METHODS OF USING A COLLOIDAL SILVER-BASED COMPOSITION IN REDUCING OR PREVENTING MICROBIAL CONTAMINATION IN PLANTS OR EXPLANTS IN TISSUE CULTURE PROCESSES

Abstract

This disclosure provides methods of using an eco-friendly colloidal silver-based composition in preventing and reducing contamination caused by microorganisms in the plant tissue culture processes. The composition comprises colloidal silver, a copolymer, surfactant, a base, and water. The methods allow cultivating plants or explants in a plant tissue culture medium under non-aseptic conditions and thus obviate the need of the cumbersome and laborious procedures to sterilize the culture medium and the culture vessel in order to create aseptic conditions for plant or explant cultivation.

Claims

1. A method for reducing or preventing microbial contamination in plants or explants in a tissue culture process, comprising cultivating a plant or explant in a plant tissue culture medium formed from a colloidal silver-based composition comprising colloidal silver, a copolymer, a surfactant, a base, and water.

2. The method of claim 1, further comprising pre-treating the plant or explant with the composition.

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

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

5. The method of any one of the preceding claims, comprising inoculating the plant or explant in the plant tissue culture medium followed by applying the composition on top of the plant or explant, thereby forming a barrier that protects the plant or explant against microbial contamination.

6. The method of any one of the preceding claims, wherein the copolymer is a methyl vinyl ether copolymer.

7. The method of any one of the preceding claims, wherein the surfactant is a polyoxyethylene octylphenyl ether.

8. The method of any one of the preceding claims, wherein the base is used to neutralize the composition.

9. The method of any one of the preceding claims, wherein the base is sodium hydroxide or potassium hydroxide.

10. The method of any one of the preceding claims, wherein the composition comprises: 0.1-20% (w/v) colloidal silver, 1.75-4.38% (w/v) methylvinyl 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 one of the preceding claims, comprising: about 5.00% (w/v) colloidal silver, about 3.50% (w/v) methylvinyl ether copolymer, about 0.099% (w/v) polyoxyethylene octylphenyl ether, about 0.032% (w/v) sodium hydroxide, and about 91.36% (w/v) water.

12. The method of any one of the preceding claims, wherein the water is distilled or deionized.

13. The method of any one of the preceding claims, wherein the colloidal silver has the following characteristics: (a) it is silver suspended in distilled water and produced by dispersion according to published guidelines (NIST, 2012) or by electrical methods of 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 at 15° C. of 10.49 g/mL; (f) it is not attacked by water or atmospheric oxygen; (g) it is obscured by ozone and hydrogen sulfide; (h) it is inert to many acids and reacts easily with dilute nitric acid and hot sulfuric acid; and (i) it is not sensitive to light in their metallic form.

14. The method of any one of the preceding claims, wherein particles of colloidal silver have an average particle size of between about 60 nm and about 140 nm.

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

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

17. The method of any one 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, 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 one of the preceding claims, wherein the composition further comprises an acceptable carrier for the tissue culture process.

21. The method of any one of the preceding claims, wherein the composition further comprises at least one of a second fungicidal agent and a second bactericidal agent.

22. The method of any one of the preceding claims, wherein the composition is provided in a concentrated form, in a powder form, or in a hydrogel form.

23. The method of any one of the preceding claims, wherein the composition is prepared by: (a) placing 89.2-95.7 Kg of water to a 100 L stainless steel vessel; (b) slowly adding 2.50-6.25 Kg the colloidal silver concentrate while stirring; (c) adding 50-120 grams of polyoxyethylene octyl phenyl ether; (d) adding 1.75-4.38 Kg of methyl vinyl ether copolymer, wherein the methyl vinyl ether copolymer was previously polymerized in a stainless steel vessel containing 1.75-4.38 L of deionized water at 40° C.; (e) adding 1 L of a 20-40 g/L sodium hydroxide solution; and (f) agitating continuously until the resultant mixture becomes transparent.

