MXPA97009379A - Compositions and methods to prevent microbial control of plantable detention media - Google Patents

Abstract

The present invention encompasses compositions and methods for reducing or preventing microbial growth in tissue culture media of plants, which comprises adding a chemical agent comprising methylchloroisothiazolinone, methylisothiazolinone, magnesium chloride and magnesium nitrate to a plant culture medium, a concentration that reduces or prevents microbial contamination of the plant tissue culture medium, which permits essentially normal germination of seeds or the essentially normal growth or development of plants, plant organs, plant tissues or plant cells. The chemical agent may also comprise potassium sorbate or sodium bensoate, or both. The present invention further provides a case for the germination of plant seeds or for the cultivation of plants, plant organs, tissues or plant cells in a plant tissue culture medium comprising the chemical agent.

Description

"COMPOSITIONS AND METHODS TO PREVENT MICROBIAL CONTAMINATION OF PLANT TISSUE CULTIVATION MEDIA" 1. FIELD OF THE INVENTION The present invention relates to compositions and methods for the prevention or inhibition of microbial growth in culture media for tissue cultures of plants that normally require maintenance of sterile conditions. Specific chemical agents are used in the culture medium at concentrations that reduce or prevent microbial contamination but which allow essentially normal germination of seeds or the essentially normal growth or development of plants, plant organs, plant tissues or plant cells. The disclosed compositions and methods are useful for researchers in the cultivation of plant tissue and plant molecular biology, as well as those in charge of plant breeding and plant nursery operators, and in a variety of commercial applications. 2. BACKGROUND OF THE INVENTION 2.1. IN VITRO PLANT CULTIVATION SYSTEMS The growth and development of plants are fundamental biological processes of great educational and commercial scientific interest. These biological processes can be observed and manipulated for seed germination and the propagation of whole plants, plant organs, plant tissues and plant cells in vitro, in sterile culture in various types of culture media. The practice of growing plants in vitro is well developed. See, for example, from T.A. Thorpe (editor), Plant Tissue Culture: Methods and Applications in Agriculture, Academic Press, Inc., New York (1981); Evans et al. (Editors), Handbook of Plant Cell Culture, Volume 1, MacMillan Pubishing Co. , New York, London, (1983); and R.A. Dixon, (editor) and R.A. Dixon (editor), Plant Cell Culture: A Practical Approach, IRL Prees, Oxford, Washington DC (1985). For normal growth and development, plants that are grown in vitro require, as a minimum, a medium that contains essential mineral salts. See, for example, F.B. Salisburry, and C.W. Ross, Plant Physiology, Third Edition, Wadsworth Publishing Co. (1985). In addition, cultured plant tissues and cells typically require various combinations of plant hormones (phytohormones), vitamins and one or more simple sugars. See Thorpe, supra.

Unfortunately, these culture media also provide a rich mix of nutrients that can support the rapid growth of bacteria and fungi. Once these contaminants are established in the crop, they usually grow rapidly, depleting the nutrient medium and producing toxins that can affect the growth of, and eventually the death of, the tissues of cultivated plants. Therefore, the use of sterile techniques has been a strict requirement for all manipulations of in vitro plant culture. For example, the preparation and maintenance of a normal tissue culture system requires sterilization of the culture medium, either by heat or by filtration, sterilization of the culture container, surface sterilization of the seed or plant tissues that go to be cultivated, sterilization of any of the instruments used to handle or manipulate the tissues of plants. In addition, any subsequent manipulation of plant tissues should typically be carried out in a filtered air environment, e.g., in a laminar flow hood. Despite the strict use of sterile techniques by an expert artisan, however, contamination of plant crops remains a persistent problem that can result in losses ranging from small numbers of crops to catastrophic loss of lots. whole of the culture medium and tissue cultures. Contamination by bacteria and fungi is an insidious process that continuously threatens plant tissue cultures through the duration of the culture period. Despite the fact that the plant tissue cultures can be sterile when started, microorganisms can often contaminate crops at any point during subsequent manipulations of tissue culture. Therefore, it would be useful to provide a chemical agent that reduces or prevents microbial contamination of plant tissue culture media and maintains the sterility of the media throughout the duration of the culture period. 2. 2 ANTIMICROBIAL AGENTS IN PLANT FABRIC CULTIVATION Various types of chemical antimicrobial agents have been tested on plant tissue cultures. Antibiotics have been extensively tested to determine their ability to inhibit or prevent the growth of bacteria in plant crops. However, the use of antibiotics has certain limitations. For example, antibiotics are expensive, they are only effective against bacteria and not against fungi, their effectiveness scale against types of bacteria is often indicated are usually thermolabile and are often phytotoxic or otherwise, capable of altering the behavior of the tissues of cultivated plants. For example, R. Phillips et al., Plant. Sci. Lett. , 21: 235-240 (1981), describe the tests of six antibiotics, ie benzyl penicillin, fosfomycin, chloramphenicol, estroptomycin, rifampicin and nalidixic acid, to prevent bacterial infection of Jerusalem artichoke (Helianthus tuberosus) cultures ). Only rifampicin was able to control bacterial contamination without affecting the regimes of plant cell division, cell differentiation or simple DNA synthesis in cultured implantations. However, rifampicin activated an increase in protein synthesis. K. Pollock et al., Plant Cell Reports, 2: 36-39 (1983), describe the toxicity of more than twenty different antibiotics in the efficiency of plate culture of cells derived from protoplast of Neither cotiana pl umbagini folia. Even though the beta-lactams, ie, the penicillins and the cephalosporins had little observable toxic effect on the efficiency of plaque culture of the cells, these antibiotics stimulated the growth of the cell colony of the plant. The aminomiglycosides, e.g., streptomycin and kanamycin, in contrast, were clearly toxic to plant cells. Erythromycin was relatively non-toxic to plant cells while tetracyclines were highly inhibitory in long-term toxicity tests. J.E. Gilbert and others, Ann. Appl. Biol., 119: 113-120 (1991) describe the use of antibiotics to control latent bacterial contamination in potato cell cultures. Two different combinations of antibiotics, either penicillin, streptomycin and amphotericin or erythromycin, streptomycin and carbenicillin were tested. Each combination when added to the media used to grow microplantules, reduced plant growth and induced chlorosis at higher concentrations. Not only were the mixtures phytotoxic, but they failed to eliminate contamination. However, when the first antibiotic mixture was added to enzyme media used to prepare protoplasts, the contamination was apparently eliminated. In addition to antibiotics, chemical insecticides have been tested due to solubility to inhibit or prevent microbial contamination in plant culture. For example, T. Macek et al., Biotechnology Techniques, 8 (12): 885-888 (1994), describe the use of diethylpyrocarbonate (DPC) to chemically stabilize nutritive media for plant cell cultures. The DPC killed all the contaminating microorganisms without changing the growth characteristics of the cells of the cells of the cultivated plants. However, since DPC decomposes in ethanol and carbon dioxide over a period of several hours when contacted with water, DPC can not serve to maintain the stability of plant cell cultures. through the duration of the growing period. Japanese Patent Number 4-311326 discloses a method for removing fungal contamination in tissue cultures of Cyclamen plants comprising immersing a slice of the tissue of a Cyclamine plant in a solution containing an imidazole or triazole fungicide or Cultivate a slice of the tissue in the culture medium containing fungicide. However, this treatment does not serve to inhibit bacterial contamination.

