JPH08182494A - Solid culture medium - Google Patents

Abstract

PURPOSE: To obtain a solid culture medium, using a nonionic water absorbing resin as a gelling agent, excellent in chemical stability without being influenced by an electrolyte and inhibiting the growth and differentiation of various microorganisms, cells, etc., and capable of providing a suitable culture environment. CONSTITUTION: A nonionic water absorbing resin which is a cross-linked polymer or copolymer, etc., of a compound, represented by the formula (R<1> and R<2> are each H or methyl) (e.g. N-vinylacetamide) and having >=10 timed water absorption ratio of an aqueous solution at a pH within the range of 2-11 in amount of 0.1-4% (wt./vol.) based on a solid culture medium is contained therein to absorb an aqueous solution of a nutrient culture medium. Thereby, the resultant objective solid culture medium is excellent in chemical stability without being influenced by an electrolyte such as inorganic salts and causing inhibition of the growth and differentiation to various plant tissues, microorganisms, animal cells, etc., and capable of manifesting constant water holding effects and providing a suitable culture environment and suitable for culturing thermophilic, acidophilic, alkalophilic or halophilic microorganisms.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is applied to various plant tissues, microorganisms, animal cells and the like, which are nonionic, have excellent chemical stability, and show a certain water retention effect without being affected by electrolytes such as inorganic salts. The present invention relates to a solid culture medium and a plant tissue culture method using a water-absorbent resin that provides a suitable culture environment without inhibiting growth and differentiation.

[0002]

2. Description of the Related Art Agar, agarose, agaropectin, gellan gum, κ-carrageenan and the like have been used as gelling agents for conventional culture media. However, it is known that when these naturally-derived gelling agents are used, unidentified trace components contained therein often inhibit cell growth and differentiation. Therefore, in order to remove these inhibitory substances, measures such as adding activated carbon to the medium have been taken. Further, since it is a natural substance, its quality varies depending on lots, manufacturers, etc. depending on its raw material, refining method, etc., and it is expensive, which makes it difficult to use.
Furthermore, the higher the salt concentration, the higher the NH ₄ ⁺ , H ₂ PO ₄ ⁻ ions, and the lower the pH of the medium (pH is about 5 or less), the harder the agar is to solidify. It was necessary and needed to find the optimum concentration of agar.

In order to gel agar, it was heated once to 85 ° C. or higher and then cooled to 38 ° C. or lower. Therefore, for example, in order to prepare a gel in a petri dish, it is necessary to pour it into a petri dish while the medium is still in a liquid state, which requires time and labor for preparation, and is particularly unsuitable for large-scale culture. Since the whole medium of agar is gelled, the diffusion rate of waste substances and harmful substances produced in the culture tissue is small, and depending on the type of cells, the toxic effect may make the culture completely difficult. Even when it is necessary to mix a substance that is unstable in temperature into the medium, it is difficult to use agar because the temperature becomes high during the preparation process. The agar gel was opaque and hindered the observation of cells. Furthermore, the agar medium once hardened cannot be frozen and stored, and the agar was frozen and stored in a state of being dispersed in the medium, and was heated and gelated each time it was used. In addition, when the agar gel was left to stand, free water was generated (splitting phenomenon) over time, which affected cell growth.

On the other hand, gelation of gellan gum and κ-carrageenan requires divalent metal ions such as Ca ²⁺ and Mg ²⁺ , and the state of the gel is changed by the metal ions in the medium. In addition, the size of the medium could not be changed freely, and it was impossible to absorb water during the culture. Furthermore, in recent years, a method of adding a water-absorbing polymer to a medium has been proposed (Japanese Patent Laid-Open No. 3-94624). However, this method is mainly intended to reduce the cost of using agar by adding a small amount to the medium, and is not a complete replacement for agar. That is, none of the drawbacks of agar have been solved.

[0005]

Under the circumstances, the present invention solves the above-mentioned problems of the prior art, is excellent in gel stability, and is constant even under various normal environments including acidity, alkalinity and high temperature. By using a nonionic water-absorbing resin showing a water absorption capacity as a gelling agent for the medium, there is no inhibition of cell growth and differentiation, and a solid medium containing low and high pH and high concentrations of salts and nutrients. Preparation is easy, nutrients and solutions such as chemicals can be added in the middle of culture, and since it is an artificial gelling agent, deterioration of reproducibility due to trace impurities found in natural gelling agents can be suppressed. In addition, it makes it possible to prepare a medium in a much shorter time, easily and cheaply than agar and the like. In particular, it enables a solid medium for tissue culture of plants and for culturing microorganisms that require special conditions.

[0006]

[Means for Solving the Problems] In view of such circumstances, as a result of intensive studies, as a result, the N-vinylacetamide-based crosslinked product was practically used as a gelling agent for the medium without inhibiting the growth and differentiation of cells. The present invention has been completed by finding that it exhibits an excellent effect.

That is, the present invention relates to a solid medium characterized by using a nonionic water-absorbent resin as a gelling agent, particularly for tissue culture of plants, thermophilic bacteria, eosinophils, alkalophilic bacteria or It relates to a solid medium for culturing halophilic bacteria. Further, the above water-absorbent resin has the general formula (1) CH ₂ ═CHNR ¹ COR ² (1) [In the formula, R ¹ and R ² may be the same or different and each represents a hydrogen atom or a methyl group. . ] It is related with the solid culture medium which is the (co) polymer crosslinked product of the compound represented by this.

