Methods of using a colloidal silver-based composition in reducing or preventing microbial contamination in plants or explants in tissue culture processes
Patent Information
- Authority / Receiving Office
- TW · TW
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2021-01-21
- Publication Date
- 2021-12-16
Abstract
Description
[Technical Field]
[0001] Field of Invention
[0002] This disclosure generally relates to methods for reducing or preventing microbial contamination of plants or explants during a tissue culture process, and more particularly to methods for using a composition based on colloidal silver to reduce or prevent microbial contamination of plants or explants during tissue culture processes (including microparticle propagation, cell culture, suspension culture, callus culture, germplasm preservation, etc.) under aseptic or non-aseptic conditions. [Previous Technology]
[0003] Background of the Invention
[0004] Tissue culture is a technique for growing plant cells by cultivating sheet-like plant tissues (also known as explants) in a suitable nutrient medium. This technique allows for the in vitro culture of plant cells and organs that can divide and regenerate into callus tissue or new plant organs. These explant systems are taken from any part of the plant and used as a starting point by introducing them into a nutrient medium to promote their growth.
[0005] During the tissue culture process, a sterile environment is generally required to avoid microbial contamination. The nutrient medium used presents a combination of micronutrients or macronutrients that can serve as a nutrient source for the explant. However, the same nutrients can also be used by microorganisms that enter the system during the introduction process or attached to the explant. Plant cell division occurs at a much slower rate than that of bacteria and fungi, which can easily proliferate on the plant tissue located within the container used for tissue culture. For this reason, in order to provide sterile materials and conditions, all materials used (glassware, containers, instruments, and the culture medium itself) must be sterilized or disinfected by methods such as autoclaving and laminar flow hoods to remove all viable microorganisms.
[0006] Generally, the sterilization of culture media and culture containers is performed using an autoclave, while the inoculation of plant tissues is performed on a clean bench equipped with a flow hood in a sterile room. Therefore, after sterilization, the culture medium or similar material must be removed from the autoclave and transferred to the sterile room. This task is cumbersome because it requires considerable care to prevent the introduction of microorganisms. Another problem is that the capacity of the autoclave or sterile room limits the amount of plant tissue that can be cultured, making it impossible to culture a large number of plant tissue lines simultaneously.
[0007] Despite the existence of methods to provide sterility, microbial contamination remains a persistent problem. The presence of endogenous microorganisms poses a significant challenge to the culture of certain explants and / or plant species. Commercial tissue culture operations often suffer a loss of a proportion of their production due to microbial contamination.
[0008] Therefore, there is an urgent need for methods and reagents for preventing and reducing contamination levels at different stages and phases of plant tissue culture. [Summary of the Invention]
[0009] Summary of the Invention
[0010] In many respects, this disclosure addresses the needs mentioned above. In one aspect, this disclosure provides a method for reducing or preventing microbial contamination of a plant or explant during a tissue culture process. The method comprises culturing a plant or explant in a plant tissue culture medium formed from a composition primarily composed of colloidal silver. The silver-based composition comprises colloidal silver, a copolymer [e.g., methyl vinyl ether copolymer], a surfactant [e.g., polyoxyethylene octylphenyl ether], an alkali (e.g., sodium hydroxide, potassium hydroxide), and water.
[0011] In some specific examples, the method further includes using the composition to pretreat the plant or explant. In some specific examples, the method includes applying the composition to the surface of a plant or explant. In some specific examples, the composition is applied by means of a spray, mist, or dropper. In some specific examples, the method includes inoculating the plant or explant in a plant tissue culture medium and then applying the composition to the plant or explant, thereby forming a barrier (e.g., a microfilm) that protects the plant or explant from microbial contamination.
[0012] In some specific examples, 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 specific examples, the composition comprises: approximately 5.00% (w / v) colloidal silver, approximately 3.50% (w / v) methyl vinyl ether copolymer, approximately 0.099% (w / v) polyoxyethylene octylphenyl ether, approximately 0.032% (w / v) sodium hydroxide, and approximately 91.36% (w / v) water.
[0013] In some specific examples, the water system is distilled or deionized. In some specific examples, the alkali (e.g., sodium hydroxide, potassium hydroxide) is used to neutralize the composition.
[0014] In certain specific examples, the colloidal silver has the following properties: (a) it is silver suspended in distilled water and is produced by dispersion according to published guidelines (NIST, 2012) or by electrical methods using silver electrodes; (b) it has an atomic mass of 107,868 g / mol; (c) it has a melting point of 960.5 °C; (d) it has a boiling point of 2000 °C; (e) it has a density of 10.49 g / mL at 15 °C; (f) it is not corroded by water or atmospheric oxygen; (g) it becomes dull by ozone and hydrogen sulfide; (h) it is inert to many acids and readily reacts with dilute nitric acid and hot sulfuric acid; and (i) it is not sensitive to light in its metallic form.
[0015] In some specific examples, the colloidal silver particles have an average particle size between about 60 nm and about 140 nm. In some specific examples, at least 50% of the colloidal silver particles have a particle size between about 60 nm and about 140 nm. In some specific examples, at least 90% of the colloidal silver particles have a particle size between about 60 nm and about 140 nm.
[0016] In some specific examples, the composition further comprises an acceptable carrier for the tissue culture process. In some specific examples, the composition further comprises at least one of a second fungicide and a second bactericide.
[0017] In some specific examples, the composition is provided in a concentrated form, in a powder form or in a hydrogel form (or concentrated gel form).
[0018] In certain specific examples, the composition is prepared by: (a) placing 89.2-95.7 kg of water into a 100 L stainless steel container; (b) slowly adding 2.50-6.25 kg of colloidal silver concentrate while stirring; (c) adding 50-120 g of polyoxyethylene octylphenyl ether; (d) adding 1.75-4.38 kg of methyl vinyl ether copolymer, wherein the methyl vinyl ether copolymer was previously polymerized at 40°C in a stainless steel container containing 1.75-4.38 L of deionized water; (e) adding 1 L of a 20-40 g / L sodium hydroxide solution; and (f) continuously stirring until the resulting mixture becomes transparent.
[0019] In some specific examples, the composition is prepared by: (a) placing 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 g of polyoxyethylene octylphenyl ether; (d) adding 3.5 kg of methyl vinyl ether copolymer, wherein the methyl vinyl ether copolymer was previously polymerized at 40°C in a stainless steel container containing 3.5 L of deionized water; (e) adding 1 L of a 35 g / L sodium hydroxide solution; and (f) continuously stirring until the resulting mixture becomes transparent.
