JP3092878B2 - New synthetic medium - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel synthetic medium which is easy to handle and is excellent in quality stability and water content stability, which is used for culturing bacteria, animal and plant tissues.

[0002]

2. Description of the Related Art In recent years, with the development of biotechnology, various bacterial cells, plant tissues,
It has been widely practiced to culture animal tissues in large quantities and extract and obtain useful substances. In addition, when useful species are genetically heterologous in plant cultivation, it becomes possible to culture a large number of heterologous species by tissue culture,
The development of culture technology is remarkable.

Hitherto, a solid medium containing agar as a base material and containing various culture solutions has been widely used for culturing these bacteria, animal and plant tissues. However, the hardness of the medium varies depending on the purity or concentration of the agar used, causing differences in the properties and growth of the cultured tissue, and because agar is obtained from natural products, there is concern about supply, There were variations in the trace components contained due to many factors such as the harvest time and the harvest time, which hindered the growth and differentiation of the cultured tissue. Furthermore, when purifying agar,
There are various drawbacks, such as difficulty in purification due to complicated removal of impurities and difficulty in determining the effects of residues in agar on the culture target. In addition, since moisture is contained when used as a culture medium, deterioration of the base material and evaporation of water may occur during long-term storage, resulting in inconvenience such as cracking of the culture medium due to drying.

[0004] As a substrate to be replaced with agar having such a defect, Japanese Patent Publication No. 54-30710 and US Pat.
340706, JP-A-51-112787, JP-A-54-14689, JP-A-52-27455, JP-A-53-82666 and the like disclose a crosslinked acrylic acid-based synthetic resin and other water absorbing agents. There has been proposed a culture medium in which various nutrients are contained using a basic resin as a base material. However, since the water absorption mechanism of the polymers mentioned above is based on the osmotic pressure of ions caused by sodium salts derived from carboxyl groups contained in the polymer molecules, cultures containing various organic substances and nutrients are used. When a culture solution is used, there are various restrictions in selecting a culture solution, such as a decrease in the amount of polymer absorbed by an influence of ions contained in a nutrient solution. In addition, since carboxylate and the like are contained in the molecule to ensure osmotic pressure, growth inhibition of cultured tissue, other salt damage may be caused, and there are restrictions on use conditions, and a sufficiently satisfactory medium is used. It wasn't. Japanese Patent Publication No. 33554/1986 discloses a similar medium.
It must be combined with another support such as crepe paper with increased wet paper strength.

[0005] An object of the present invention is to solve the above-mentioned problems, except for the drawbacks of the conventional synthetic medium, and to stabilize the water content without affecting the ions in the culture solution with stable quality. An object of the present invention is to provide a synthetic medium having excellent properties.

[0006]

Means for Solving the Problems The present inventors have made intensive studies to achieve the above object, and as a result, have reached the present invention.
That is, the gist of the present invention is as follows: (1) A crosslinked polyalkylene oxide having a water absorption of 10 to 50 times its own weight obtained by urethane crosslinking of a polyalkylene oxide having a weight average molecular weight of 1,000 to 6,000,000 is used as a base material. and then, the cross-linked polyalkylene oxide in synthetic medium, characterized in that it consists of water and / or obtained by absorbing the culture liquid gel, (2) the crosslinked polyalkylene oxide is one which is processed into a sheet ( (3) The synthetic medium according to (1), wherein the crosslinked polyalkylene oxide is processed into a nonwoven fabric, and (4) the polyalkylene oxide is ethylene oxide homopolymer or ethylene oxide-propylene oxide. At least one selected from the group consisting of a polymer and a propylene oxide homopolymer (1) to (3)
The invention relates to the described synthetic medium.