Further, the above water-absorbent resin has a pH of 2-11.
Of the aqueous solution having a liquid absorption capacity of 10 times or more, and the water-absorbent resin is 0.1 to 10 times the solid medium.
4% (w / v) solid medium. Further, the present invention is a polymer cross-linked product or copolymer cross-linked product of N-vinylacetamide, wherein a water-absorbing resin having an absorption capacity of 10 times or more of an aqueous solution in the range of pH 2 to 11 is used as a gelling agent. The present invention relates to a tissue culture method for plants, which comprises 0.1 to 4% (w / v) with respect to a solid medium. Hereinafter, the present invention will be described in detail. By using the above water-absorbent resin as a gelling agent for a solid medium, there is no inhibition on the growth and differentiation of cells, and various environmental conditions (including harsh conditions such as heat, acid, alkali and salt) The present invention provides a solid medium and a tissue culture method that can be used in.

By using the gelling agent of the present invention, there is no change in the physical properties of the gel due to the components such as salts and nutrients in the medium and the pH, so that various media can be easily gelled. Become. This can be said to be most suitable for culturing microorganisms having an acidophilicity, a basophile, highly halophilic bacteria, marine microorganisms, algae and the like. Moreover, the present invention enables the preparation of a gradient plate containing a high concentration of salt. Furthermore, since it does not liquefy at high temperatures like agar, it is also suitable for culturing hyperthermia. According to the present invention, the hardness of the gel can be freely changed depending on the concentration of the water absorbent resin added. Since the growth of cells is significantly affected by the hardness of the medium, the effect of being able to easily adjust the hardness of the medium is great. In addition, a solution of nutrients and drugs can be added during the culture, and the size of the medium can be freely changed. Further, since it is an artificial gelling agent, deterioration of reproducibility due to trace impurities found in a natural gelling agent can be suppressed, and the gel becomes transparent so that cells can be easily observed.

On the other hand, since it has heat resistance against temperature conditions such as normally used sterilization, there is no change in the physical properties of gel due to sterilization. For example, when it is desired to add a heat-labile substance to the medium, the gel can be sterilized as a powder, and a liquid component containing the substance can be sterilized by filtration and added. Further, since the size of the resin can be freely set, for example, by reducing the particle size, it is not necessary to gel the whole like agar. That is, the gel can be agitated during the culture, and the waste substances and harmful substances produced in the culture tissue can be diffused during the culture. It is also possible to substitute gelatin used for puncture culture. Furthermore, it is also possible to prepare a high-layer culture medium and easily culture an anaerobic microorganism. With agar, a medium in which agar was once solidified could not be frozen and preserved, and agar was frozen and preserved together with the medium, and was heated and gelated each time it was used. However, when the water absorbent resin of the present invention is used, the medium can be stored frozen while being frozen. In addition, the water-absorbent resin is particularly suitable for culturing plant cells because it is resistant to decomposition by light and is stable. Furthermore, the medium can be produced at a much lower cost than agar and the like.

As the water absorbent resin used in the present invention, polyvinyl alcohol type, polyacrylic acid amide type, poly-
N-vinylcarboxylic acid amide-based nonionic crosslinked products are suitable, and among them, poly-N-vinylcarboxylic acid amide-based crosslinked products are preferable.

In particular, the above general formula (1) [in the formula, R ¹ and R
² is as described above. The polymer cross-linked product or the copolymer cross-linked product of) is preferable, and as the compound of the general formula (1), N-
Examples thereof include vinylformamide, N-vinylacetamide, N-methyl-N-vinylformamide, and N-methyl-N-vinylacetamide. Among them, N-vinylacetamide polymer cross-linked products or copolymer cross-linked products have excellent effects. Exert.

As the raw material compound, only the N-vinylamide compound represented by the general formula (1) may be polymerized in the presence of a crosslinking agent, but other ethylenically unsaturated compounds may be copolymerized. At this time, it is important that one or more N-vinylamide compounds of the general formula (1) are contained in an amount of 50 mol% or more of the entire raw material compounds. If it is less than 50 mol%, the liquid absorption capacity in the presence of salts, metal ions, etc. in the medium decreases and becomes chemically unstable (for example, decomposed by light), resulting in a liquid absorption capacity. descend. The polymer obtained by using the compound represented by the general formula (1) as a main component is
Unlike polycarboxylic acid-based water-absorbing resin, which is a conventional polymer electrolyte, it is a nonionic polymer, so the water absorption capacity does not decrease significantly due to the presence of ions contained in the medium, and it swells too much. None, that is, it is characterized in that it does not show a large change in the physical properties of the gel depending on the components in the medium.

The compound which can be copolymerized with the compound represented by the general formula (1) in the present invention includes, for example, methyl acrylate, ethyl acrylate, propyl acrylate, 2-hydroxyethyl acrylate, 2-hydroxypropyl acrylate and N. , N-dimethylaminoethyl acrylate, acrylamide, N, N-dimethylaminopropyl acrylamide, acrylonitrile, methyl vinyl ketone, ethyl vinyl ketone, propyl vinyl ketone, vinyl acetate, methyl vinyl ether,
Ethyl vinyl ether, propyl vinyl ether, methyl methacrylate, ethyl methacrylate, propyl methacrylate, 2-hydroxyethyl methacrylate,
2-hydroxypropyl methacrylate, N, N-dimethylaminoethyl methacrylate, methacrylamide,
N, N-dimethylaminopropyl methacrylamide, N
Compounds such as vinyl-2pyrrolidone.