[0020] In some specific cases, the microbial contamination is caused by fungi or bacteria. In certain specific examples, these fungal systems are selected from the group consisting of the following genera: Blumeria, Sphaerotheca, Phytophthora, Rhizoctonia, Fusarium, Penicillium, Aspergillus, Colletotrichum, Botrytis, Magnaporthe, Pythium, Puccinia, Erysiphe, Alternaria, Pseudoperonospora, Plasmodiophora, Sclerotinia, Fulvia, Peronospora, Ustilago, and Rhizopus. In certain specific examples, these bacterial strains are selected from the group consisting of: Corynebacterium, Bacillus, Staphylococcus, Escherichia, Pseudomonas, Xanthomonas, Erwinia, Clavibacter, Ralstonia, Burkholderia, and Agrobacterium.
[0021] The foregoing summary of the invention is not intended to define every aspect of the disclosure herein, while other aspects are described in other sections, such as the detailed description below. This entire document is intended to be presented as a single, unified disclosure, and it should be understood that all combinations of features described herein are contemplated, i.e., combinations of such features are not found together in the same sentence, paragraph, or section of this document. Other features and advantages of the invention will become apparent from the detailed description below. However, it should be understood that this detailed description and the specific examples, while indicating specific examples of the disclosure herein, are given by way of illustration only, as various variations and modifications falling within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
Implementation Method
[0022] Detailed Description of the Invention
[0023] This disclosure provides a method for reducing or preventing microbial contamination of a plant or explant during a tissue culture process. The method involves culturing a plant or explant in a plant tissue culture medium formed from a composition primarily composed of colloidal silver. The silver-based composition comprises colloidal silver, a copolymer (e.g., a methyl vinyl ether copolymer), a surfactant (e.g., polyoxyethylene octylphenyl ether), a pH adjuster (e.g., an alkali), and water.
[0024] The method of this invention permits the culture of plants or explants in a plant tissue culture medium under non-sterile conditions. Therefore, these methods eliminate the need for cumbersome and laborious procedures for sterilizing the culture medium and culture containers in order to create a sterile environment for the culture of plants or explants.
[0025] The antimicrobial properties of silver are widely recognized, and its efficacy as an antimicrobial agent has been demonstrated. To meet the need for the development of effective antimicrobial agents responsive to the ubiquitous and undesirable contamination caused by bacteria and fungi during tissue culture, this disclosure provides a method based on a colloidal silver-based composition that possesses the ability to prevent and control infections caused by microorganisms during tissue culture, for example, through a contact mode of action (e.g., non-systemic).
[0026] This composition contains colloidal silver as an active / effective component, wherein the colloidal silver is mixed with a polymer. When this composition is added to the nutrient medium for tissue culture, or applied directly to such explants, or used as part of the sterilization process of the explants before their introduction into the nutrient medium or at any stage of the tissue culture process, it prevents the development of contamination caused by fungi and bacteria, either by its presence within the nutrient medium or by forming a microfilm on the surface of the explants or by aiding in the sterilization process, without any side effects on the explants or their development. Due to the nature of the composition and its non-systemic application, it is non-toxic to the host explants, plants, and humans, and it does not pollute the ecosystem. This composition can be used as part of the sterilization process of explants before their introduction. This composition can be used to introduce and / or proliferate plant tissues in a non-sterile state. The composition includes an article containing colloidal silver, which acts as a fungicide and a bactericide to prevent and control various contaminations caused by fungi and bacteria during tissue culture.
[0027] The mechanisms involved in the antimicrobial activity of colloidal silver may include: (1) altering and damaging the membrane structure of a microorganism, which increases its permeability and disrupts its transport function, leading to cell death; (2) the permeation of a microorganism and its interaction with compounds containing phosphorus and sulfur (such as DNA and proteins); (3) loss of the ability of the DNA to replicate; (4) deactivation of specific enzymes; (5) attack on the respiratory chain; and (6) generation of hydrogen peroxide and free radicals. The antimicrobial activity exerted by the composition is multifaceted, utilizing the principles of microfilm technology and surface chemistry: (1) when applied to such explants, the composition forms a microfilm that acts as an effective physical barrier against microorganisms (e.g., bacteria, fungi). The composition is an aqueous suspension containing colloidal silver in a matrix of a methyl vinyl ether copolymer with a protective effect, wherein the colloidal silver associated with the methyl vinyl ether copolymer produces an effective protective microfilm. The microfilm is produced by the polymer and after the microfilm is formed, the encapsulated colloidal silver can come into direct contact with microorganisms (e.g., bacteria, fungi) on the surface of the explant; (3) when applied to fungi and bacteria on the surface of the explant or to the nutrient culture medium or to the solution used to disinfect the explant, the composition neutralizes the enzymes used by microorganisms to metabolize oxygen, and (4) at the same time, it alters the permeability of the cell membrane of the single-celled organism, inducing effective asphyxiation of the microorganism.
[0028] One of the advantages of the disclosed composition is that the methyl vinyl ether copolymer used in the formulation creates an invisible net that holds the components together without interfering with the normal function of the explant tissue and allowing cell regeneration and proliferation.
[0029] In some specific examples, the method may include obtaining the explant, which may be derived from another in vitro cultured plant, a living plant, a fragment of a living plant, a seed, or another form of plant tissue, which may be selected from a maintained plant species used as a culture starting point. The plant tissue may be disinfected using a substance that helps remove microbial contamination already present on or within the explant. This process often begins with scrubbing with soap and water, followed by treatment with a disinfectant. In some specific examples, the composition may be used in a disinfectant in addition to or as an alternative to commonly used disinfectants. The disinfected explant may undergo preparation for inoculation in a culture medium. This preparation consists of dissecting the explant to expose suitable tissue and removing unwanted tissue. This preparation can be performed in a sterile environment, such as in a single-layer flow hood. However, when the composition of the present invention is used, this operation can be performed without the use of a single-layer flow hood. In some specific examples, the method may further include inoculating the prepared plant explants into a culture medium using sterile containers. This step can be performed in a sterile environment, such as in a single-layer flow hood. However, when the components of the present invention are used, this operation can be performed without the use of a single-layer flow hood. The containers containing the plant explants can be transferred to an environment with suitable growth conditions, such as a culture chamber providing appropriate temperature and light conditions. Additionally and / or selectively, after the explants have been inoculated into the culture medium, a liquid solution of one of the components described herein can be added to the explants, thereby forming a shield (e.g., a microfilm) that will protect the explants from microbial contamination.