[0007] The synthetic medium of the present invention is based on a crosslinked polyalkylene oxide which itself has processability. Examples of the polyalkylene oxide as a raw material before crosslinking include ethylene oxide homopolymer and ethylene oxide-propylene. At least one selected from the group consisting of oxide copolymers and propylene oxide homopolymers can be used. The weight average molecular weight of such a polyalkylene oxide is usually 1,000
It is 0 to 6,000,000, preferably 10,000 to 4,000,000. If the weight average molecular weight is less than 1,000, water absorption is poor even if a crosslinking reaction is carried out, and if it is more than 6,000,000, gelation becomes difficult, which is not preferable.

The method for crosslinking the polyalkylene oxide is not particularly limited, and includes known methods. Examples thereof include those described in JP-B-46-12155 and JP-B-50-36280. A method by chemical cross-linking of polyethylene oxide and diisocyanates, a method of radiation cross-linking of polyethylene oxide, and the like can be appropriately applied. Among them, tetramethylene diisocyanate, hexamethylene diisocyanate, phenylene diisocyanate, tolylene diisocyanate, a diisocyanate such as diphenylmethane diisocyanate is reacted with the polyalkylene oxide, and a cross-linked polyalkylene oxide obtained by urethane cross-linking is used in the synthetic medium of the present invention. It is preferably used. The crosslinked polyalkylene oxide thus obtained has a pure water absorption of usually 10 to 50 times, preferably 30 to 50 times its own weight.
It is suitable that the gel having a moderate hardness can be obtained without dissolving the polyalkylene oxide by water absorption up to 50 times. If the amount of pure water absorption is less than 10 times, it is not enough to hold the amount of liquid required for culture,
On the other hand, if it exceeds 50 times, the strength of the gelled solid after water absorption will be weak.

The crosslinked polyalkylene oxide used in the present invention differs from the conventionally used polyacrylic acid-based water-absorbing resin in that the mechanism of water absorption does not depend on the osmotic pressure of ions. Therefore, the amount of water absorbed by the polymer is hardly affected by organic substances such as sugars, various vitamins and amino acids, and various nutrients such as nitrates, calcium salts and phosphates contained in the culture solution. For example, MS medium (Murashig)
It is capable of stably absorbing a large amount of various culture solutions including e-Skoogm, a basic medium containing inorganic salts, trace elements, and vitamins, and holding it in a large amount. In addition, since the crosslinked polyalkylene oxide has a water absorption mechanism based on affinity with water, it is not necessary to include salts such as sodium for securing osmotic pressure unnecessary for culturing in the gel. For this reason, it is possible to prevent salt damage such as growth inhibition caused by the salts contained in the culture, and it is also convenient for controlling the pH of the medium because the base material does not contain salts.

[0010] The crosslinked polyalkylene oxide used in the present invention has thermoplasticity, which is also very advantageous when used as a base material for a synthetic medium. This is because, when producing a synthetic medium base material, molding can be performed in the same manner as in a normal thermoplastic. The crosslinked polyalkylene oxide after the molding process is used as a synthetic medium, and therefore has sufficient strength even after being absorbed and gelled.

When the synthetic medium of the present invention is processed into a sheet or a film, it is usually unnecessary to reinforce the blending of fibers and fillers, and the crosslinked polyalkylene oxide can be prepared by a commonly used method. It is processed into a sheet or film by itself. When the synthetic medium of the present invention is processed into a nonwoven fabric, a crosslinked polyalkylene oxide may be formed by a method used in ordinary nonwoven fabric production such as an air pulp method, a wet method, a spunlace method, a melt blow method, a spun bond method, and a flash spinning method. Is processed into a nonwoven fabric. In the present invention, it is also easy to process these into a shape suitable for use, such as by punching them into a circle in accordance with a culture dish serving as a culture vessel. In terms of the quality after processing, since the base material is synthetic and different from natural products, uniform quality can be stably produced in large quantities, and various physical properties can be easily controlled during production.