The cross-linking agent used in the present invention has 2
It is a compound having at least one unsaturated bond and exhibiting good copolymerizability with N-vinylcarboxylic acid amide, and examples thereof include the following. Examples of the compound having two or more allyl groups in one molecule include acetals such as tetraallyloxyethane, pentaerythritol tetraallyl ether, pentaerythritol triallyl ether, pentaerythritol diallyl ether, trimethylolpropane triallyl ether and triaryl ether. Methylolpropane diallyl ether, ethylene glycol diallyl ether, diethylene glycol diallyl ether, triethylene glycol diallyl ether, diallyl ether, monosaccharides, disaccharides, polysaccharides, cellulose and other compounds derived from compounds having two or more hydroxyl groups in one molecule Ethers such as polyallyl ether, tetraallyl pyromellitic acid, triallyl trimellitate, triallyl citrate, diallyl oxalate, diallyl succinate, Diallyl dipin, diallyl maleate, diallyl fumarate, diallyl terephthalate, diallyl isophthalate, diallyl phthalate, esters such as polyallyl esters derived from compounds having two or more carboxyl groups in the molecule, and diallylamine , Triallyl isocyanurate, triallyl cyanurate and the like. Further, examples of the compound having two or more vinyl ester structures in one molecule include:
Divinyl oxalate, divinyl malonate, divinyl succinate, divinyl glutarate, divinyl adipate, divinyl pimelic acid, divinyl maleate, divinyl fumarate, trivinyl citrate, trivinyl trimellitate,
A compound having two or more vinyl ester structures such as tetravinyl pyromellitic acid.

Further, N, N'-methylenebisacrylamide, ethylene glycol di (meth) acrylate, diethylene glycol di (meth) acrylate, triethylene glycol di (meth) acrylate, polyethylene glycol di (meth) acrylate, trimethylolpropane tri ( Compounds having multiple acrylamide structures or (meth) acrylic groups, such as (meth) acrylate and pentaerythritol tri (meth) acrylate;
Examples thereof include compounds having two or more unsaturated groups in one molecule, such as divinylbenzene, divinyl ether, and allyl (meth) acrylate. These cross-linking agents may be used alone or in combination of two or more.

The amount of the crosslinking agent used is based on the starting compound.
The cross-linking agent / monomer ratio is a molar ratio of 10/90 to 0.00
Selected from the range 01 / 99.9999, but 1/9
The range of 9 to 0.0005 / 99.99995 is particularly preferable. The amount of the cross-linking agent is 10/9 in terms of the cross-linking agent / monomer molar ratio.
When it is more than 0, the cross-linking density of the resulting water-absorbent resin becomes too high, so that the swelling rate becomes extremely small and the effect as the water-absorbent resin cannot be substantially exhibited. Further, the cross-linking agent / monomer ratio is 0.0001 / 99.9999 in molar ratio.
If the amount of the cross-linking agent is less than that, the production of water-soluble or hydrophilic polymer which is not involved in the cross-linking increases, and similarly, the effect as a substantial water-absorbent resin cannot be exhibited.

The polymerization process is not particularly limited, but it is usually preferable to use an aqueous solution polymerization method, a reverse phase suspension polymerization method, a reverse phase emulsion polymerization method or the like. For example, as an aqueous solution polymerization method, a monomer component and a cross-linking agent are uniformly dissolved in water or a mixed solvent of a hydrophilic organic solvent such as methanol, tetrahydrofuran, or acetone that can be uniformly mixed with water, and vacuum degassing or After removing the dissolved oxygen in the system by replacement with an inert gas such as nitrogen, a polymerization initiator is added to carry out copolymerization. The polymerization initiation temperature is usually -10 to 60
C., and the polymerization time is about 0.5 to 20 hours. As the polymerization initiator, a peroxide, an organic or inorganic peracid or a salt thereof, which can be uniformly dissolved in a polymerization solvent, or an azobis compound alone or in combination with a reducing agent, a redox compound is used.

The reaction product is a gel containing the solvent used in the reaction and is usually cut with a rotary cutter or the like.
The solvent is removed by a method such as heating or decompression, followed by drying, and then, if necessary, pulverizing and classifying to obtain a powder having a particle size of several μm to 3 mm. As the reverse phase suspension polymerization method and the reverse phase emulsion polymerization method, a monomer component and a crosslinking agent are uniformly dissolved in water and suspended with a dispersant and a surfactant in an organic solvent that is not uniformly mixed with water. Emulsify and polymerize. As the polymerization initiator, not only water-soluble ones but also those soluble in an organic solvent are used. Examples of the organic solvent include hexane, cyclohexane, heptane, octane,
Hydrocarbons such as benzene, toluene, xylene and ethylbenzene, halogenated hydrocarbons such as carbon tetrachloride and dichloroethane, and mineral oil such as Isopar are used.

In the reverse phase suspension polymerization method and the reverse phase emulsion polymerization method, a surfactant is used as a dispersant, and a protective colloid is used in combination as necessary. Typical examples thereof include sorbitan monostearate, sorbitan monopalmitate, polyvinyl alcohol, methyl cellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose and the like.

The method of removing dissolved oxygen in the system and the amount of the polymerization initiator used are the same as described above. Further, in the case of the reverse phase suspension polymerization method, the polymer is usually obtained in the form of particles of 100 μm to several mm. Therefore, the polymer may be filtered, dried and, if necessary, pulverized after the polymerization. It is also possible to remove water from the polymer by utilizing azeotropic distillation of hydrocarbon and water after the polymerization. In the case of reverse phase emulsion polymerization, the particle size of the polymer is usually about several μm, and the polymer is obtained as an emulsion with a hydrocarbon. This emulsion can be used as it is, or can be used after precipitating a polymer with an inorganic salt or acetone, filtering and drying. The polymerization reaction conditions are not particularly limited, but are generally as follows. The amount of the solvent used is the same as the aqueous monomer solution to 20 times, preferably the same amount to 10 times, particularly preferably the same amount to 5 times, the polymerization initiation temperature is about −10 ° C. to 90 ° C., and the reaction time is 0.5 to 20. It's about time.