24. The method of claim 23, wherein the composition is prepared by: (a) placing 91.4 Kg of water to a 100 L stainless steel vessel; (b) slowly adding 5 Kg the colloidal silver concentrate while stirring; (c) adding 99 grams of polyoxyethylene octyl phenyl ether; (d) adding 3.5 Kg of methyl vinyl ether copolymer, wherein the methyl vinyl ether copolymer was previously polymerized in a stainless steel vessel containing 3.5 L of deionized water at 40° C.; (e) adding 1 L of a 35 g/L sodium hydroxide solution; and (f) and agitating continuously until the resultant mixture becomes transparent.

Description

EXAMPLES

Example 1

[0082] A preparation of the disclosed composition was added to a standard Murashige & Skoog tissue culture nutrient media and used for the introduction of Musa sp. explants. The introduction was performed according to standard in vitro introduction protocols for this plant species. Different treatments were used and prepared by adding amounts of the composition of the invention to the culture media which ranged from 0.5% (w/v) to 2% (w/v). After 25 days of incubation at the recommended conditions, it was observed an incidence of microbial contamination of 0%, compared to 27% in the control group. Using 0.5% (w/v) of the preparation of the disclosed composition according to the invention, only 3% of the material evaluated was observed to present signs of microbial contamination. The incidence of the bacterial and fungal infection was greatly reduced in the explants introduced in nutrient media containing the composition, and no new development of the infection was observed during the following steps of the tissue culture process.

Example 2

[0083] A solution was prepared by adding one 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 plants of the Cordyline genus, which were taken from in vitro introductions and transferred to in vitro nutrient media intended for shoot multiplication. The application was done by using a sprayer to coat the surface of the in vitro plants and the method proved applicable and practical. No symptoms of phytotoxicity were observed on the treated in vitro plants. The application of the composition of the invention showed significant increases in the multiplication of the plants, with no signs of microbial contamination.

Example 3

[0084] According to one embodiment of the invention, a liquid formulation was prepared to contain 5% (w/v) of the composition of the invention and used as the disinfection solution to prepare explants of a species of bamboo (Guadua angustifolia) prior to the in vitro introduction process. A formulation of the invention was diluted in distilled water to obtain the desired concentration and placed on an ultrasonicator water bath. Explants obtained from plants growing in greenhouse conditions were placed in the water bath and incubated to 15 minutes. Subsequently, the explants were introduced to nutrient culture media under a laminar flow hood. Controls were subjected to a disinfection method using chlorine, as suggested in the literature. A two-fold decrease was observed in microbial contamination at the end of the introduction process in the explants disinfected using the composition of the invention, compared to controls. None of the explants treated with the composition of the invention presented signs of chemical damage.

Example 4

[0085] Somatic embryos of Citrus obtained from suspended cell cultures were placed on agar nutrient media containing 1% (w/v) of the composition of the invention. Additionally, the inoculated suspensions were coated with a solution prepared to contain 0.5% (w/v) of the composition of the invention. After the periods and conditions of incubation recommended in the literature, we observed an incidence of microbial contamination 70% lower, compared to controls without the treatment of the composition of the invention, using standard procedures for the technique.

Example 5

[0086] Dried seeds of several plant species (including mustard, pumpkin, and radish) were purchased from commercial sources and introduced into standard Murashige & Skoog tissue culture nutrient media prepared with 10% (w/v) of the composition of the invention. The nutrient media was placed in glass vials with no sterilization measures, such as autoclaving. The seeds were placed on the unsterilized nutrient media and incubated using standard growth conditions. The entire introduction process was performed on a non-sterile laboratory bench, without using a laminar flow hood. After 8 days of incubation, over 80% percent of the glass vials showed successful seed germination with no signs of microbial contamination, compared to the controls using media without the addition of the composition of the invention, where 100% of the glass vials showed excessive signs of microbial contamination.

[0087] 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 the examples, while indicating specific embodiments of the invention, are given by way of illustration only. Additionally, it is contemplated that changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.