Japanese Patent Number 4-63589 discloses the use of allyl isothiocyanate (CH2 = CHC1-2N = C = S) as a sterilizing agent in plant culture media, wherein the chemical agent eliminated the need to subject autoclave and no abnormality was observed in the tissue cultures of the carnation plants. Combinations of aiotics and chemical insecticides have been tested to determine their ability to inhibit microbial contamination in plant crops. For example, D.A. Francko, Aquatic Botany, 26: 113-117 (1986), describes the use of aiotics (penicillin G and streptomycin sulfate) in combination with a fungicide (Captan) in an attempt to obtain seed axenic plants from the Nelumbo Lutes aquatic plant (Willd.). The tests indicated that when these agents included in the incubation media, more or less half of the seed cultures lacked detectable contaminants after two weeks of growth. However, only 11 percent of the initial cultures remained free of contaminants four weeks after transfer to new media lacking the aodies and the fungicide. JH Haldeman, et al., Hortscience, 22 (2): 306-307 (1987), describe the use of a combination of a fungicide (benomyl) and an aiotic (rifamopicin) as a treatment to control persistent fungal and bacterial contamination in Camellia sprouts tip plant implantations of plants that have grown in the field. A 24-hour treatment of the tips of the shoots with this mixture after normal disinfestation treatments in a bleaching solution, considerably reduced, but did not eliminate the contamination. W. Kneifel, and W. Leonhardt, Plant Cell, Tissue and Organ Culture, 29: 139-144 (1992), describe the tests of different combinations of aiotics and chemical insecticides against gram-positive and gram-negative bacteria isolated from the cultures of plant tissues. A mixture of Imipenem ™ (N-Formimidoyl-thienanicine hydrochloride, Merck, USA) and ampicillin, and a mixture of Imipenem ™ and penicillin G, were found to be very effective in inhibiting bacterial contamination and had no obvious effect on the regimen. of growth or growth of the root, nor was there any flux damage to chlorophyll. A mixture of IMIPENEM ™ and KATHON ™ (5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one; Rohm &Haas, Austria), however, led to reduced root growth of two species of plants tested. A variety of other chemicals are recognized in the art as having general preservative or insecticidal activity. Four of these chemical substances that were useful in the present inven are potassium sorbate, sodium benzoate, methylchloroisothiazolinone and methylisothiazolinone. The Merck Index (Tenth Edition, 1983, Entry Number 7555) lists potassium sorbate as an inhibitor of mold and yeast, and sodium benzoate (Entry Number 8413) as a preservative, especially for food products. D. Ryu, and D.L. Holdt, J. Food Protection, 56 (10): 862-867 (1993), demonstrates that potassium sorbate inhibits the growth of Penicillium expansum in fungus and in apples. DJ. Hall, Proc. Fia. State Hort. Soc, 101: 184-187 (1988), demonstrates that potassium sorbate and sodium benzoate each are effective as post-harvest fungicides in citrus fruits. O.E. Idise, and Izuagbe, Microbios Lett., 28: 117-121 (1985), show that sodium benzoate effectively inhibits the growth of bacteria and yeast in bottled palm wine. 0.0. Sodeko et al., Mcrobios, 51: 133-143 (1987), demonstrate that sodium benzoate effectively inhibits bacteria and fungi in orange juice. U.S. Patent No. 3,122,432 to Biggs discloses a composition for preserving cut flowers comprising various ingredients, including sodium benzoate which is described as a fermentation and mold inhibitor. Methylchloroisothiazolinone and methylisothiazolinone, together with the magnesium salts as stabilizers, comprises the KATHONTM CG insecticide (Rohm & amp;; Haas, USA), which is used extensively in a wide range of cosmetics, paints, air conditioning units, etc. B. Gruvberger et al., Contact Dermatitis, 15: 24-27 (1986), describe chromatographic methods for detecting KATHON ™ CG in cosmetics. T.K. Haack and E.F. Warwick, in: Pesticide Formulations and Application Systems, D.G. Devisetty, et al. (Editors), Am. Soc. Testing and Materials, Philadelphia (1993), pages 105-115, provide data related to the Legend MK ™ agricultural condom which is a different commercial form of methylchloroisothiazolinone and methylisothiazolinone, and which is describes therein as an effective antimicrobial agent for aqueous fluent pesticidal formulations. P.N. Green, Lett. Appl. Microbiol. , 17: 158-161 (1993) demonstrates that of five commercial insecticides tested, the mixture of methylchloroisothiazolinone and methylisothiazolinone was highly effective against microbial blms generated in the laboratory of Legionella bozemanii. J.S., Chapman, Am. Clin. Lab., Pages 13-14 (August 1994), describes how a third commercial form of the mixture of methylchloroisothiazolinone and methylisothiazolinone, sold as ProClin ™ (Rohm &Hass, distributed by Supelco Inc.) is highly effective against a broad spectrum of at least 14 different species of gram-negative bacteria, 9 different species of gram-positive bacteria and 18 different species of fungi. In addition, U.S. Patent Nos. 3,523,121; 3,761,488; 4,105,431; 4,243,403; 4,252,694; 4,265,899 and 4,279,762, assigned to Lewis disclose novel isothiazolones which are insecticidally active against a wide range of microorganisms, including bacteria, algae and fungi in a wide range of applications, including soaps, detergents, coatings, cosmetics, oils or lubricants for blades , and as fungicides in seeds that are going to be planted in the ground. In addition, U.S. Patent Nos. 4,454,146; 4,499,071; 4,540,570 and 4,555,400 issued to Borovian disclose synergistic preservative compositions for inhibiting the growth of microorganisms, whose compositions comprise at least two components, the first of which is selected from a group of one or more traditional preservatives, including benzoic acid, acid sorbic acid and a mixture of methylchloroisothiazolinone and methylisothiazolinone, and a second component which is a polycyclic compound defined therewith which, in combination with the first component, kills or inhibits microorganisms synergistically. Finally, the North American Patent Nos. 5,028,620 and 5,100,905 issued to Hsu disclose a synergistic insecticidal composition that decreased the sensitization potential, the first component of which is methylchloroisothiazolinone within the range of about 1.2 percent to 25.4 percent, and the second component of which is methylisothiazolinone within the range of about 74.6 percent to about 98.8 percent. Even though these four components, namely, potassium sorbate, sodium benzoate, methylchloroisothiazolinone and methylisothiazolinone, are known to have preservative or insecticidal activities and there is no teaching or suggestion in the art that these chemicals or any combination thereof would be Useful for reducing or preventing microbial contamination in tissue cultures of plants without adversely affecting the tissues of plants. In fact, the report by W. Kneifel and W. Leonhardt, supra, of reduced root growth in a mixture of IMIPENEM ™ and KATHON ™ reveals that by moving away from the use of KATHON ™ containing agents in tissue cultures of plants. It would be beneficial to provide additional chemical agents that could be added to the tissue culture media of plants, whose agents reduce or prevent bacterial and fungal contamination throughout the duration of the culture period, and which allow essentially normal seed germination or growth of essentially normal development of seeds, plants, plant organs, plant tissues or cells of cultivated plants.