As described above, the water-absorbent resin of the present invention is primarily obtained in the form of powder having a particle size of several μm to several mm, but in the state of absorbing a liquid, it is in the form of beads or dispersion, cream, paste-like viscosity. It may be a viscous material or the like, and may be molded into a string shape, a film shape, a sheet shape, a plate shape, or various kinds of moldings, and may be shaped according to the shape of the culture container. The water-absorbent resin of the present invention can absorb deionized water 10 to 2000 times its own weight. It is preferable to absorb liquid 10 times or more in the range of pH 2 to 10. Therefore, a medium of various callus, microorganisms or the like having a pH of 2 to 11 is preferable,
Can be used in the medium. As a method for measuring the liquid absorption ratio, 0.20 g of the crosslinked polymer was put into 2 L of deionized water to be sufficiently swollen, and then the swollen crosslinked polymer was filtered in a box made of a 200-mesh wire mesh. , Can be calculated by the following formula. Liquid absorption ratio = (weight of swollen polymer cross-linked product / 0.20)
-1.0

The amount of the water absorbent resin used in the present invention is 0.1% to 4% in w / v% in the culture solution.
A particularly preferable range is 0.4% to 1.5%. 0.1
If it is less than 4%, the gel will be too soft and the shape retention will be poor, and if it is more than 4%, the gel will be too hard, and in any case, the growth and differentiation of cells will be affected. To obtain a solid medium using the gelling agent of the present invention, for example, the culture solution and the gelling agent are separately sterilized under high pressure steam, and then mixed under aseptic conditions, the culture solution and the gelling agent are mixed in advance, A method of sterilizing both can be considered.

In the present invention, if necessary, a conventional gelling agent such as agar, agarose, agaropectin, gellan gum, κ-carrageenan can be added. At the time of gelation, other water-absorbent resin may be added if necessary. Examples of the water absorbent resin include starch-polyacrylonitrile graft copolymer, crosslinked polyalkylene oxide,
Examples thereof include saponified vinyl ester-ethylenically unsaturated carboxylic acid copolymers, self-crosslinked polyacrylic acid salts, reaction products of polyvinyl alcohol-based copolymers with cyclic acid anhydrides, and crosslinked polyacrylic acid salts. The amount of these water-absorbent resins added is 0.1% or less relative to the medium, and if the amount is more than this, the differentiation and growth of cells will be affected. Examples of cultivated by tissue culture of plants include, but are not limited to, moss plants, mushrooms, trees, algae, pollen, vegetables, flowers, grains, potatoes and the like.

Typical synthetic mediums used for plant tissue culture include White medium, Murashige & Skoog medium, and Rinsmeier medium.
Linsmeier &Skoog's medium, Nadson's medium, Gautre's medium, Ericsson's medium, Tsukamoto Kano,
Examples include Nitsch's medium, Gamborg's medium, Heller's medium, and others whose composition is changed based on these mediums.

Specific examples of the inorganic components of the inorganic synthetic medium include copper, nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, molybdenum, chlorine, sodium, iodine and cobalt. Specifically, potassium nitrate, sodium nitrate, calcium nitrate, ammonium nitrate, sodium dihydrogen phosphate, sodium ethylenediaminetetraacetate, potassium chloride, calcium chloride, magnesium sulfate, sodium sulfate, ammonium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate. ,
Zinc sulfate, boron, copper sulfate, sodium molybdate,
Examples thereof include molybdenum trioxide, potassium iodide, cobalt chloride and the like.

Specific examples of the saccharide include carbohydrates such as sucrose, inositol and glucose, and their derivatives. Specific examples of plant growth preparation substances include indole acetic acid (IAA), α-naphthalene acetic acid (NAA),
Examples include auxins such as 2,4-dichlorophenoxyacetic acid (2,4-D) and indolebutyric acid (IBA), cytokinins such as kinetin, zeatin, dihydrozeatin, benzyladenine (BA), and isobenenyladenine. . Specific examples of vitamins include biotin, thiamine, pyridoxine, pantothenic acid, ascorbic acid (vitamin C), nicotinic acid, paraaminobenzoic acid, folic acid and the like.

Specific examples of amino acids include glycine, glutamic acid, aspartic acid and its amide, and arginine. Specific examples of the natural substances include casein hydrolyzate, coconut milk, yeast extract, malt extract, tomato and potato extract (shibori juice), and the like, other than the above-mentioned specific examples, substances necessary for culture as appropriate. May be added. On the other hand, useful components that can be produced by plant cell culture using the present solid medium include, for example, lignan, lopan alkaloid, saponin, peroxidase, pigment, nicotine, fragrance, cardiac glycoside, indole alkaloid, illin, vinblastine, and cloxin. But not limited to this.

As a method for culturing a tissue of a plant using the present solid medium, for example, an appropriate amount of the present water-absorbent resin is placed in a culture vessel, which is impregnated with a required amount of liquid medium and then autoclaved. It can be performed by a method such as sterilization by a method such as that described above, and naturally cooling, and then placing a previously disinfected plant tissue section on the main medium material in the culture container. Also,
The control method during this culture is the same as when using agar, but when using this solid medium, in the operation under aseptic conditions, the addition of liquid medium without transplanting the cultured tissue, Addition of phytohormonal agents, washing and replacement of liquid medium can be performed.