[0030] In some specific examples, the method includes pretreating the plant or explant with the composition. Surface sterilization of the plant material before introduction into a sterile culture medium is a critical step in establishing guidelines for plant tissue culture procedures. Severe microbial contamination present in plants and explants makes the establishment of in vitro cultures problematic. This contamination is typically overcome by effective surface sterilization of the plant material, among other aseptic techniques. The explant system is treated for surface sterilization by immersing the explant in an appropriate concentration of a chemical sterilizing agent or disinfectant for a specified period of time. This treatment results in the establishment of a culture with minimal or reduced contamination levels. Various types of disinfectants can be used, including but not limited to: ethanol, isopropanol, sodium hypochlorite (NaOCl) / calcium (Ca(ClO)2), hydrogen peroxide (H2O2), mercuric chloride (HgCl2), silver nitrate (AgNO3), and bromine water. The type, concentration, and exposure time of the required disinfectant vary depending on the plant species and plant part. Ethanol is a very potent sterilizing agent but is also phytotoxic, causing damage to the explant and reducing the prospects of successful introduction. It generally requires subsequent treatment with other disinfectants. Even at micromolar concentrations, hypochlorite and related chemical compounds are very effective in significantly reducing the microbial community in the explant, although they are also aggressive towards plant tissue.
[0031] Effective surface pretreatment of one of the plant tissues to be introduced into the culture medium significantly increases the success rate. Reducing the microbial load in the explant will reduce the emergence of microbial contamination in subsequent steps of the process.
[0032] In some specific examples, the method includes applying the composition to the surface of a plant or explant. In some specific examples, the composition is applied by means of a spray, mist, or dropper.
[0033] In some specific examples, 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 specific examples, the composition comprises: approximately 5.00% (w / v) colloidal silver, approximately 3.50% (w / v) methyl vinyl ether copolymer, approximately 0.099% (w / v) polyoxyethylene octylphenyl ether, approximately 0.032% (w / v) sodium hydroxide, and approximately 91.36% (w / v) water.
[0034] In some specific examples, the water is distilled or deionized. The colloidal silver contained in the composition has high preservation properties and can therefore be used in a diluted state in tap water or distilled water. As a result, the composition is easier to handle and has reduced costs.
[0035] In certain specific examples, the copolymer may be any of the following: methyl vinyl ether / maleic anhydride, poly(methyl methacrylate), PMMA, polyether-silicone, iso-dimethylsilicone, methylene diphenyl diisocyanate, phenylenediamine, polyvinyl acetate, ethylene-vinyl acetate (EVA), polyvinyl alcohol, polyvinylpyrrolidone, vinyl chloride, vinylidene chloride copolymers, calcium lignosulfonates, acrylic copolymers, polyvinyl acrylates, and polyethylene oxide. Oxide), acylamide, polyhydroxyethyl acrylate, and similar substances.
[0036] In certain specific examples, the surfactant may be any of the following: polyoxyethylene glycol octylphenol ethers, polyoxyethylene glycol alkylphenol ethers, polysorbate, sorbitan alkyl esters, 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); organosilicone surfactants include Silwet L77 (UAP), Silikin (TERRA), Dyne-Amic (HELENA), Kinetic (HELENA), and Sylgard 309. (WILBUR-ELLIS), Century (PRECISION), and similar items.
[0037] In certain specific examples, a pH adjuster, such as sodium hydroxide, may be used to neutralize the composition. It will be understood 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.
[0038] In certain specific examples, the colloidal silver has the following properties: (a) it is silver suspended in distilled water and is produced by dispersion according to published guidelines (NIST, 2012) or by electrical measurement using a silver electrode; (b) it has an atomic mass of 107,868 g / mol; (c) it has a melting point of 960.5 °C; (d) it has a boiling point of 2000 °C; (e) it has a density of 10.49 g / mL at 15 °C; (f) it is not corroded by water or atmospheric oxygen; (g) it becomes dull by ozone and hydrogen sulfide; (h) it is inert to many acids and readily reacts with dilute nitric acid and hot sulfuric acid; and (i) it is not sensitive to light in its metallic form.
[0039] In some specific examples, the composition is provided in a concentrated form (e.g., a stock solution), a powder form, or a hydrogel form (or concentrated gel form), which may further, for example, be formulated into a plant tissue culture medium by dilution in a liquid. The liquid may be water, such as distilled or deionized water.
[0040] In some specific examples, the composition used in the method disclosed above can be prepared by a multi-step process. The process begins with the preparation of the 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 to a colloidal silver generator, which is connected to a 110 V voltage source. The generator consists of a pump for controlling the amount of water entering the electrodes and another for regulating the amount of water leaving the system. A second pump, connected to a 110 V power outlet, recirculates the water containing the colloidal silver in the system for 1 minute to increase the silver concentration and thus ensure that the final product contains the required silver concentration. The controlled size of the colloidal silver is obtained by filtration. A solution was then prepared by placing 89.2-95.7 kg of water into a 100 L stainless steel container and slowly adding 2.50-6.25 kg of colloidal silver concentrate while stirring; then adding 50-120 g of polyoxyethylene octylphenyl ether and 1.75-4.38 kg of methyl vinyl ether copolymer, wherein the methyl vinyl ether copolymer was previously polymerized at 40°C in a stainless steel container containing 1.75-4.38 L of deionized water; then adding 1 L of a 20-40 g / L sodium hydroxide solution and stirring continuously until the resulting mixture became transparent.
[0041] In some specific examples, the composition is prepared by: (a) placing 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 g of polyoxyethylene octylphenyl ether; (d) adding 3.5 kg of methyl vinyl ether copolymer, wherein the methyl vinyl ether copolymer was previously polymerized at 40°C in a stainless steel container containing 3.5 L of deionized water; (e) adding 1 L of a 35 g / L sodium hydroxide solution; and (f) continuously stirring until the resulting mixture becomes transparent.
[0042] The process for preparing this composition can utilize a generator, such as a Robey device. This generator uses a 110 V voltage source, a current controller, two silver electrodes, and a cellulose filter. The cellulose filter restricts the passage of silver colloids having an average particle size of less than approximately 60 nm and greater than approximately 600 nm.
[0043] In some specific examples, the colloidal silver particles have an average particle size between about 60 nm and about 600 nm. In some specific examples, 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 specific examples, at least 90% of the colloidal silver particles have a particle size between about 60 nm and about 600 nm. In some specific examples, 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.
[0044] In some specific examples, the colloidal silver particles have an average particle size between about 60 nm and about 140 nm. In some specific examples, at least 50% of the colloidal silver particles have a particle size between about 60 nm and about 140 nm. In some specific examples, at least 90% of the colloidal silver particles have a particle size between about 60 nm and about 140 nm.
[0045] In some specific examples, the particles of such colloidal silver may have a maximum size (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).