COMPENDIUM OF THE INVENTION It is an object of the present invention to provide compositions and methods for reducing or preventing microbial contamination of tissue cultures of in vitro plants throughout the culture period. This object can be achieved by incorporating a chemical agent in a plant growth medium at a concentration that effectively reduces or prevents the growth of bacteria and fungi and that permits essentially normal germination of seed or the essentially normal growth or development of plants , plant organs, plant tissues or plant cells. A further object of the present invention is to provide a plant tissue culture medium comprising a chemical agent in a concentration that is effective to reduce or prevent microbial contamination throughout the culture period and which allows essentially normal seed germination. or the essentially normal growth or development of plants, plant organs, plant tissues or plant cells. A further object of the present invention is to provide a kit for growing seeds, plants, plant organs, plant tissues or plant cells in a plant tissue culture medium comprising a chemical agent in a concentration that is effective to reduce or preventing microbial contamination through the culture period and allowing essentially normal germination of seeds or the essentially normal growth or development of plants, plant organs, plant tissues or plant cells whose kit comprises a culture package comprising a plant tissue culture medium comprising the chemical agent, wherein the culture medium is either ready, that is, ready for immediate use, ready to be prepared, for example, by the addition of water. The kit further comprises one or more seeds of plants which have preferably been sterilized on their surface, one or more plants, plant organs, plant tissues or plant cells. 4. DETAILED DESCRIPTION OF THE INVENTION The present invention provides compositions and methods for reducing or preventing microbial contamination of plant cultures in vitro through the culture period. A chemical agent is incorporated into a plant growth medium in a concentration that is effective to reduce or prevent the growth of bacteria and fungi and that permits essentially normal seed germination or the essentially normal growth or development of plants, plant organs , plant tissues or plant cells. The chemical agent useful for practicing the present invention preferably comprises a mixture of methylchloroisothiazolinone (5-chloro-2-methyl-4-isothiazolin-3-one), (methylisothiazolinone (2-methyl-4-isothiazolin-3-) ona), magnesium chloride and magnesium nitrate.Most preferably, the chemical agent comprises a mixture of methylchloroisothiazolinone, methylisothiazolinone, magnesium chloride, magnesium nitrate and potassium sorbate or sodium benzoate.Most preferably, the chemical agent comprises A mixture of methylchloroisothiazolinone, methylisothiazolinone, magnesium chloride, magnesium nitrate, magnesium sorbate and sodium benzoate Applicants have surprisingly discovered that these combinations of chemicals, on a specific scale of concentrations, are effective in reducing or preventing microbial contamination. in plant tissue cultures through the length of the tissue culture period, but allow the generation n essentially normal seed development or growth or essentially normal plants, plant organs, plant tissues or plant cells. The relative concentrations of the individual components comprising the chemical agent can be varied to produce a mixture that is optimally effective to practice the method of the present invention for any specific plant culture medium, plant species, seed of plants, plants, plant organ, plant tissue or plant cell. However, a preferred mixture of chemical agent components comprises: methylchloroisothiazolinone in a concentration range of about 2.0 to about 2.6 grams per liter; methylisothiazolinone in a concentration range of about 0.6 to about 0.8 gram per liter; magnesium chloride in a concentration range of about 15.0 to about 30 grams per liter; and magnmesium nitrate in a concentration range of about 15.0 to about 30 grams per liter. An especially preferred mixture of the components in the chemical agent further comprises: potassium sorbate in a concentration range of about 15 to about 25 grams per liter, or sodium benzoate in a concentration range of about 13 to about 27 grams per liter . An especially preferred mixture of the components in the chemical agent further comprises, potassium sorbate in a concentration range of about 15 to about 25 grams per liter; and sodium benzoate on a concentration scale of about 30 percent to about 27 grams per liter. In all cases, the components of the chemical agent are mixed to form a solution of the chemical agent using any liquid in which the components are dissolved, but preferably in water, and de-1! especially preferred in distilled or deionized water. As used in the present application, the terms "plant tissue culture" or "plant tissue culture" refers to any process that is carried out in vitro where the seeds are germinated or the plants, organs of plants, plant tissues or plant cells are propagated, differentiated, subcultured or otherwise maintained in a culture medium, defined or undefined, which is typically maintained in a sterile condition (syn: aseptic, axenic), i.e. free of microbial contamination and usually incubated under controlled environmental conditions. As used in the present application, the term "microbial contamination" refers to the growth of any of the unwanted microorganisms, eg, bacteria or fungi in a plant tissue culture As used in the present application, the term " plant tissue culture medium "," plant growth medium "," culture medium "and" medium "refer to a solid substrate or liquid solution in which a seed of the plant will germinate or a plant can be maintained or To grow, an isolated plant organ or plant tissue may be maintained, propagated or differentiated or one or more isolated plant cells, aggregates of plant cells or protoplasts of plant cells may be maintained, propagated or differentiated, and where maintains a sterile condition, that is, essentially free of microbial contamination The terms "plant tissue culture medium," "plant growth medium", "culture medium" and "medium" are designated In addition to being reference to water containing an appropriate mixture of mineral salts. The culture medium may also incorporate, in appropriate concentrations phytohormones, including for example auxins, cytokinins or gibberellins, vitamins such as one or more of B vitamins, one or more sources or sources of carbon, including for example sucrose or glucose, and one or more undefined growth enhancers such as coconut milk. The components of the mineral salt mixtures can be selected and prepared in accordance with the requirements of the specific plant species being propagated. The appropriate composition of the mineral salts can either be determined empirically or selected from general salt compositions known in the plant tissue culture technique and prepared accordingly. Alternatively, the mineral salts may be selected from any number of commercially available mixtures (eg, from Sigma Chemical Co., of St. Louis, Mo.). Mixtures of the mineral salt useful in the practice of the present invention include, for example, Hoagland basic salt mixture, Gambor B-5 basic salt mixture, Heller basic