Further, the present solid medium can be used as a conditioned medium for acclimatizing young plants, if necessary in combination with a commercially available liquid fertilizer. When used in combination with a liquid fertilizer for this purpose, for example, the liquid fertilizer adjusted to a required concentration is supplied to the present solid medium by a method such as drip or spray or a method that also serves as irrigation. And in order to acclimate the seedlings using the conditioned medium thus prepared, for example, remove the medium or rooting medium used for the tissue culture from the seedlings obtained by tissue culture,
After washing with water, the roots of young plants are planted by wrapping them in an appropriate amount of a conditioned medium stored in a cultivation container, moderately protected from light and moisture, and acclimated in a greenhouse for about 1 week to 1 month. The management method at the time of acclimatization is the same as the conventional case using perlite, vermiculite, etc., but since the solid medium can be prepared to have an appropriate water retention amount, it is usually used for hippocampus or the like. It is not necessary and the seedlings that have been acclimated can be used for ordinary soil cultivation or solution cultivation without removing the medium. When acclimation is performed using the present solid medium in this manner, it is possible to always provide the roots of young plants with appropriate water retention and air permeability, and since the porosity is high, the medium remains in the medium even after irrigation. In addition, a large amount of gas phase remains to prevent root oxygen deficit. In tissue culture, it is often necessary to change the medium components depending on the growth stage of callus, and by using the solid medium of the present invention, the necessary components are appropriately added without impairing the performance of the solid medium. It can be made into a desired medium component.

As described above, the present solid medium is not only used as a solid medium for growth, differentiation and rooting of tissue culture, but also as a conditioned medium used for acclimation of seedlings cultivated in this tissue culture. Since it can be used, from the growth, differentiation and rooting of plants to the acclimation of seedlings obtained by this, the liquid medium used in each process is washed off with water and the next liquid medium is added without transplanting operation. Can be done consistently. Then, by consistently performing plant growth, differentiation, rooting to acclimation in this way, it is possible to significantly reduce damage to seedlings, and it is possible to improve the yield at the time of seedling production by the tissue culture method, Moreover, the growth rate of the plant after acclimation can be improved.

Further, the acclimated plant can be used for ordinary soil cultivation, hydroponics, sand cultivation, gravel cultivation, rock wool cultivation and the like without removing the solid medium. In addition, using the solid medium of the present invention, plant appreciation, plant nutrition,
It is also possible to carry out research on pathology and physiology, raising seedlings, breeding, and breeding. This eliminates the need for maintenance such as watering and fertilizing. Furthermore, when the solid medium of the present invention is used for appreciation, it is transparent and looks good, but further coloring makes it possible to achieve color variety.

The solid medium of the present invention is, for example,
It is also used for culturing various microorganisms such as fungi such as yeast, mold, mushrooms and actinomycetes, or bacteria such as aerobic bacteria and anaerobic bacteria. As the medium used, for example,
There are various basal media, or media in which other additives are added to the basal media. For example, sorbitan-casein-digest (SCD) medium, soybin-casein-digest medium, etc. medium for general bacterial count measurement, lactose broth medium, MacConkey medium, desoxycholate medium, EMB medium, etc. E. coli test medium, brilliant green medium , Xylose / lysine / desoxycholate medium,
Bismuth-sulfite medium, triple-sugar-iron medium, sulfite-indole-motility medium, lysine-iron medium and other salmonella test medium, NAC medium, cetrimid medium, Pseudomonas aeruginosa medium for fluorescein detection, Pseudomonas for detection of pyocyanin Pseudomonas aeruginosa test medium such as bacterial culture medium, Vogel-Johnson medium, Bird Parker medium, yellow vinegar test medium such as egg yolk-added Mannit salt medium, cellulose medium, corn meal medium, Czapek medium, malt extract medium, malt extract -Yeast extract medium, synthetic mucor medium, M40Y medium, oatmeal medium, oatmeal wheat malt medium, potato carrot medium, potato glucose medium, potato sucrose medium, Sabouraud glucose medium, V-8 juice medium, Weitzman-Silva -Hunter culture , Fungal medium such YpSs medium, Davis medium Spizizen medium, various minimal medium such as Burkholder medium, various selection media, there are various bacteria culture media and the like. Compounds that can be added to the medium include water, alcohols, glucose and sucrose, maltose, sorbitol, fructose, mannose, glycerin, other polyhydric alcohols, ammonium sulfate, ammonium chloride, yeast extract, broth (meat), peptone, amino acids. Carbon and nitrogen sources such as sodium phosphate, potassium phosphate, sodium chloride, calcium chloride, magnesium chloride, magnesium sulfate, sodium citrate,
Inorganic salts such as Co, Mo, Cu, Fe, Mn, Zn (ammonium salts, sulfates, etc.), antibiotics such as penicillin, chloramphenicol, streptomycin, kanamycin, coomassie brilliant blue, bromthymol blue, rose Pigments such as Bengal, biotin, vitamins such as thiamine, potato, carrot,
Examples include, but are not limited to, extracts such as onions, nucleic acid components, and pH adjusters.