[0046] These colloidal silver particles can have a variety of different shapes, including spheres, oblate spheroids, cylinders, ovals, ellipsoids, shells, cubes, cuboids, cones, pyramids, rod-shaped bodies (e.g., cylinders or elongated structures with a square or rectangular cross-section), tetrapods [particles with four leg-like appendages], triangular bodies, prisms, etc.
[0047] In certain specific examples, one or more substantially homogeneous groups of such colloidal silver particles are used, for example, two, three, four, five or more substantially homogeneous groups having distinguishable properties (e.g., size, optical properties). It will be understood that a combination of one of two or more groups having distinguishable properties can be considered a single group.
[0048] As used herein, “tissue culture process,” “tissue culturing process,” or “culturing plant tissue” means any process that causes seeds to germinate or causes plants, plant organs, plant tissues, or plant cells to proliferate or differentiate. In some specific examples, plant tissue cultures in a culture medium (e.g., a solid medium or a liquid medium) may be further subcultured. Such plant tissue cultures may be maintained in a defined or undefined culture medium, typically under sterile (aseptic) conditions. However, the methods disclosed above advantageously allow tissue cultures to be maintained under non-sterile conditions.
[0049] Plant tissue culture media may contain a suitable mixture of one of the following: plant hormones or mineral salts in appropriate concentrations. Examples of plant hormones may include: auxins, cytokinins or gibberellins, vitamins (such as one or more B vitamins), one or more carbon sources (for example, sucrose or glucose), and one or more undefined growth enhancers (such as coconut milk).
[0050] The components of the mineral salt mixture can be selected according to the specific plant species being propagated and the intended requirements. The appropriate composition of these mineral salts can be determined empirically or selected from mineral salts previously known in plant tissue culture techniques. Alternatively, these mineral salts can be selected from commercially available mixtures (e.g., from Sigma Chemical Co., St. Louis, MO, USA). Furthermore, other macronutrients and vitamin components can be combined in various ways to generate a culture medium suitable for the plant variety being propagated.
[0051] According to the methods disclosed herein, the composition based on colloidal silver is added to the plant tissue culture medium at a concentration that reduces or prevents the growth of bacteria or fungi or both, and allows for normal seed germination or the reproduction of the plant, plant organs, plant tissues or seeds cultivated by the plant and promotes normal cell growth and development (e.g., substantially normal seed germination).
[0052] In certain specific examples, plant tissue culture media can be prepared by combining its components according to established procedural guidelines for the plant species being used. The culture medium 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 supplements, growth regulators, and solidifying agents.
[0053] Several commonly used basal media with a known composition exist. These include Murashige and Skoog (MS) medium, Linsmaier and Skoog (LS) medium, Gamborg (B5) medium, and Nitsch and Nitsch (NN) medium. The medium used for a particular process may be one of these basal media formulations, with one or more of the following components specifically added: macronutrients (such as N, P, K, S, Mg, and Ca), micronutrients (such as Cu, Fe, Zn, Mn, B, and Mo), vitamins, amino acid or nitrogen supplements, carbon sources, organic supplements, growth regulators, and solidifying agents.
[0054] The amount (or final concentration) of the component described herein to be used in culture media, disinfectant solutions, or other applications may vary in each use case. For example, among other variables, it may depend on the plant species used (some are more susceptible to contamination), the source of the explant (some are dirtier than others), and the technique used. In some specific examples, a culture medium may contain between about 0.01% (w / v) and about 25% (w / v) of the component. Similarly, when applied using a dropper, as a disinfectant solution, or used in other applications, the component may be used at a concentration between about 0.01% (w / v) and about 25% (w / v).
[0055] In certain specific examples, compared with a control culture medium lacking the component, the method of using a composition based on colloidal silver in a plant tissue culture process reduces 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%).
[0056] As used herein, substantially normal germination of a seed is defined as a germination rate of at least 50% of a control group in which the component is not present in the culture medium.
[0057] In certain specific examples, the use of such methods with a composition based primarily on colloidal silver in a plant tissue culture process has resulted in an increased growth rate or an increased cell division rate of a plant cell, plant organ, or plant tissue that is at least 20% higher than that of a corresponding control group that does not contain the composition (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%).
[0058] In certain specific examples, the effectiveness of the method may be investigated using morphological, anatomical, physiological, and biochemical analyses known in the art. For example, a morphological analysis may include comparing roots, branches, leaves, or reproductive organs, or parts in terms of shape, size, or number. An anatomical analysis may include, for example, comparing the size, shape, morphology, or differentiation of cells, such as the number, location, or maturity of vascular tissue, trichomes, or stomata, or the presence or absence of actively dividing meristematic tissue. A physiological analysis may be applied, for example, comparing respiration, photosynthesis, stomatal resistance, or the rate of ethylene production. A biochemical analysis may include, for example, comparing protein or DNA synthesis, chlorophyll degradation, or the presence, absence, or quantity of other pigments. These analyses may also be used to determine or select the optimal concentration of the component in a tissue culture medium, thereby applying the method to a specific plant species.
[0059] In certain specific instances, the microbial contamination may be caused by any undesirable microorganism (such as fungi or bacteria).
[0060] This fungus may be one of the fungi that cause contamination during tissue culture, including but not limited to: *Erysticercus*, *Monophyllum*, *Phytophthora*, *Rhizoctonia*, *Fusarium*, *Penicillium*, *Aspergillus*, *Colletotrichum*, *Botrytis*, *Gastrodia*, *Pythium*, *Pseudomonas*, *Erysticercus*, *Alternaria*, *Pseudomonas*, *Plasmodiophora ...seudomonas*, *Plasmodiophora*, *Pseudomonas*, *Plasmodiophora*, *Sclerotinia*, *Ceratophyllum*, *Peronospora*, *Ustilago*, and *Rhizopus*. This composition inhibits the growth and development of both Gram-positive and Gram-negative bacteria.
[0061] In certain specific examples, exemplary bacteria include, but are not limited to: Corynebacterium, Bacillus, Staphylococcus, Escherichia, Pseudomonas, Xanthomonas, Evans, Corynebacterium, Ralstonia solanacearum, Burkholderia, and Agrobacterium.
[0062] In some specific examples, the composition further includes a carrier that is compatible or acceptable to the tissue culture process. The compatible or acceptable carrier may be a nutrient or a surfactant. Also within the scope of this disclosure is a tissue culture nutrient medium, formulated from the composition as described, for controlling contaminating microorganisms. In a specific example, the nutrient medium may further include an acceptable carrier for the tissue culture process. Also within the scope of this disclosure is a disinfectant solution, formulated from the composition as described, for controlling contaminating microorganisms in the explant before introducing the explant into the nutrient medium.