salt mixture, Murashige basic salt mixture and Skoog, Nitsch's basic salt mix, White's basic salt mix and variations thereof. In addition, the various macronutrients, micronutrients and vitamin components known in the art can be combined in various ways in order to produce an appropriate culture medium for the species of plant that is spreading. According to the method of the present invention, the chemical agent is added to the plant tissue culture medium in a concentration which will reduce or prevent the growth of bacteria or fungi or both, and which allows the germination of essentially normal seed and the growth or essentially normal development of a plant, plant organs, plant tissues or plant cells grown therein. As used in the present invention, a chemical agent is effective to reduce or prevent microbial growth in a plant growth medium if to the addition of the chemical agent to the plant growth medium at a concentration that allows the germination of seeds essentially normal or essentially normal growth of plants, parts of plants, plant tissues or plant cells, reduces the amount of bacterial or fungal contamination by at least 80 percent compared to control media lacking the chemical agent. In accordance with the present invention, essentially normal seed germination is defined as a percentage of germination that is at least 50 percent of the germination percentage in the control culture medium that does not contain the chemical agent. According to the present invention, the essentially normal growth of a plant, organ of plants, plant tissues, or plant cells is defined as a growth regime or the regimen of division of plant cells, plant organ, tissues of plants or plant cells, which is at least 50 percent of the growth regimen or the regimen of cell division in a corresponding plant, plant organ, plant tissue or plant cell in the control culture medium that does not contain the chemical agent. According to the present invention, the essentially normal development of a plant, organ of plants, plant tissues or plant cells is defined as when one or more of the development events occur in the plant, plant organs, plant tissues. or plant cells that are essentially the same as those that occur in a plant, plant organs, plant tissues or corresponding plant cells in the control culture medium that does not contain the chemical agent. In general, the chemical agent useful in the method of the present invention will be effective within a scale of concentrations in the culture medium that allow essentially normal germination of seeds or the essentially normal growth or development of a plant, organs of plants, plant tissues or plant cells grown therein. A preferred scale of concentrations of the chemical agent in the culture medium is from about 0.01 percent to about 0.20 percent (volume / volume). A more preferred scale of concentrations of the chemical agent in the culture medium is from about 0.02 percent to about 0.10 percent (volume / volume). The especially preferred concentration scale of the chemical agent in the culture medium is from about 0.3 percent to about 0.05 percent (volume / volume). For those seeds of plants, plants, plant organs, plant tissues or plant cells of any species for which the aforementioned scales are not satisfactory, the optimum effective concentration of the chemical agent in the culture medium to be used in the method claimed can be determined empirically by growing seeds, plants, plant organs, plant tissues or plant cells in a tissue culture medium of plants at a scale of chemical agent concentrations, and selecting one or more concentrations at which Microbial contamination is reduced or prevented, but it allows seed germination to be essentially normal or to allow essentially normal growth or development of the plants, plant organs, plant tissues or plant cells. This empirical determination can be carried out without undue experiments using standard techniques for seed germination or preparation of plants, plant organs, plant tissues or plant cells in combination with routine selection techniques known to those skilled in the art. . For example, plant tissue culture media can be prepared according to any standard recipe for tissue culture media of plants known in the art, with the chemical agent added on a scale of concentrations for selection as well as without the chemical agent (control). The seeds of plants, can be "planted" in these media, incubated properly and examined after a sufficient period of time to determine germination percentages. In addition, an evaluation of the growth or development of the roots and shoots of the germinated seeds as determined for example using any of the morphological, anatomical or biochemical characteristics can be done by comparing the germinated seeds in culture media comprising the chemical agent with respect to to seeds germinated in control media lacking the chemical agent. The skilled artisan will be aware and will be able to apply the different morphological, anatomical, physiological and biochemical tests that are useful to determine the effects of chemical agents on the growth of plants. For example, a morphological analysis may comprise a comparison of the shape, size or number of roots, shoots, leaves or reproductive organs or part of them. An anatomical analysis may comprise, for example, a comparison analysis of the size, shape, pattern, or differentiation of cells, such as, for example, the quantity, location or maturation of vascular tissues, trichomes or stomata, or the presence or absence of meritallos that are actively divided. A physiological analysis may comprise, for example, a comparative analysis of respiration regimens, photosynthesis, stomach resistance or ethylene production. A biochemical analysis may comprise, for example, a comparison analysis of proteins or DNA synthesis, chlorophyll degradation or the presence, absence or quantity of other pigments. One more of these assays, all of which are known to a skilled artisan, can contribute to an empirical determination of the optimal concentration of the chemical agent in the plant tissue culture medium to practice the method of the present invention in a specific plant species. Once the optimum concentration of the chemical agent for carrying out the method of the present invention is determined, it can be added to a culture medium which is then to be used in liquid form for liquid cultures, including for example, suspension cultures of cells or protoplast cultures, or i.e., subsequently solidified by the addition of a gelling agent. The culture medium is then typically adjusted to an appropriate pH, for example, a pH of 5.8, using appropriate acidic or basic solutions, e.g., hydrochloric acid (HCl) or potassium hydroxide (KOH). In those cases where the culture medium is to be solidified, a gelling agent, for example, a gel of PHYTAGEL ™ gel (Sigma Chemical Company) can be added to the culture medium at an appropriate concentration scale for example of 0.2 percent. at 0.3 percent (by weight / volume) for gelatin gum. The culture medium must then be heated sufficiently eg by undergoing autoclaving to dissolve the gelling agent. The presence of the chemical agent, however, makes the autoclaving unnecessary for sterilization purposes. The antimicrobial activity of the culture medium comprising the chemical agent is not reduced by