The solid medium of the present invention is for culturing microorganisms,
In particular, it is useful against extreme environmental microorganisms such as thermophiles, acidophiles, alkalophilic bacteria, and halophilic bacteria. Sulfol as an example
Obusacidocaldarius has a maximum temperature of 85 ° C. and a pH of 2 to 3 is optimal, and it is difficult to produce an agar plate under these conditions. Examples of such thermophilic thermophiles include archaea, Thermoplasma acidophilum (optimum culture conditions 55 to 59 ° C pH 1-2), and Cyanidium caldar.
ium, Bacillus acidocaldarius (optimum culture conditions 65-
70 ° C., pH 2-4) and the like. Also used for the research of viruses such as phages, φNS40, NS
It can be used for phages of thermophilic thermophiles such as 11. Examples of halophilic bacteria include Halobacterium sarinarium and Halobacterium isolated from salted fish, salted leather, salt lake, and sun salt.
cutirubrum, Halobacterium halobium, Halobacterium saccharovorum isolated from salt fields, Halobacterium vallismortis isolated from saltwater pools, Halobacterium volcanii isolated from Dead Sea sediments, Halobacterium mediterranei isolated from Spanish salt fields, and single species from the Dead Sea Separated Halobacterium sodomense Halobacterium pharaonis isolated from an alkaline salt lake in Egypt Halococus morrhuae isolated from salted fish and salted meat, Natronobacterium pha isolated from an alkaline soda lake in Kenya
raonis, Natronobacterim gregoryi, Natronococcus oc
cultus, Haloarcula sinaiie isolated from salt fields in the Red Sea
Haloarcula isolated from salt fields in Nsis, California
californiae, Ectothiorhodospira halochloris isolated from Egyptian alkaline soda lake, Ectothiorhodospira halophila isolated from salt lake mud, Ectothiorhodospira abdelmalekii isolated from alkaline soda lake
, Vibrio costicola isolated from salted bacon, Paracoccus halodenitrificans isolated from salted meat, Spirochaeta halophila isolated from salt lake mud, Pseudomonas sp. 101 isolated from crude salt, single salted anchovy Separated Bacteroides halosmophilus, Pediococ
cus halophilus, Microcus isolated from soy sauce brewing salt
halobius, Micrococcus var isolated from soy sauce moromi
ians subsp. halophilus, Diploco isolated from salted fish
Plano isolated from ccus gardidarum, a conserved strain of halobacterium
coccus halophilus, Bacil isolated from soy sauce brewing salt
lus sp., Nitrosococcus mob isolated from saltwater of the North Sea
Haloa, ilis, isolated from Great Salt Lake sediments
naerobium praevalens, H isolated from Dead Sea sediments
Examples include alobacteroides halobius, Clostridium lortetti, soy sauce yeast Staphylococcus aureus, and salt-tolerant yeast Zygosaccharomyces rouxii.

Since the water absorbent resin of the present invention is nonionic, these low halophilic bacteria (growing optimum salt concentration 0.2 to 0.5
M), moderate halobacterium (optimum salt concentration 0.5-2.5M), border zone highly halobacterium (optimal salt concentration 1.5-4M), highly halophilic bacterium (optimum salt concentration 2.5-) It is a suitable material for the following medium used when culturing 5.2 M) or salt-tolerant bacteria. Sehgal and Gibbons complex medium, Dundas, Sri
nivasan & Halvorson synthetic medium, Webber medium, Katzen
elson and Lochhead medium, Brown and Gibbons medium, Ab
ram and Gibbons medium, enrichment medium for separating carbohydrate-utilizing highly halophilic bacteria, enriching medium for separating alkalophilic highly halophilic bacteria,
Examples include a box-type accumulation medium for separating highly halophilic bacteria, a photosynthetic autotrophic accumulation medium for separating highly halophilic bacteria, and Halo TYEG medium. Similarly, at the pH of the medium, Thiobacillus and the like have a pH of 2.
Since the optimum pH for growth is 0 to 3.0, this water absorbent resin exerts its power.

The solid medium of the present invention is also used for culturing various animal cells. Examples of the medium to be used include various basal mediums, and mediums in which other additives are added to the basal medium. For example, BME, MEM, DMEM, F12, F10, I
MDM, L-15, McCoy-15A, 199, α-MEM, JMEM, SMEM, RPMI1640,
There is a basal medium such as William-E. Examples of substances that can be added include amino acids, vitamins, essential nutrients, inorganic salts, cell growth factors, cell adhesion factors, serum and FBS. The animal cells to be cultured include, for example, epithelial cells, muscle cells, osteoblasts, fibroblasts, endothelial cells, normal cells such as nerve cells, cancer cells, hybridomas and the like. Specifically, for example, armyworm, larvae-derived Sf9 cells, bovine-derived KU-1 cells, flesh fly-derived CAF cells, hamster-derived CHL cells, human-derived HAL-01 cells, BALL-1 cells, HeLa
Cells, NCU-F5 cells, medaka-derived OLF-136 cells, cynomolgus monkey-derived JTC-12 cells, African green monkey-derived Verots S3 cells, mouse-derived FM3A cells, NIH3T3-3 cells, pig-derived PK15 cells, rabbits Examples include, but are not limited to, RC2 cells derived from MT, MT-9 cells derived from rat, and HNS2 cells derived from salamander.

[0037]

The present invention will be described in more detail with reference to the following examples, but it goes without saying that the technical scope of the present invention is not limited to these examples.

Example 1 N-vinylacetamide (200 g) and tetraallyloxyethane (0.48 g) were dissolved in 750 g of water, and the mixture was placed in a 1 L three-necked separable flask. Set the nitrogen inlet tube, thermometer holder and exhaust tube in the three-necked flask,
It was immersed in a constant temperature bath at 30 ° C. Nitrogen at a rate of 1 L / min 3
After bubbling for 0 minute to remove dissolved oxygen, a solution obtained by dissolving 0.40 g of 2,2′-azobis (2-amidinopropane) dihydrochloride in 49.6 g of water was added to the reaction solution. Place the flask in a heat-insulated container and set the nitrogen flow rate to 0.1.
It was reduced to L / min and allowed to stand. After 16 hours, the gel content was taken out, subdivided with a mincer (household meat grinder), dehydrated with acetone, and dried at 105 ° C for 5 hours. The dried gel thus obtained is crushed and classified to 48 to 10
The product was adjusted to 0 mesh and used as a final product (hereinafter, referred to as Example 1. The same applies hereinafter).