[0063] "Acceptable carrier for tissue culture process" or "carrier compatible or acceptable for tissue culture process" means any substance other than water that can be added to a composition without causing or having an adverse effect on the plant, explant, or the like. In some specific examples, the carrier can be a solid or liquid carrier and can be in various forms (including microspheres, powders, emulsions, and the like). The carrier can be one or more of a group of carriers that impart various properties (such as increased stability, wettability, or dispersibility). In some specific examples, the carrier can contain glycerol, cellulose, PEG, natural substances or extracts (such as protein hydrolysates, coconut milk, yeast extracts, malt extracts), activated charcoal, and coagulants (such as agar, agarose, and gellan gum).
[0064] In certain specific examples, exemplary embodiments of the carrier may include, but are not limited to: alginate, gum, starch, lecithin, formononetin, polyvinyl alcohol, alkali formononetinate, hesperetin, polyvinyl acetate, cephalins, 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, gluconate, polystyrene, polyethylene, carboxymethyl cellulose, gum ghatti, and polyoxyethylene-polyoxybutylene block copolymers. The carrier may be a non-naturally occurring compound, such as polymers and copolymers. For example, non-limiting examples of polymers that can be used as an adhesive 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, acrylamide polymers and copolymers, hydroxyethyl polyacrylate, methacrylamide monomers, and polychloroprene. In some specific examples, the composition may also contain a surfactant. Non-limiting examples of surfactants include: nitrogen-based surfactant blends [such as Prefer 28 (CENEX), SurfN (US), Inhance (BRANDT), P-28 (WILFARM), and Patrol (HELENA)]; esterified seed oils including SunIt II (AMCY), MSO (UAP), Scoil (AGSCO), Hasten (WILFARM), and Mes-100 (DREXEL); and organosilicone surfactants including Silwet L77 (UAP), Silikin (TERRA), Dyne-Amic (HELENA), Kinetic (HELENA), Sylgard 309 (WILBUR-ELLIS), and Century (PRECISION).The surfactant is present at a concentration between 0.001% v / v and 10% v / v (e.g., between 0.001% v / v and 1% v / v). In some specific examples, the composition may contain a stabilizer. Such a stabilizer may contain 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).
[0065] In some specific examples, the composition further comprises a second fungicide and / or a second bactericide. In some specific examples, it may be advantageous for the composition to contain an agent such as a fungicide, a bactericide, or a nutrient. Ideally, the agent is one that does not pose a safety concern for human, animal, or industrial use [e.g., there are no safety concerns or the compound is sufficiently degradable that commercial plant products derived from tissue culture processes contain a negligible amount of the compound]. As used herein, a "fungicide" comprises a commercially available synthetic chemical compound designed to protect plants and explants from pathogenic fungi. Examples of such fungicides may include, but are not limited to: 2-(thiocyanato methylthio)-benzothiazole, 2-phenylphenol, 8-hydroxyquinoline sulfate, meetoctradin, amisulbrom, antimycin, Ampelomyces quisqualis, azaconazole, azoxystrobin, Bacillus subtilis, benalaxyl, benomyl, benthiavalicarb-isopropyl, and benzylaminobenzenesulfonate (BABS). Salts, bicarbonates, biphenyls, bismerthiazol, bitertanol, bixafen, blasticidin-S, borax, Bordeaux mixture, boscalid, bromuconazole, bupirimate, calcium polysulfide, captafol, captan, carbendazim, carboxin, carpropamid, carvone, chloroneb, chlorothalonil, chlozolinate, Coniothyrium minitans, copper hydroxide, copper octanoate, basic copper oxychloride.oxychloride, copper sulfate, basic copper sulfate [copper sulfate (tribasic)], cuprous oxide, cyazofamid, cyflufenamid, cymoxanil, cyproconazole, cyprodinil, dazomet, debacarb, diammonium ethylenebis-(dithiocarbamate) [diammonium ethylenebis-(dithiocarbamate)], dichlofluanid, dichlorophen, diclocymet, diclomezine, dichloran, diethofencarb, difenzoquat ion, diflumetorim, dimethomorph, dimoxystrobin, diniconazole, dacryl-M (diniconazole-M), dinobuton, dinocap, diphenylamine, dithianon, dodemorph, dodemorph acetate, dodine, dodine free base, edifenphos, enestrobin, epoxiconazole, ethaboxam, ethoxyquin, etridiazole, famoxadone, fenamidone, fenarimol, fenbuconazole, fenfuram, fenhexamid, fenoxanil, fenpiclonil, fenpropidin, fenpropimorph, fentin, fentin acetate acetate, triphenyltin hydroxide (fentin)hydroxide), ferbam, ferimzone, fluazinam, fludioxonil, flumorph, fluopicolide, fluopyram, fluoroimide, fluoxastrobin, fluquinconazole, flusilazole, flusulfamide, flutianil, flutolanil, flutriafol, fluxapyroxad, folpet, formaldehyde, fosetyl, fosetyl-aluminium, fuberidazole, furalaxyl, furametpyr, guazatine, guazatine acetate Acetates), GY-81, hexachlorobenzene, hexaconazole, hymexazol, imazalil, imazalil sulfate, imibenconazole, iminoctadine, iminoctadine triacetate, iminoctadine tris(albesilate), ipconazole, iprobenfos, iprodione, iprovalicarb, isoprothiolane, isopyrazam, isotianil, kasugamycin, kasugamycin hydrochloridehydrate), 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, methimazole Metasulfocarb, iodomethyl, methyl isothiocyanate, metiram, metominostrobin, metrafenone, mildiomycin, myclobutanil, nabam, nitrothal-isopropyl, nuarimol, octhilinone, ofurate, oleic acid (fatty acid), orysastrobin, oxadixyl, oxine-copper, oxpoconazole fumarate, oxycarboxin, pefurazoate, penconazole, pencycuron, penflufen, pentachlorophenol, pentachlorophenyl laurate, penthiopyrad, phenylmercury acetate, phosphonic acid, phthalide, picoxystrobin, polyoxin B, polyoxins, polyoxorim, potassium bicarbonate, potassium hydroxyquinoline sulfate, probenazole, prochloraz, procymidone, propamocarb, propamocarbhydrochloride, propiconazole, propineb, proquinazid, prothioconazole, piraclostrobin, pirametostrobin, piraoxystrobin, pyrazophos, pyribencarb, pyributicarb, pyrifenox, pyrimethanil, pyroquilon, quinoclamine, quinoxyfen, quintozene, Reynoutria sachalinensis extract, sedaxane, silthiofam, simeconazole, sodium 2-phenylphenol, sodium bicarbonate, sodium pentachlorophenate. pentachlorophenoxide), spiroxamine, sulfur, SYP-Z071, SYP-Z048, tar, tebuconazole, tebufloquin, tetraconazole, thiabendazole, thifluzamide, thiophanate-methyl, thiram, tiadinil, tolclofos-methyl, tolylfluanid, trimethoprim (t