autoclaving (121 ° C, 15 minutes). In addition, applicants have discovered that the culture medium of the plant comprising the chemical agent that has been autoclaved is less inhibitory for germination of the plant seeds or for the growth or development of the plants, organs of plants, plant tissues or plant cells, as compared to the same culture medium comprising the chemical agent that has not been autoclaved. However, this does not mean that autoclaving is strictly required for the practice of the invention, only that the scale of concentrations of the chemical agent in the culture medium useful for practicing the present invention may be lower in some cases. wherein the culture medium has not undergone autoclaving. After the autoclave treatment, the culture medium comprising the chemical agent and the dissolved gelling agent, can then be transferred to individual culture containers of any kind, useful in the art and the medium is allowed to cool and solidify. An additional benefit of the chemical agent is that, when autoclaving is not needed to dissolve a gelling agent, the heat-labile components of the culture medium, such as vitamins and sugars, will no longer require filter sterilization. By means of culture prepared in this way it can be stored for a prolonged period of time or used immediately. The batches of culture media can be subdivided by transfers to a mass in culture bases useful in the art, including flasks, beakers, square-sided containers, screw-cap jars, culture tubes or any of the other containers that They are useful for growing seeds, plants, plant organs, plant tissues or plant cells. The culture containers can be composed of glass including, for example, borosilicate glass or soda glass, as well as plastics including, for example, polystyrene or transparent polypropylene. The culture containers, generally must be clean, that is to say, exempt of erasure, dust or chemical residues; however, the addition of the chemical agent to the culture medium makes it unnecessary to sterilize the culture containers by heat, radiation or other chemical means. Any plant seed, whole plant, isolated plant organ, plant tissue or plant cell can be grown in the plant tissue culture medium containing the chemical agent. For example, the seeds of any plant species can be germinated in the culture medium of the invention. These seeds can come from any species of dicotyledonous, monocotyledonous or gymnosperm plants, including any species of woody plant that grows as a tree or shrub, any herbaceous species or any species that produces edible fruits, seeds or legumes, or any species that produces flowers in colors or aromatic. For example, the seed, whole plant, isolated plant organ, plant tissue or plant cell, can be selected from a plant species of the group consisting of cucumber, morning glory, balsam, pepper, eggplant, marigold, lotus, cabbage, margarita and carnation. Further, for the purposes of this invention, the term "plant seed" encompasses the spores of ferns and other lower vascular plants as well as the spores of non-vascular plants, such as mosses, hepatics and hops. Despite the anti-microbial nature of the chemical agent, it is preferred that before placing a seed, plant or plant organ in the culture medium to initiate a new culture, the seed, plant or plant organ must be sterilized on its surface to remove fungal or bacterial spores that are often initially present in relatively high density thereon. Techniques for sterilizing the surface of seeds, plants and plant organs are well known in the art and can be achieved using any sterilization agent generally obtainable, but a household bleach (sodium hypochlorite solution) diluted in water is preferred. For example, one or more of the seeds can be sterilized on their surface effectively by immersion in a diluted solution of the domestic bleach in water at a concentration, for example, from about 5 percent to about 20 percent (by volume / volume ) of the bleach, with or without a surfactant for a period of time sufficient to stabilize the seed on its surface, eg, from about 10 minutes to about 120 minutes. The seeds are then preferably rinsed three times with water. Organs of plants that can be grown according to the method of the present invention include, but are not limited to, leaves, stems, roots, vegetative buds, flower buds, meritals, embryos, cotyledons, endosperm, sepals, petals, pistils, carpels, stamens, pollen, pollen tubes, ovules, ovaries and fruits or sections, slices or discs that are taken from them. Plant tissues that can be grown in accordance with the present invention include, but are not limited to callus tissues, terrestrial tissues, vascular tissues, storage tissues, meristematic tissues, leaf tissues, bud tissues, root tissues, tissues of gall, tissue of the plant and reproductive tissues. Plant cells that can be grown in the culture medium of the present invention include, but are not limited to, isolated cells with cell walls, aggregates of various sizes thereof and protoplasts. Any of the plants, plant organs, plant tissues or plant cells that have been grown in vitro in the culture medium of the invention, can be transferred ie subcultured into a new culture medium comprising the chemical agent during appropriate periods of time for growth, development or continuous differentiation in vitro. The present invention provides an additional benefit since subculturing the tissues of plants or plant cells can be carried out without having to strictly adhere to sterile techniques or the use of a sterile working environment., such as a laminar flow hood. The present invention proposes that plants, plant organs, plant tissues or cells of cultivated plants can be transferred from the culture medium of the present invention to a culture medium that does not comprise the chemical agent or vice versa as appropriate. The present invention further proposes that an intact plant or intact plant organ that has been cultivated or otherwise regenerated or maintained in the culture medium of the present invention, can subsequently be removed from the tissue culture environment in vitro and transferred to a substrate, for example, to soil or vermiculite, for continuous growth and development outside the in vitro environment. The present invention further provides a kit for growing seeds, plants, plant organs, plant tissues or plant cells in the plant tissue culture medium comprising a chemical agent in a concentration that is effective in reducing or preventing contamination microbial through the culture period and allowing essentially normal germination of seeds or the essentially normal growth or development of plants, plant organs, plant tissues or plant cells whose case comprises a culture container consisting of a medium tissue culture of plants comprising the chemical agent, wherein the culture medium is either prepared, that is, ready for immediate use or ready to be prepared, for example, by the addition of water. The kit further comprises one or more seeds of plants that have been sterilized on their surface preferably, or one or more plants, plant organs, plant tissues or plant cells. Having now generally described the invention, it will be more readily understood by reference to the following examples which are provided by way of illustration and which are not intended to limit the present invention.