Example-2 100 g of N-vinylformamide and 100 g of acrylamide, N, N'-diacetyl-N, N'-divinyl-1,4-bisaminomethylcyclohexane 44.80
The reaction was carried out in exactly the same manner as in Example 1 using g, and the polymer was post-treated.

Example 3 139.5 g of N-vinylacetamide and 60.5 g of methyl acrylate were dissolved in 750 g of water, and 0.023 g of divinylbenzene was dissolved in 1 L of water. It was put in a separable flask. A nitrogen inlet tube, a thermometer holder and an exhaust tube were set in the three-necked flask and immersed in a constant temperature bath at 30 ° C. Nitrogen was bubbled at a rate of 1 L / min for 30 minutes to remove dissolved oxygen, and then 2,2′-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride was added to 0.4
What melt | dissolved 0 g in 49.6 g of water was added to the reaction liquid. Put the flask in a heat-insulated container and set the nitrogen flow rate to 0.1 L /
It decreased to a minute and was left to stand. Then, the polymer was post-treated in the same manner as in Example 1.

Comparative Example-1 81.5 g of N-vinylacetamide and 118.5 g of acrylonitrile, N, N'-diacetyl-N, N'-
A reaction of 0.1 g of divinyl-1,4-bisaminomethylcyclohexane was carried out in the same manner as in Example 1 to carry out a post-treatment of the polymer.

Comparative Example-2 71 g of N-vinylformamide and acrylamide 1
29 g, N, N'-diacetyl-N, N'-divinyl-
1,4-bisaminomethylcyclohexane 123.2 g
Was subjected to the same reaction as in Example 1 to carry out a post-treatment of the polymer.

Comparative Examples-3 to 6 The same procedure as in Example 1 was carried out for three commercially available water-absorbent resins. The water absorbent resin is shown below. Comparative Example-3 Sulfone Hydrochloric Acid Acrylic Water-Absorbent Resin (Aqualic CS: Nippon Shokubai Chemical Co., Ltd.) Comparative Example-4 Starch-acrylic acid graft copolymer neutralized product (Sunwet IM-1500: Sanyo Chemical Industries) Comparative Example-5 Na salt of saponified vinyl acetate-methyl acrylate copolymer (Sumikagel S-50: Sumitomo Chemical Co., Ltd.) Comparative Example-6 Acrylate salt cross-linking water-absorbent resin (Diawet AL-III: Mitsubishi Petrochemical) )

Experimental Example 1 Absorption capacity of each polymer 0.5 g of the polymer was dispersed in each solution, allowed to stand for 2 hours, filtered through a 200-mesh wire net, and the weight of the swollen gel was weighed. However, the liquid absorption ratio is given by the following formula. Liquid absorption ratio = (weight of swollen gel-weight of dry gel) /
Weight of dry gel 1) Medium solution Artificial seawater (Jamarine Laboratory), MS medium,
Selective medium for halophilic bacteria (NaCl 300 g, peptone 1)
0g distilled water 1000ml)

[0045]

[Table 1]

2) pH buffer (Britton-Robinson universal buffer 0.12 mol) pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11

[Table 2] Agar did not solidify at a pH of 3 or less, and was colored brown at a pH of 8 or more.

Experimental Example 2 Plant tissue culture 1) Plant tissue Tobacco cultured cells Nicotiana tabacum cell line
XD-6 Rice cultured cells Derived from Yamahikari seedling roots 2) Container A 100 ml Erlenmeyer flask with two aluminum foil caps was used.

3) Culture solution Murashige-Skoog solution (MS solution) 4) Agar As a control, 0.8% (g / 10) agar for plant medium was always used.
0 ml) was used. 5) Culture of callus The medium was fully swollen with a culture solution, and then autoclaved (120 ° C., 2 atm for 15 minutes) for sterilization, and allowed to cool. Then, callus was transplanted and kept at a darkness of 26 to 28 degrees. It was grown in place and observed.

Experimental Example 2-1 Effect of Polymer Type 1) Test Samples Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2,
Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6, agar (0.
8%) 2) Tested concentration 3 g / 100 ml 3) Test method Tobacco callus and rice callus, medium 20 ml, culture period of about 3 weeks 4) Results Callus growth, microscopic observation, fresh weight, dry weight, comparison with control agar Therefore, although Examples 1 to 3 showed almost the same growth as the agar medium, Comparative Examples 1 to 6
Then, the callus hardly grew.

Experimental Example 2-2 Effect of Polymer Concentration 1) Tested Concentration (g / 100 ml) 0.05, 0.1, 0.8, 3, 4, 10 2) Tested Resin Example 1, Example 2, Example 3 and agar (0.8%) 3) Test method Tobacco callus, medium 20 ml, culture period about 3 weeks 4) Results (table) Growth of callus / microscopic observation, fresh weight, dry weight, control agar From the comparison with the above, when the amount of resin added is low, the callus will fall apart and will not grow much and the color will become light.On the contrary, if there is more resin, it will grow at the same level as agar or more at first, but gradually grows. It became bad and stiff. Also, many microscopic observations showed deformation and abnormal cells, and both fresh weight and dry weight were low.