riadimefon, triadimenol, triazoxide, tricyclazole, triridemorph, trifloxystrobin, triflumizole, triforine, triticonazole, validamycin, valifenalate, valiphenal, vinclozolin, zineb, ziram, zoxamide, Candida oleophila, Fusarium oxysporum*Streptomyces griseoviridis*, *Trichoderma*, *(RS)-N-(3,5-dichlorophenyl)-2-(methoxymethyl)-succinimide, 1,2-dichloropropane, 1,3-dichloro-1,1,3,3-tetrafluoroacetone hydrate, 1-chloro-2,4-dinitronaphthalene dinitronaphthalene), 1-chloro-2-nitropropane, 2-(2-heptadecyl-2-imidazolin-1-yl)ethanol, 2,3-dihydro-5-phenyl-1,4-dithi-ine 1,1,4,4-tetraoxide, 2-methoxyethylmercury acetate, 2-methoxyethylmercury chloride, 2-methoxyethylmercury silicate, 3-(4-chlorophenyl)-5-methylrhodanal [-methylrhodanine], 4-(2-nitroprop-1-enyl)phenyl thiocyanate, ampropylfos, anilazine, azithiram, barium polysulfide, Bayer 3239432394), benodanil, benquinox, bentaluron, benzamacril, benzamacril-isobutyl, benzamamorf, binapacryl, bis(methylmercury) sulfate, bis(tributyltin) oxide, buthiobate, cadmium calcium copper zinc chromate sulfate sulfate, carbamorph, CECA, chlobenthiazone, chloraniformethan, chlorfenazole, chlorquinox, climbazole, cyclafuramid, cypendazole, cyprofuram, decafentin, dichlone, dichlozoline, diclobutrazol, dimethoprim Methirimol, Diocton, Dinosulfon, Dinoterbon, Dipyrithione, Ditalimfos, Dodicin, Drazoxolon, EBP, ESBP, Etaconazole, Etem, Ethirim, Fenaminosulfonium, Fenapanil, Fenitropan, 5-fluorocytosine and its pre-fungicides (pro) fungicides, fluotrimazole, furcarbanil, furconazole, furconazole-cis, furmecyclox, furophanate, glyodine, griseofulvin, halacrinate, Hercules 39443944), hexylthiofos, ICIA0858, isopamphos, isovaledione, mebenil, mecarbinzid, metazoxolon, methfuroxam, methylmercury dicyandiamide, metsulfovax, milneb, mucochloric acid anhydride anhydride, myclozolin, N-3,5-dichlorophenyl-succinimide, N-3-nitrophenylitaconimide, natamycin, N-ethylmercurio-4-toluenesulfonanilide, nickel bis(dimethyldithiocarbamate), OCH, phenylmercury dimethyldithiocarbamate, phenylmercury nitrate, phosdiphen, picolinamide UK-2A and its derivatives, prothiocarb, prothiocarb hydrochloride. hydrochloride, pyracarbolid, pyridinitril, pyroxychlor, pyroxyfur, quinacetol, quinacetol sulfatesulfate), quinazamid, quinconazole, rabenzazole, salicylanilide, SSF-109, sultropen, tecoram, thiadifluor, thicyofen, thiochlorfenphim, thiophanate, thioquinox, tioxymid, triamiphos, triarimol, triazbutil, trihlamide, urbacid, XRD-563, zarilamide, and IK-1140. Examples of bactericides may 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. (xil), Cefazolin, Cefalotin, Cefalothin, Cefalexin, Cefacolor, Cefamandole, Cefoxitin, Cefprozil, Cefouroxime, Cefixime, Cefdinir, Cefditoren, Cefooperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, seriphen (1Cefepime, Ceftaroline fosamil, Ceftobiprole, Teicoplanin, Vancomycin, Telavancin, Clindamycin, Lincomycin, Daptomycin, Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, Spiramycin cin), Aztreonam, Furazolidone, Nitrofurantoin, Linezolid, Posizolid, Radezolid, Torezolid, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Dimethoxybenzylpenicillin (2 Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Temocillin, Ticarcillin, Amoxicillin / clavulanate, Ampicillin / sulbactam, Piperacillin / tazobactam, Ticarcillin / clavulanate, Bacitracin, Colistin, Polymyxin BB) Ciprofloxacin, Enoxacin, Gatifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, Temafloxacin, Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilimide (archaic), sulfasalazine, sulfisoxazole, trimethoprim / sulfamethoxazole (TMP-SMX), sulfonamidochrysoi dine (traditional), demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, ripanmycin (3) Rifampicin (known as Rifampin in the US), Rifabutin, Rifapentine, Streptomycin, Arsphenamine, Chloramphenicol, Fosfomycin, Fusidic acidThe following are listed: acid, metronidazole, mupirocin, platensimycin, quinupristin / dalfopristin, chloramphenicol, tigcycline, tinidazole, and trimethoprim.
[0066] In certain specific examples, the method may include applying the composition to the surface of a plant or explant [e.g., leaf, fruit, flower, stem, root, meristem, callus tissue, branch, embryo, seed]. At very low concentrations, the method can selectively control fungi and / or bacteria in the plant. Furthermore, once the composition is applied, the preventative effect can last for 1-3 weeks or longer. The composition controls both spores and hyphae, and even when applied at high concentrations, it does not cause chemical harm and is harmless to humans and plants.
[0067] In some specific examples, the method may include application to the nutrient medium of the tissue culture process. In some specific examples, the method may include application of the composition, such as by spray, mist, or dropper. The composition may be mixed with an acceptable carrier or diluent and thus formulated into various different formulations (including nutrient media or additives for the tissue culture process). Furthermore, the composition may be mixed with an additional component or surfactant or other known reagent, such as to control microbial contamination. The term "diluent" means an acceptable liquid or solid added to the composition so that it can be readily used or diluted to a desired active concentration. Exemplary examples of diluents include talc, kaolin, zeolite, xylene, diatomaceous earth, water, etc.
[0068] A formulation for use in a spray form, such as a water-dispersible concentrate or wet powder, may further include a wetting agent, a dispersant, a surfactant, etc. In addition to the diluent and the surfactant, a stabilizer, an inactivating agent, an adhesion improver, a colorant, a coagulant, an infiltrating agent, and a defoamer may be additionally included. The disclosed components can be formulated in various different forms. For example, the wet powder form prepared together with kaolin or diatomaceous earth can be diluted with water before being used as a spray liquid, and thus can be sprayed onto explants. Furthermore, the components can be mixed with an emulsifier to obtain a concentrate, which is then diluted with water before being applied to explants. Definition
[0069] To aid in understanding the detailed description of the components and methods disclosed herein, certain specific definitions are provided to facilitate an unambiguous disclosure of one of the various aspects of the 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 pertains.