. EXAMPLE: INHIBITORY EFFECT OF THE CHEMICAL AGENT ON MICROBIAL POLLUTION The following experiments were carried out to test the antimicrobial effect of increasing the concentrations of the chemical agent in a plant tissue culture medium. A normal plant culture medium consisting of Murashige and Skoog mineral salts, 2 percent (w / v) sucrose, 0.4 percent (w / v) Gelrite ™ adjusted to a pH of 5.8 with KOH was tested. , either or without the chemical agent, as will be described below. In the first set of experiments, a chemical agent comprising the following components was prepared to be incorporated into the culture medium before being subjected to autoclaving: Methylchloroisothiazolinone 2.3 grams Methylisothiazolinone 0.7 grams Magnesium chloride 23.0 grams Magnesium nitrate 23.0 grams Potassium sorbate 20.0 grams Sodium benzoate 20.0 grams Distilled water to a total volume of one liter. The different concentrations of the above-mentioned chemical agent in the culture medium were tested to determine the optimal concentrations for antimicrobial activity. For example, a scale of chemical agent concentrations of 0.2 percent to 1.0 percent (volume / volume) in the culture medium was tested in increments of 0.1 percent. A chemical agent concentration scale of 0.1 percent to 0.2 percent (volume / volume) in the culture medium and increments of 0.025 percent was tested. Finally, a scale of chemical agent concentrations of 0.01 percent to 0.1 percent of the culture medium was tested in increments of 0.01 percent. After the addition of the chemical agent, the culture medium was autoclaved (15 minutes, 121 ° C) to dissolve the gelling agent. All subsequent manipulations were carried out under non-sterile conditions. The culture media containing the chemical agent were emptied into non-sterile polystyrene containers, covered with non-sterile closures and allowed to solidify. The culture medium that does not contain the chemical agent was emptied in either non-sterile containers (control A) or in sterile containers (control B), with the containers capped with non-sterile and sterile closures respectively and allowed to solidify. Ten containers containing the culture medium comprising the agent the chemical at each concentration and ten containers of the control medium A were incubated for 7 days at 28 ° C in the dark. After one week, no microbial contamination was evident in any container containing the culture medium comprising the chemical agent. In contrast, microbial contamination was evident in all control A packages containing an average of 6.5 fungal colonies and bacteria in total per container. In a second experiment, 10 containers containing a culture medium comprising the chemical agent at each concentration and 10 control containers B were uncovered and left exposed to the outside air for 8 hours. The containers were recapped and incubated for 7 days at 28 ° C in the dark. No microbial contamination was evident in any container containing the medium with the chemical agent, with the exception of two containers containing the lowest concentration of the chemical agent, ie, 0.01 percent (volume / volume), each of which contained a single fungal colony on the surface of the medium. In contrast, all control B containers were contaminated containing an average of 2.5 fungal and bacterial colonies, in total, per container. Additional experiments were carried out to test the effectiveness of the different formulations of the chemical agent. A first chemical agent was prepared comprising all six components used in the chemical agent formulated above, namely methylchloroisothiazolinone, methylisothiazolinone, magnesium chloride, magnesium nitrate, potassium sorbate and sodium benzoate, each in the same concentration as before. A second chemical agent comprising the same components in the same concentrations as the previous ones but lacking sodium benzoate was prepared. A third chemical agent was prepared comprising the same components in the same concentrations as the previous ones, but lacking potassium sorbate. A fourth chemical agent comprising the same components in the same concentrations as before, but lacking both potassium sorbate and sodium benzoate, was pre-prepared. A fifth chemical agent comprising only potassium sorbate and sodium benzoate was prepared in the same concentrations as above. A sixth chemical agent comprising only potassium sorbate in the same concentration as above was prepared. A seventh chemical agent comprising only sodium benzoate in the same concentration as above was prepared. Plant culture media were prepared as above. The seven different chemical agents prepared as above were incorporated into different batches of plant culture medium as above to a final concentration of 0.035 percent (volume / volume) in all media except the controls. All the culture media of plants comprising the different chemical agents were emptied into 10 non-sterile polystyrene containers each and covered with non-sterile covers. The control culture medium without the chemical agent was also prepared as above. The containers were incubated for 14 days at 27 ° C in the dark. After incubation, the 10 containers with the control culture medium had a total of 82 microbial colonies (0 percent effective). The culture medium comprising the first chemical agent (all six components present) did not have microbial colonies (100 percent effective). The culture medium comprising the second chemical agent lacking only sodium benzoate had 7 microbial colonies (91 percent effective). The culture medium comprising the third chemical agent lacking only potassium sorbate had 10 microbial colonies (88 percent effective). The culture medium comprising the fourth chemical agent that lacked both potassium sorbate and sodium benzoate had 13 microbial colonies (84 percent effective). The culture medium comprising the fifth chemical agent consisting only of potassium sorbate and sodium benzoate had 31 microbial colonies (62 percent effective). The culture medium comprising the sixth chemical agent comprising only potassium sorbate had 55 microbial colonies (33 percent effective). Finally, the culture medium comprising the seventh chemical agent comprising only sodium benzoate had 65 microbial colonies (21 percent effective). These results indicate that the different formulations of the chemical agent are effective to reduce or prevent the growth of bacteria and fungi in plant culture media under non-sterile conditions. However, to make effective in accordance with the present invention, the components of methylchloroisothiazolinone, methylisothiazolinone, magnesium chloride and magnesium nitrate must be included in the chemical agent. 6. EXAMPLE: EFFECT OF THE CHEMICAL AGENT ON THE GERMINATION OF SEEDS The following experiments were carried out to test the effects of increasing the concentrations of the chemical agent in a plant culture medium during germination of the seeds and the subsequent growth of shoots and roots. The chemical agent used for these experiments comprised methylchloroisothiazolinone, methylisothiazolinone, magnesium chloride, magnesium nitrate, potassium sorbate and sodium benzoate and was formulated as described above for the first set of experiments in Section 5, and incorporated in the culture medium as above. The culture media were then subjected to autoclaving (121 ° C, 15 minutes). The seeds of 10 plant species were sterilized on their surface or not sterilized on their surface. Seeds to be sterilized on the surface were submerged in 20 percent to 40 percent (volume / volume) of a solution of household bleach for 30 minutes and rinsed 3 times with water from the regular non-sterile tap. The seeds were then inserted approximately up to .254 cm into the solidified culture medium comprising the chemical agent at each concentration. One hundred seeds were tested for each plant species at each concentration of the chemical agent. The containers were incubated for 7 days at 28 ° C in the dark and selected for both microbial contamination and seed germination.