[0051]

[Table 3]

[0052]

[Table 4]

Experimental Example 2-3 Culture solution addition test 1) Tested polymer concentration 3 g / 100 ml 2) Tested resin Example 1, agar (0.8%) 3) Test method Tobacco callus, medium 40 ml, culture period 5 weeks Without control addition Method of addition: 2 every 7 days regardless of the amount of medium decreased
A total of 8 ml of water or MS medium was added by a syringe so as not to directly contact the callus. (The addition amount is about 1g
The callus was cultured for 5 weeks to grow to about 6-15 g, and the amount of medium absorbed was taken into consideration. )

4) Results

[Table 5] Note: The larger the number of +, the better. *: The additive solution was only slightly absorbed, and separated and stayed on the medium, and the callus became partly immersed and growth was often poor. . **: The added solution was absorbed promptly, the growth became very good, and both the fresh weight and the dry weight increased. After the 4th week, bleaching was observed in order of good growth, but in the control, it was bleached before that.

[0055]

[Table 6]

[0056]

[Table 7] It was found that in the polymer medium, the addition of water or MS solution resulted in better callus growth and continued culture. It also grew faster than the agar medium.

Experimental Example 3 Microorganism culture test 1) Microorganism Strong acid-resistant filamentous fungus HAPM (Penicillium genus) Optimum pH: 2, Culture temperature: 25 ° C, Mycelium: White, spores:
Deep grey-green 2) Container Petri dish (50 x 11 cm) 3) Culture solution Xylan or glucose (C source) 1%, polypeptone 0.1%, yeast extract 0.02%, KH ₂ PO ₄
0.002%, final pH 2.0 4) Agar As a control, agar for bacterial culture was used at a concentration of 2.0% (w / v).

5) Culturing method Concentration of water-absorbent resin 3.0 g / 100 ml Preparation of medium Agar and xylan are decomposed by autoclave at low pH, and thus prepared as follows. (Final pH of 2.0 when 100 ml was prepared) A: 1 g of xylan or glucose was suspended and dissolved in 50 ml of water. B: Polypeptone 0.1 g, yeast extract 0.02 g,
Dissolve 0.002 g of KH ₂ PO ₄ in 50 ml of water,
The pH was adjusted to 1.7 with N--H2 SO4.

A. Agar medium (2.0 g / 100 ml) 1. A to which 2.0 g of agar was added and B were autoclaved separately. 2. After allowing to cool, the temperature was lowered to several tens of degrees, B was added to A, and they were quickly mixed. 3. In a dish that has been ice-cooled in advance, 2. Was quickly dispensed and allowed to set. b. Medium using water absorbent resin (when polymer is not autoclaved at low pH) 1. 2.5 g of polymer added to A and swelled, and B
Were autoclaved separately. 2. B was added to A, mixed well and dispensed. c. Medium using water absorbent resin (when autoclaving is performed at low pH of polymer) 1. 2.5 g of a polymer was added to A + B to swell, and autoclaved to dispense.

Experimental Example 3-1 Cultivation of filamentous fungus 1) Test samples Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2,
Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6, Agar 2) Test Method A solid medium was prepared as described above and inoculated with filamentous fungal spores in order to compare the growth of hyphae, The cells were cultured and observed. 1. The spores and hyphae of the filamentous fungus were suspended in sterile water, the spores were removed from the hyphae, and the suspension was filtered to obtain a spore-only suspension. (Separation of spores) 2. Using a hematometer, the spores were diluted to a few cells. 3. To the prepared flat medium, 2. 0.05 diluted with
Each ml was added and spread with a conradi stick.

[0061]

[Table 8] Note) The larger the number of +, the faster the growth rate and the wider the growth range. In addition, in the comparative example, the solid medium was not obtained (sol rather than gel), and the test could not be performed. Comparative Examples 1 and 2
It was difficult to confirm the clear zone on the xylan medium because the hyphae had abnormal thickness and morphology and showed unevenness, meandering, and crimping.

[0062]

[Table 9]

[0063]

EFFECTS OF THE INVENTION Since the solid medium of the present invention can be added with a solution of nutrients, drugs, etc. during the culture, it can be used as a solid medium having a different purpose and condition from those of agar and the like. Further, it is easy to prepare a solid medium containing low pH, high pH and high concentration of salts and nutrients.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical display location C12R 1:80)

Claims (7)

[Claims] 1. A solid medium comprising a nonionic water-absorbing resin as a gelling agent. 2. A solid medium for tissue culture of plants, which comprises a nonionic water-absorbing resin as a gelling agent. 3. A solid medium for thermophilic bacterium, acidophilic bacterium, alkalophilic bacterium or halophilic bacterium, which comprises a nonionic water-absorbent resin as a gelling agent. 4. The water-absorbent resin is represented by the general formula (1) CH ₂ ═CHNR ¹ COR ² (1) [wherein R ¹ and R ² may be the same or different and each represents a hydrogen atom or a methyl group. Show. ] The solid culture medium according to claim 1, which is a polymer cross-linked product or a copolymer cross-linked product of the compound represented by the following. 5. The solid medium according to claim 1, 2, 3, or 4, wherein the water-absorbent resin has an absorption capacity of 10 times or more for an aqueous solution having a pH range of 2-11. 6. The water-absorbent resin is 0.1 to 0.1% relative to the solid medium.
4. Containing 4% (w / v), 1, 2, 3, 4 or 5.
The solid medium described. 7. A N-vinylacetamide polymer cross-linked product or copolymer cross-linked product, wherein a water-absorbing resin having an absorption capacity of 10 times or more of an aqueous solution in the range of pH 2 to 11 is used as a gelling agent and is solid. A method for tissue culture of a plant, which comprises 0.1 to 4% (w / v) of the medium.