafenide, sulfacetamide, sulfadiazine,Silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilimide (initial stage), sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole (co-trimoxazole) (TMP-SMX), sulfonamide chrysoidine (initial stage), demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, clofazimine, dapsone, capreomycin These include cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin (known as rifampin in the US), rifabutin, rifapentine, streptomycin, arsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin / dalfopristin, thiamphenicol, tigecycline, tinidazole, and trimethoprim.

[0063] In some embodiments, the method can include applying the composition to the surface of a plant or explant (e.g., leaves, fruits, flowers, stems, roots, meristems, calluses, shoots, embryos, seeds). The method can selectively control fungi and / or bacteria in plants at very low concentrations. Additionally, once the composition is applied, the protective effect can last for 1-3 weeks or longer. The composition can control both spores and hyphae, and even when applied at high concentrations, it is non-chemically damaging and non-toxic to humans and plants.

[0064] In some embodiments, the method may include applying the composition to a nutrient medium of a tissue culture process. In some embodiments, the method may include applying the composition as a spray, mist, or drops. The composition can be mixed with an acceptable carrier or diluent and thus formulated into a variety of formulations, including as an additive to a nutrient medium or tissue culture process. In addition, the composition can be mixed with additional ingredients or surfactants or other known agents for controlling microbial contamination. The term "diluent" refers to an acceptable liquid or solid that is added to the composition so that it can be used immediately or diluted to a desired active concentration. Examples of diluents include talc, kaolin, zeolite, xylene, diatoms, water, etc.

[0065] Formulations for use in spray form, such as water-dispersible concentrates or wettable powders, can further include wetting agents, dispersants, surfactants, etc. In addition to diluents and surfactants, stabilizers, deactivators, adhesion improvers, colorants, coagulants, wetting agents, and defoamers can be additionally included. The disclosed compositions can be formulated in various forms. For example, wettable powder forms prepared with kaolin or diatoms can be diluted with water before use as a spray and can therefore be sprayed onto explants. Furthermore, the compositions can be mixed with emulsifiers to obtain concentrates, which are subsequently diluted with water before application to explants.

[0066] definition For the purpose of aiding in the understanding of the detailed description of the compositions and methods according to the present disclosure, some specific definitions are provided to facilitate a clear disclosure of the various aspects of the present disclosure. 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 disclosure belongs.

[0067] The terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the probability of developing a disorder or condition in a subject (e.g., a plant) that does not have the disorder or condition but is at risk of or susceptible to developing the disorder or condition.

[0068] The terms "reduce," "reducing," "reduction," "decrease," or "inhibit" are all used broadly herein to mean a decrease by a statistically significant amount. However, for the avoidance of doubt, "reduced," "reduction," or "reduce" or "inhibit" means a decrease of at least 10% compared to a reference level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%; or a decrease of up to and including 100% (e.g., an absent level compared to a reference sample), or any decrease between 10-100% compared to a reference level.

[0069] The terms "treating" or "treatment" refer to the administration of a compound or agent to a subject (e.g., a plant) having or at risk of developing a disorder, for the purpose of curing, alleviating, mitigating, treating, delaying the onset of, preventing, or ameliorating the disorder, a symptom of the disorder, a disease state secondary to the disorder, or a predisposition to the disorder.

[0070] The word "substantially" does not exclude "completely", for example, a composition that is "substantially free" of Y may be completely free of Y. If necessary, the word "substantially" may be omitted from the definition of the invention.

[0071] As used herein, the term "approximately" or "about," when applied to one or more values ​​of interest, refers to a value similar to a specified reference value. In some embodiments, the term "approximately" or "about" refers to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the specified reference value in either direction (greater than or less than) unless otherwise specified or clear from the context (except where such number exceeds 100% of the possible value). Unless otherwise indicated herein, the term "about" is intended to include values ​​(e.g., percent by weight) that approximate the specified range that are equivalent with respect to the functionality of the individual component, composition, or embodiment.

[0072] As disclosed herein, numerous ranges of values ​​are provided. It is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of such a range is also specifically disclosed, unless the context clearly dictates otherwise. Each smaller range between any stated value or intervening value of a stated range, and any other stated value or intervening value within a stated range, is encompassed within the scope of the invention. The upper and lower limits of these smaller ranges may independently be included or excluded, and each range in which either limit, neither limit, or both limits are included in the smaller range is also encompassed within the scope of the invention, subject to any specifically excluded limit within the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0073] It is hereby noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The terms "including," "comprising," "containing," or "having" and variations thereof are meant to encompass the items listed thereafter and equivalents thereof, as well as additional subject matter unless otherwise noted.

[0074] The phrases "in one embodiment," "in various embodiments," "in some embodiments," etc. are used repeatedly. Such phrases do not necessarily refer to the same embodiment, but they may, unless the context dictates otherwise.