With respect to microbial contamination, chemical agent concentrations of 0.4 percent (volume / volume) and less, colonies of bacteria and fungi that appeared in approximately 86 percent of the seeds had not been sterilized on their surface, while the seeds sterilized on their surface remained free of any microbial contamination at all concentrations. The effect of the concentration of the chemical agent on the percentage of seed germination varied with the plant species. For example, at a concentration of 1.0 percent (volume / volume) of the chemical agent, cucumber seeds sterilized on the surface germinated to a high percentage (72 percent), while the germination of the lotus seeds sterilized in its surface was dramatically reduced (5 percent) (Picture 1). The germination percentage of seed sterilized on the surface for all species remained high (> 50 percent) at chemical agent concentrations up to and including 0.2 percent (volume / volume). These results indicate that the addition of the chemical agent to the culture medium to a concentration of 0.2 percent (volume / volume) allows seed germination to be essentially normal in all tested species, but that some species, eg, the cucumber, morning morning glory, balsam, etc. they exhibit essentially normal germination up to a concentration of the chemical agent in the middle of 1.0 percent (volume / volume). In addition, these results indicate that sterilization of the seed surface is preferred so that the chemical agent in the culture medium expresses its optimum effect to reduce or prevent microbial contamination of plant tissue cultures due to the high density of the plants. bacterial or fungal spores normally present on the seeds of most plant species. It is worth mentioning that the seeds that were not sterilized on their surface usually had higher percentages of germination than the seeds of the same species that were sterilized on their surface (Table 1). This difference was more evident in media containing chemical agent concentrations of 0.2 percent (volume / volume) and higher, it can be the result of the bleach making the seed coat more permeable, thus making the seed more sensitive to the chemical agent. In relation to the subsequent growth of buds and roots of germinating seeds, at chemical agent concentrations of 0.3 percent (volume / volume) and higher, for all species either no roots of the germinating seeds grew or the roots that grew stopped in their growth and development (Table 2). The normal growth and development of both shoots and roots was observed in all plant species, in culture media containing a chemical agent concentration of 0.05 percent or less. These results indicate that even when a concentration of 0.05 percent (volume / volume) of the chemical agent or less in the culture medium allows the growth and essentially normal development of the shoots and roots in all species tested, some species , that is, cucumber and cabbage can produce essentially normal shoots and roots in the culture medium containing the chemical agent in a concentration of up to 0.2 percent (volume / volume). As described above in section 4, the optimum concentration of the chemical agent for other plant species can be determined empirically and without undue experimentation. 7. EXAMPLE: EFFECT OF THE CHEMICAL AGENT ON ORGANOGENESIS OF A CALLO TISSUE The following experiments were carried out to test the effects of increasing the concentrations of the chemical agent in culture medium in callus tissue organogenesis. The chemical agent used for these experiments comprised methylchloroisothiazolinone, methylisothiazolinone, magnesium chloride, magnesium nitrate, potassium sorbate and sodium benzoate, as formulated above. The eggplant leaves (cv. Black Beuty) of the plants that grew in vitro were cut into small squares (approximately one centimeter by one centimeter) using sterile techniques. Ten leaf squares were incubated in the culture medium containing Murashige and Skoog mineral salts, vitamins, including thiamin, pyridoxine and nicotinic acid, 3 percent sucrose (weight / volume), 2 milligrams per liter of naphthaleneacetic acid (NAA) ) and 0.8 percent agar (weight / volume), (control medium) at a pH of 5.8, which medium would be subjected to autoclaving (121 ° C, 15 minutes) before use. Ten leaf squares, each were incubated in the same type of culture medium as before, but also comprised either 0.03 percent or 0.04 percent (volume / volume) of the chemical agent. The cultures were incubated in the dark at 27 ° C. After 30 to 40 days, approximately 50 milligrams of the white callus tissue had proliferated on all the leaf squares in all media. The callus tissue was separated from each of the leaf frames and subcultured in a fresh culture medium prepared as above, with the exception that trans-zeatin (2 milligrams per liter) replaced NAA. The callus tissues were subcultured at 27 ° C with a light treatment of 283.20 cubic meters for 10 hours per day. After 37 days, callus tissues in all media regenerated an average of 28 shoots per callus tissue. The shoots were separated from the callus tissues and inserted into a new culture medium prepared as above, but lacking any of the phytohormones. All the outbreaks generated roots in all the culture media. These results indicate that the addition of an antimicrobially effective concentration of the chemical agent to the culture medium did not negatively affect callus induction, callus proliferation or regeneration of the plant. Even though this invention has been described in relation to specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any of the variations, uses or adaptations of the invention, generally following the principles of the invention and including those deviations from the present disclosure that remain within the known or customary practice within the art to the which relates the invention and which can be applied to essential features indicated in the foregoing as given below in the scope of the appended claims. All of the aforementioned publications are incorporated herein by reference.