The synthetic medium of the present invention comprises a gel obtained by absorbing water and / or a culture solution into the above-mentioned crosslinked polyalkylene oxide. The water and / or culture solution used at this time is not particularly limited, and its components can be appropriately changed depending on the culture object. For example, sterilized distilled water, SSS medium, MS medium, Rei medium
A nat-White medium, a B5 medium, or the like can be used. The gelation is performed by directly adding the water and / or the culture solution to the crosslinked polyalkylene oxide contained in the container, or dispersing a material such as an inorganic salt for preparing a culture medium in the crosslinked polyalkylene oxide in advance. It is carried out by adding sterile distilled water or the like, and the total absorption of water and / or culture solution at that time is usually 10 to 50 times the weight of the crosslinked polyalkylene oxide. If the amount is less than 10 times, the culture solution may be separated into an absorbed part and an unabsorbed part,
If it exceeds twice, the culture solution that cannot be absorbed is released, which is not preferable. The crosslinked polyalkylene oxide used at this time may be a single type or a mixture of two or more types.

The synthetic medium of the present invention can be used for culturing various bacterial cells, plant tissues, animal tissues, plant cultivation, and the like. At this time, methods such as culturing and cultivation are the same as those performed on a normal agar medium.

[0014]

EXAMPLES The present invention will be described in detail with reference to examples below, but the present invention is not limited to these examples.

Production Example 1 (Method for producing crosslinked polyethylene oxide) Polyethylene oxide (weight average molecular weight: 20,000)
0) was dehydrated at 100 ° C. for 2 hours under reduced pressure of 5 mmHg. Take 100 g of this in 70 ml of toluene at 70 ° C.
While stirring, 6 g of 1,4-phenylene diisocyanate and 0.2 g of triethylamine were added. The mixture was further stirred at 80 ° C. for 3 hours. After the reaction, the resin solution was poured into 3 liters of hexane to obtain a resin precipitate. The resin precipitate was dried under reduced pressure to obtain a water-insoluble crosslinked polyethylene oxide powder. The pure water absorption of the obtained crosslinked polyethylene oxide was 40 g per 1 g of the resin.
Met.

Production Examples 2 to 3 (Method for producing crosslinked polyethylene oxide) The polyethylene oxide has a weight average molecular weight of 30.
A crosslinked polyethylene oxide powder was produced in the same manner as in Production Example 1 except that the amount of 1,4-phenylene diisocyanate used was 1 g and 0.5 g, respectively. The pure water absorption of the obtained crosslinked polyethylene oxide was 20 g and 18 g, respectively, for 1 g of the resin.
g.

Examples 1 to 3 Each of the crosslinked polyethylene oxides obtained in Production Examples 1, 2 and 3 was heated to 160 to 190 ° C. and melted to a thickness of 1 mm.
And a 4 cm diameter circle (weight 0.5
g), a circular petri dish made of styrene resin (130
mm φ13 mm), put the Petri dish into a steel pressure-resistant container equipped with a heating device, and purge the air inside the mixture with an ethylene oxide-carbon dioxide gas mixture (ethylene oxide 20
vol.), and sterilized at a temperature of 55 ° C. and a pressure of 2.3 atm (gauge) for 10 hours.

Next, when 20 ml of the MS medium solution having the composition shown in Table 1 is added to the above-mentioned three Petri dishes containing the crosslinked polyethylene oxide sheet, the sheet instantly absorbs the added solution and becomes a gel. Sheet-shaped synthetic media 1, 2, and 3 were obtained.

[0019]

[Table 1]

Test Example 1 (Preparation of Explants) A 6 cm length of a carrot radish that had been in contact with the soil on the surface of the radish was thoroughly washed.
The slices were cut into small pieces and immersed in a 1% sodium hypochlorite solution for about 15 minutes to sterilize. After washing with sterilized water, a 4 mmφ circular rod-shaped carrot tissue was obtained using a sterilized cork borer. A portion excluding both ends of the carrot tissue was cut into a thickness of about 5 mm to obtain a disc-shaped explant. (Callus Induction) A total of 30 synthetic media 1, 2, and 3 in the Petri dishes obtained in the Examples were prepared,
The disc-shaped explants obtained above were planted in a petri dish with tweezers three per dish. The petri dishes with the explants were cultured in a thermostat at 25 to 27 ° C. In any of the synthetic mediums 1, 2, and 3, the explants grew softly in about 2 weeks after the start of the culture, and the cells grew to callus. During this time, no growth inhibition or the like was observed.