[0070] The terms “prevent”, “preventing”, “prevention”, “prophylactic treatment” and similar terms mean reducing the probability of developing a hazard or disease in a subject (e.g., a plant) that does not have a hazard or disease but is at risk of developing one or is sensitive to one or the disease.
[0071] The terms “decrease,” “reduced,” “reduction,” “decrease,” or “inhibit” are generally used herein to mean a reduction that is statistically significant. However, for the avoidance of doubt, “decrease,” “reduction,” “decrease,” or “inhibit” means a reduction of at least 10% compared to a reference level, for example, a reduction of at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, or a reduction of up to and including a reduction of 100% (e.g., a level that is not present compared to a reference sample), or any reduction between 10% and 100% compared to a reference level.
[0072] The term “treating” or “treatment” means the application of a compound or reagent to a subject (e.g., a plant) that has an obstacle or is in danger of developing such an obstacle, with the aim of curing, alleviating, relieving, remedying, preventing or improving, or delaying the onset of: the obstacle, the symptoms of the obstacle, the disease state secondary to the obstacle, or the predisposing factors to the obstacle.
[0073] The word "substantially" does not exclude "completely," for example, a composition that "substantially does not contain" Y can be completely free of Y. Where necessary, the word "substantially" may be omitted from the definition of this invention.
[0074] As used herein, the terms “approximately” or “about”, when applied to one or more values of interest, mean a value similar to the stated reference value. In certain specific instances, the terms “approximately” or “about” mean a range of values falling 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 any direction (greater or less) of the stated reference value, unless otherwise stated or otherwise apparent from the context (except when the value would exceed 100% of a possible value). Unless otherwise indicated herein, the term “about” is intended to include values (e.g., weight percentages) adjacent to the stated range, which are equivalent in terms of the functionality of the individual components, compositions, or specific examples.
[0075] As disclosed herein, numerous numerical ranges are provided. It is understood that each midpoint value between the upper and lower limits of that range [to the tenth of the unit of the lower limit] is also specifically disclosed, unless the context explicitly specifies otherwise. Each smaller range between any setpoint or midpoint value in a given range and any other setpoint or midpoint value within that range is encompassed in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range, and each range containing no, one, or two limits is also encompassed in this invention, subject to any specifically excluded limit in the stated range. Where the given range contains one or both of these limits, the range excluding either or both of those included limits is also encompassed in this invention.
[0076] It should be noted herein that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. The terms “including,” “comprising,” “containing,” or “having,” and variations thereof, are intended to cover the items subsequently listed and their equivalents as well as other subject matter, unless otherwise noted.
[0077] Phrases such as “in one specific instance,” “in various different specific instances,” “in certain specific instances,” and similar phrases are used repeatedly. Such phrases do not necessarily refer to the same specific instance, but they may, unless the context specifies otherwise.
[0078] The terms “and / or” or “ / ” refer to any one of the items associated with the term, any combination of such items, or all of such items.
[0079] As used herein, the term "each" is intended to identify an individual item in a collection of items when used with reference to that collection, but does not necessarily refer to every item in the collection. Exceptions may occur if the explicit disclosure or context otherwise clearly specifies otherwise.
[0080] Unless otherwise requested, the use of any and all exemplary or illustrative phrases (e.g., “such as”) provided herein is intended only to better illustrate the invention and not to limit the scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is indispensable to the implementation of the invention.
[0081] Unless otherwise specified herein or clearly contradicted by the context, all methods described herein are performed in any suitable order. With respect to any of the provided methods, the steps of the method may occur simultaneously or sequentially. When the steps of the method occur sequentially, they may occur in any order unless otherwise stated.
[0082] In the case where a method includes a combination of steps, each and every combination or subcombination of those steps is covered within the scope of the disclosure herein, unless otherwise stated herein.
[0083] The chapter titles used in this document are for organizational purposes and are not intended to limit the subject matter described.
[0084] Every publication, patent application, patent, and other reference cited herein is incorporated herein by reference on its entirety and to the extent that it is not inconsistent with the disclosure herein. Publications disclosed herein are provided only because their disclosures predate the filing date of this invention. Nothing herein is to be construed as an admission that this invention does not enjoy prior art by virtue of publications preceding such inventions. Furthermore, the publication dates provided may differ from the actual publication dates, which may require independent verification.
[0085] It is understood that the exemplary and specific examples described herein are for illustrative purposes only, and various modifications or variations thereof will be suggested to those skilled in the art and are included within the spirit and scope of this application and the scope of the appended patent application. Exemplary Example 1
[0086] An article containing the disclosed composition was added to a standard Murashige and Skoog tissue culture nutrient medium and used for the introduction of explants of a Musa sp. species. The introduction was performed according to standard in vitro introduction guidelines for this plant species. Different treatments were used and were prepared by adding amounts ranging from 0.5% (w / v) to 2% (w / v) of the composition of the present invention to the medium. After 25 days of culture under the recommended conditions, the incidence of microbial contamination was observed to be 0%, compared to 27% in the control group. Using 0.5% (w / v) of the article containing the composition disclosed according to the present invention, only 3% of the evaluated material was observed to show signs of microbial contamination. In explants introduced into a nutrient medium containing the composition, the incidence of bacterial and fungal infections was significantly reduced, and no new development of infection was observed in the following steps of the tissue culture process. Example 2
[0087] A solution is prepared by adding one part of the composition to nine parts of distilled water to obtain a 10% (w / v) solution. Such a solution is applied to in vitro plants of the genus *Cordylinegenus*, which are obtained from in vitro introduction and transferred to an in vitro nutrient medium intended for shoot propagation. The application is accomplished by covering the surface of the in vitro plants with a sprayer, a method that has proven suitable and practical. No symptoms of phytotoxicity were observed on the treated in vitro plants. The application of the composition of the present invention showed a significant increase in the propagation of these plants, with no signs of microbial contamination. Example 3
[0088] According to one specific example of the invention, prior to the in vitro introduction process, a liquid formulation containing 5% (w / v) of the composition of the invention was prepared and used as a disinfectant solution to prepare explants of bamboo [Guadua angustifolia]. One formulation of the invention was diluted in distilled water to obtain the desired concentration and placed on an ultrasonic oscillator water bath. Explants derived from plants grown under greenhouse conditions were placed in the water bath and cultured for 15 minutes. Subsequently, these explants were introduced into a nutrient medium located in a single-layer flow hood. As suggested in the literature, a control was introduced using a chlorine disinfection method. Compared to the control, at the end of the introduction process, explants disinfected with the composition of the invention showed a two-fold reduction in microbial contamination. None of the explants treated with the composition of the invention showed signs of chemical damage. Example 4
[0089] Citrus embryos obtained from suspension cell cultures were placed on agar nutrient medium containing 1% (w / v) of the composition of the present invention. Furthermore, the inoculated suspension was covered with a solution prepared to contain 0.5% (w / v) of the composition of the present invention. Following the culture period and conditions suggested in the literature, using standard operating procedures of this technique, we observed a 70% reduction in the incidence of microbial contamination compared to a control not treated with the composition of the present invention. Example 5
[0090] Dried seeds of several plant species (including mustard, pumpkin, and radish) were purchased from commercial sources and introduced into standard Murashige and Skoog tissue culture nutrient media prepared with 10% (w / v) of the composition of the present invention. This nutrient medium was placed in glass vials without sterilization measures (such as autoclaving). The seeds were placed on unsterilized nutrient media and cultured using standard growth conditions. The entire introduction process was performed on a non-sterile laboratory bench without using a laminar flow cabinet. After 8 days of culture, more than 80% of the glass vials showed successful seed germination without signs of microbial contamination, compared to a control using a medium without the addition of the composition of the present invention (in which 100% of the glass vials showed excessive signs of microbial contamination).