TABLE 1 TABLE I (Continued) * 100 seeds / plant / treatment S = Surface sterilized N = Not sterilized t The chemical agent for these experiments included methylchloroisothiazolinone, methylisothiazolinone, magnesium chloride, magnesium nitrate, potassium sorbate and sodium benzoate. The culture media comprising this chemical agent were subjected to autoclaving.

Table 2" * 100 seeds per treatment t The chemical agent for these experiments included methylchloroisothiazolinone, methylisothiazolinone, magnesium chloride, magnesium nitrate, potassium sorbate and sodium benzoate. The culture media comprising this chemical agent were subjected to autoclaving. S = Normal outbreak formation N = Normal root formation, = No abnormal growth

Claims (10)

R E I V I N D I C A C I O N E S:

1. A plant tissue culture medium comprising a chemical agent, the chemical agent comprising: methylchloroisothiazolinone in a concentration range of about 2.0 to about 2.6 grams per liter; methylisothiazolinone in a concentration range of about 0.6 to about 0.8 gram per liter; magnesium chloride in a concentration range of about 15.0 percent to about 30 grams per liter and magnesium nitrate in a concentration range of about 15.0 to about 30 grams per liter; wherein the chemical agent is present in the plant tissue culture medium at a concentration that reduces or prevents microbial contamination of the plant tissue culture medium and allows essentially normal germination of seeds or the essentially normal growth or development of plants, plant organs, plant tissues or plant cells.

2. The plant tissue culture medium of claim 1, wherein the chemical agent further comprises potassium sorbate in a concentration range of about 15 to about 25 grams per liter.

3. The plant tissue culture medium of claim 1, wherein the chemical agent further comprises sodium benzoate in a concentration range of about 13 to about 27 grams per liter.

4. The plant tissue culture medium of claim 1, wherein the chemical agent further comprises potassium sorbate in a concentration range of about 15 to about 25 grams per liter and sodium benzoate of a concentration scale of about 13 to approximately 27 grams per liter.

The plant tissue culture medium of claim 4, wherein the chemical agent is present in the plant tissue culture medium in a concentration range of about 0.01 percent (volume / volume) to about 0.20. percent (in volume / volume).

The plant tissue culture medium of claim 4, wherein the chemical agent is present in the plant tissue culture medium in a concentration range of about 0.02 percent (volume / volume) to about 0.10 percent. cent (in volume / volume).

7. The plant tissue culture medium of claim 4, wherein the chemical agent is present in the plant tissue culture medium in a concentration range of about 0.03 percent (volume / volume) to about 0.05 percent ( in volume / volume).

8. A case for germinating a plant seed or growing a plant, plant organ, plant tissue or in vitro plant cell, the kit of which comprises: (a) a culture container; (b) a seed of plant, plant, plant tissue, plant organ or plant cell; and (c) the plant tissue culture medium of claim 1, 2, 3, 4, 5, 6 or 7. The kit of claim 8, wherein the seed, plant, plant organ, tissue plant or plant cell is a plant species that is selected from the group consisting of cucumber, morning morning glory, balsam, pepper, eggplant, calendula, lotus, cabbage, daisy and carnation. A method for reducing or preventing microbial contamination in a plant tissue culture medium comprising adding the chemical agent of claim 1, 2, 3, 4, 5, 6 or 7, to the tissue culture medium of plants.