Examples 4 to 6 Three test tubes were filled with 1 g of the powdered crosslinked polyethylene oxide obtained in Production Examples 1, 2, and 3, respectively, and 5 ml of the same MS medium as used in Example 1 was used. After absorption, synthetic media 4, 5, and 6 of the present invention were obtained.

Test Example 2 The synthetic media 4, 5 and 6 obtained in Examples 4 to 6
Explants obtained in the same manner as in Test Example 1 from about asparagus stems were stabbed two per test tube and 25 to 27
The cells were cultured in a constant temperature incubator. In all the synthetic media, callus of asparagus was induced in about 2 weeks. Also in this case, no particular inhibition of growth was observed.

Example 7 The crosslinked polyethylene oxide obtained in Production Example 1 was extruded into a stream of air from a die heated to 100 ° C., and the towed fibers were collected on a moving screen to form a fibrous crosslink having a diameter of 500 μm. Obtain a nonwoven fabric made of polyethylene oxide,
The non-woven fabric is further rounded to a diameter of 13 cm (weight 0.5 g).
Cut into a circular petri dish made of styrene resin (130 mm
(φ × 13 mm), placed in a steel pressure-resistant container equipped with a heating device, and the air inside was replaced with an ethylene oxide-carbon dioxide gas mixture (ethylene oxide 20 vol%), at a temperature of 55 ° C. and a pressure of 2. The bacteria were sterilized at 3 atm (gauge) for 10 hours. 20 ml of sterile distilled water was absorbed into the solution to obtain a synthetic medium 7 of the present invention.

Test Example 3 The synthetic medium 7 obtained in Example 7 was used as a seedbed for germination, and 10 seeds of cracked radish were sprayed. By keeping it in a dark place at room temperature (around 25 ° C.), almost 100% of seeds germinated after 2-3 days. After this, they continued to grow well for over a week without water supply.

[0025]

The synthetic medium of the present invention is different from the conventional synthetic medium in that the quality is stable even after processing, and is excellent in water content stability without being affected by ions in the culture solution. Further, since growth inhibition and the like during culture do not occur, the processability is good, and the handling is easy, it is extremely useful for culturing various bacterial cells, plant tissues, animal tissues, and plant cultivation.

Continued on the front page (72) Inventor Hitoshi Tanaka 1st Irifune-cho, Shima-ku, Himeji-shi, Hyogo Sumitomo Seika Co., Ltd. (56) References JP-A-63-68027 (JP, A) JP-B-51-39672 (JP, B2) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 1/00 C12M 1/00 C12M 5/00

Claims (4)

(57) [Claims] 1. A crosslinked polyalkylene oxide having a water absorption of 10 to 50 times its own weight obtained by urethane crosslinking of a polyalkylene oxide having a weight-average molecular weight of 1,000 to 6,000,000 is used as a base material. Alkyleneoxy
Synthetic medium to absorb water and / or culture in de characterized by comprising the obtained gel. 2. The synthetic medium according to claim 1, wherein the crosslinked polyalkylene oxide is processed into a sheet. 3. The synthetic medium according to claim 1, wherein the crosslinked polyalkylene oxide is processed into a nonwoven fabric. 4. The polyalkylene oxide is at least one selected from the group consisting of ethylene oxide homopolymer, ethylene oxide-propylene oxide copolymer and propylene oxide homopolymer.
4. The synthetic medium according to 2 or 3.