[0075] The term "and / or" or " / " means any one of the items, any combination of the items, or all of the items with which this term is associated.

[0076] As used herein, the term "each," when used in reference to a collection of items, is intended to identify each individual item in the collection, but does not necessarily refer to every item in the collection. Exclusions may occur where clear disclosure or context clearly dictates otherwise.

[0077] Any and all examples provided herein, or the use of exemplary language (e.g., "etc.") are intended only to better clarify the invention and do not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0078] All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted otherwise by context. For any of the methods provided, the method steps may occur simultaneously or sequentially. When method steps occur sequentially, the steps may occur in either order unless otherwise indicated.

[0079] Where the method includes a combination of steps, each and every combination or subcombination of the steps is encompassed within the scope of the present disclosure unless stated otherwise herein.

[0080] As used herein, section headings are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0081] Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety to the extent not inconsistent with this disclosure. The publications disclosed herein are provided solely for their disclosure prior to the filing date of the present invention. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0082] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are to be included within the spirit and purview of the present application and the scope of the appended claims. [Example]

[0083] Example 1 Preparations of the disclosed compositions were added to standard Murashige and Skoog tissue culture nutrient medium and used for the introduction of Musa sp. explants. Introduction followed standard in vitro introduction protocols for this plant species. Different treatments were used and prepared by adding amounts ranging from 0.5% (w / v) to 2% (w / v) of the compositions of the present invention to the culture medium. After 25 days of incubation under recommended conditions, a 0% incidence of microbial contamination was observed, compared to 27% in the control group. Using a 0.5% (w / v) preparation of the disclosed compositions according to the present invention, only 3% of the evaluated materials were observed to show signs of microbial contamination. The incidence of bacterial and fungal infections was significantly reduced in explants introduced in nutrient medium containing the compositions, and no new infections were observed during subsequent steps of the tissue culture process.

[0084] Example 2 A solution was prepared by adding 1 part of the composition to 9 parts of distilled water to obtain a 10% (w / v) solution. Such a solution was applied to in vitro Cordyline plants, which were removed from the in vitro introduction and transferred to an in vitro nutrient medium intended for shoot propagation. Application was carried out by using a sprayer to coat the surface of the in vitro plants and a method that had been proven applicable and practical. No phytotoxicity symptoms were observed on the treated in vitro plants. Application of the composition of the present invention showed a significant increase in plant growth without any signs of microbial contamination.

[0085] Example 3 In accordance with one embodiment of the present invention, a liquid formulation containing 5% (w / v) of the composition of the present invention was prepared and used as a sterilizing solution to prepare explants of the bamboo plant species (Guadua angustifolia) prior to the in vitro transfer process. The composition of the present invention was diluted in distilled water to the desired concentration and placed in an ultrasonic water bath. Explants obtained from plants grown under greenhouse conditions were placed in the water bath and incubated for 15 minutes. The explants were then transferred to nutrient culture medium under a laminar flow hood. Controls were subjected to a chlorine-based sterilization method as described in the literature. Compared to the control, a two-fold reduction in microbial contamination was observed at the end of the transfer process in explants sterilized with the composition of the present invention. None of the explants treated with the composition of the present invention showed signs of chemical damage.

[0086] Example 4 Citrus somatic embryos obtained from suspension cell culture were placed on an agar nutrient medium containing 1% (w / v) of the composition of the present invention. In addition, the inoculated suspension was covered with a solution prepared to contain 0.5% (w / v) of the composition of the present invention. After the incubation period and conditions recommended in the literature, the inventors, using standard procedures for the technology, observed a 70% lower incidence of microbial contamination compared to a control not treated with the composition of the present invention.

[0087] Example 5 Dried seeds of several plant species (including mustard, pumpkin, and radish) were purchased from a commercial source and transferred to standard Murashige and Skoog tissue culture nutrient medium prepared with 10% (w / v) of the composition of the present invention. The nutrient medium was placed in glass vials without sterilization procedures such as autoclaving. The seeds were placed on the unsterilized nutrient medium and incubated using standard growth conditions. The entire transfer process was performed on a non-sterile laboratory bench without a laminar flow hood. After 8 days of incubation, more than 80% of the glass vials showed successful seed germination without signs of microbial contamination, compared to the control medium without the addition of the composition of the present invention, in which 100% of the glass vials showed signs of excessive microbial contamination.

[0088] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and examples, while indicating specific embodiments of the present invention, are given for illustrative purposes only. Additionally, it is contemplated that changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description.