[0091] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that this detailed description and the examples, while pointing to specific examples of the invention, are given by way of illustration only. Furthermore, it is contemplated that variations and modifications falling within the spirit and scope of the invention will become apparent from this detailed description to those skilled in the art.
Claims
1. A method for reducing or preventing microbial contamination of a plant or explant during a tissue culture process, comprising culturing a plant or explant in a plant tissue culture medium formed from a composition primarily composed of colloidal silver, the composition comprising colloidal silver, a copolymer, a surfactant, an alkali, and water.
2. The method of claim 1 further includes pretreating the plant or explant with the composition.
3. The method of claim 1 or 2, comprising applying the composition to the surface of a plant or explant.
4. The method of claim 3, which includes applying the composition as a spray, mist or dropper.
5. The method of any of the preceding claims, comprising inoculating the plant or explant into the plant tissue culture medium and subsequently applying the composition to the plant or explant, thereby forming a barrier protecting the plant or explant from microbial contamination.
6. The method of any of the preceding claims, wherein the copolymer is a methyl vinyl ether copolymer.
7. The method of any of the preceding claims, wherein the surfactant is a polyoxyethylene octylphenyl ether.
8. The method of any of the preceding claims, wherein the base is used to neutralize the composition.
9. The method of any of the preceding claims, wherein the base is sodium hydroxide or potassium hydroxide.
10. The method of any of the preceding claims, wherein 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.
11. The method of any of the foregoing claims, comprising: Approximately 5.00% (w / v) colloidal silver, approximately 3.50% (w / v) methyl vinyl ether copolymer, approximately 0.099% (w / v) polyoxyethylene octylphenyl ether, approximately 0.032% (w / v) sodium hydroxide, and approximately 91.36% (w / v) water.
12. The method of any of the preceding claims, wherein the water system is distilled or deionized.
13. The method of any of the preceding claims, wherein the colloidal silver has the following properties: (a) it is silver suspended in distilled water and is produced by dispersion according to published guidelines (NIST, 2012) or by electrical measurement using a silver electrode; (b) it has an atomic mass of 107,868 g / mol; (c) it has a melting point of 960.5 °C; (d) it has a boiling point of 2000 °C; (e) it has a density of 10.49 g / mL at 15 °C; (f) it is not corroded by water or atmospheric oxygen; (g) it becomes dull by ozone and hydrogen sulfide; (h) it is inert to many acids and readily reacts with dilute nitric acid and hot sulfuric acid; and (i) it is not sensitive to light in its metallic form.
14. The method of any of the preceding claims, wherein the colloidal silver particles have an average particle size between about 60 nm and about 140 nm.
15. The method of any of the preceding claims, wherein at least 50% of the colloidal silver particles have a particle size between about 60 nm and about 140 nm.
16. The method of any of the preceding claims, wherein at least 90% of the colloidal silver particles have a particle size between about 60 nm and about 140 nm.
17. The method of any of the preceding claims, wherein the microbial contamination is caused by fungi or bacteria.
18. The method of claim 17, wherein the fungi are selected from the group consisting of: *Blumeria*, *Sphaerotheca*, *Phytophthora*, *Rhizoctonia*, *Fusarium*, *Penicillium*, *Aspergillus*, *Colletotrichum*, *Botrytis*, *Gastropoda*, etc. The genera include Magnaporthe, Pythium, Puccinia, Erysiphe, Alternaria, Pseudoperonospora, Plasmodiophora, Sclerotinia, Fulvia, Peronospora, Ustilago, and Rhizopus.
19. The method of claim 17, wherein the bacteria are selected from the group consisting of: Corynebacterium, Bacillus, Staphylococcus, Escherichia, Pseudomonas, Xanthomonas, Erwinia, Clavibacter, Ralstonia, Burkholderia, and Agrobacterium.
20. The method of any of the preceding claims, wherein the composition further comprises an acceptable carrier for the tissue culture process.
21. The method of any of the preceding claims, wherein the composition further comprises at least one of a second fungicide and a second bactericide.
22. The method of any of the preceding claims, wherein the composition is provided in a concentrated form, a powder form, or a hydrogel form.
23. The method of any of the preceding claims, wherein the composition is prepared by: (a) placing 89.2-95.7 kg of water into a 100 L stainless steel container; (b) slowly adding 2.50-6.25 kg of colloidal silver concentrate while stirring; (c) adding 50-120 g of polyoxyethylene octylphenyl ether; (d) adding 1.75-4.38 kg of methyl vinyl ether copolymer, wherein the methyl vinyl ether copolymer was previously polymerized at 40°C in a stainless steel container containing 1.75-4.38 L of deionized water; (e) adding 1 L of a 20-40 g / L sodium hydroxide solution; and (f) continuously stirring until the resulting mixture becomes transparent.
24. The method of claim 23, wherein the composition is prepared by: (a) placing 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 g of polyoxyethylene octylphenyl ether; (d) adding 3.5 kg of methyl vinyl ether copolymer, wherein the methyl vinyl ether copolymer was previously polymerized at 40°C in a stainless steel container containing 3.5 L of deionized water; (e) adding 1 L of a 35 g / L sodium hydroxide solution; and (f) continuously stirring until the resulting mixture becomes transparent.