Claims

1. 1. A method for reducing or preventing microbial contamination of plants or explants in a tissue culture process, comprising: 0.1 to 20% (w / v) colloidal silver, 1.75 to 4.38% (w / v) methyl vinyl ether copolymer, 0.02 to 0.099% (w / v) polyoxyethylene octylphenyl ether, 0.02-0.04% (w / v) sodium hydroxide, and 89-95% (w / v) water 10. A method comprising culturing a plant or explant in a plant tissue culture medium formed from a colloidal silver-based composition comprising:

2. 10. The method of claim 1, further comprising pretreating the plant or explant with the composition.

3. 3. The method of claim 1 or 2, comprising applying the composition to the surface of the plant or explant.

4. 4. The method of claim 3, comprising applying the composition as a spray, mist, or drops.

5. 5. The method of any one of claims 1 to 4, comprising the step of inoculating the plant tissue culture medium with the plant or explant, followed by applying the composition onto the plant or explant, thereby forming a barrier that protects the plant or explant against microbial contamination.

6. The method of any one of claims 1 to 5, wherein the sodium hydroxide is used to neutralize the composition.

7. The colloidal silver-based composition of claim 1, Approximately 5.00% (w / v) colloidal silver, about 3.50% (w / v) methyl vinyl ether copolymer, approximately 0.099% (w / v) polyoxyethylene octylphenyl ether, about 0.032% (w / v) sodium hydroxide, and Approximately 91.36% (w / v) water The method according to any one of claims 1 to 6, comprising:

8. The colloidal silver has the following characteristics: (a) Silver suspended in distilled water, dispersed according to published guidelines (NIST, 2012), or produced by electrical methods at a silver electrode; (b) has an atomic mass of 107,868 g / mol; (c) has a melting point of 960.5°C; (d) has a boiling point of 2000°C; (e) has a density of 10.49 g / mL at 15°C; (f) not attacked by water or atmospheric oxygen; (g) Clouding due to ozone and hydrogen sulfide; (h) is inert to many acids and reacts readily with dilute nitric acid and hot sulfuric acid; and (i) insensitive to light in its metallic form The method according to any one of claims 1 to 7, comprising:

9. 9. The method of claim 1, wherein the particles of colloidal silver have an average particle size between about 60 nm and about 140 nm.

10. 10. The method of any one of claims 1 to 9, wherein at least 50% of the particles of colloidal silver have a particle size between about 60 nm and about 140 nm.

11. 11. The method of any one of claims 1 to 10, wherein at least 90% of the particles of colloidal silver have a particle size between about 60 nm and about 140 nm.

12. The method according to any one of claims 1 to 11, wherein the microbial contamination is caused by a fungus or a bacterium.

13. 13. The method of claim 12, wherein the fungus is selected from the group consisting of Blumeria, Sphaerotheca, Phytophthora, Rhizoctonia, Fusarium, Penicillium, Aspergillus, Colletotrichum, Botrytis, Magnaporthe, Pythium, Puccinia, Erysiphe, Alternaria, Pseudoperonospora, Plasmodiophora, Sclerotinia, Fulvia, Peronospora, Ustilago, and Rhizopus.

14. 13. The method of claim 12, wherein the bacterium is selected from the group consisting of Corynebacterium, Bacillus, Staphylococcus, Escherichia, Pseudomonas, Xanthomonas, Erwinia, Clavibacter, Ralstonia, Burkholderia, and Agrobacterium.

15. The method of any one of claims 1 to 14, wherein the composition further comprises a carrier that is acceptable for tissue culture processes.

16. 16. The method of any one of claims 1 to 15, wherein the composition further comprises at least one of a second fungicide and a second bactericide.

17. The composition is prepared by the following steps: (a) placing 89.2 to 95.7 kg of water into a 100 L stainless steel container; (b) slowly adding 2.50 to 6.25 kg of colloidal silver concentrate while stirring; (c) adding 50 to 120 grams of polyoxyethylene octylphenyl ether; (d) adding 1.75 to 4.38 kg of methyl vinyl ether copolymer, which has been prepolymerized in a stainless steel vessel containing 1.75 to 4.38 L of deionized water at 40°C; (e) adding 1 L of a 20-40 g / L sodium hydroxide solution; and (f) continuously stirring the resulting mixture until it becomes clear. The method according to any one of claims 1 to 16, wherein the compound is prepared by

18. The composition is prepared by the following steps: (a) Put 91.4 kg of water into a 100 L stainless steel container; (b) slowly adding 5 kg of colloidal silver concentrate while stirring; (c) adding 99 grams of polyoxyethylene octylphenyl ether; (d) adding 3.5 kg of methyl vinyl ether copolymer, which was prepolymerized in a stainless steel vessel containing 3.5 L of deionized water at 40° C.; (e) adding 1 L of 35 g / L sodium hydroxide solution; and (f) continuously stirring the resulting mixture until it becomes clear.

18. The method of claim 17, wherein the compound is prepared by