JP2006271281A - Culture medium support consisting essentially of konjak glucomannan and method for utilizing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve problems that a conventional culture medium support consisting essentially of agar or gellan gum inhibits growth, propagation and differentiation according to the kind of a cultured product. <P>SOLUTION: It is found that konjak glucomannan which has not hitherto been utilized as the culture medium support produces no inhibitory effects on the growth, etc., of the cultured product. Thereby, the culture medium support consisting essentially of the konjak glucomannan is developed. A wider range of plants, microorganisms or plant tissues can be cultured by using the new culture medium support. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a method for producing and using a solid medium using a medium support mainly composed of konjac glucomannan.

In biotechnology using plants or fungi, it is first necessary to cultivate target cells and tissues aseptically. It becomes possible to isolate and proliferate specific microorganisms by culturing, or to efficiently grow plants that are difficult to grow, and to produce useful substances with high efficiency.

In such culture, a culture solution generally called a culture medium is used. The components of the medium are various nutrients, growth control substances, inorganic salts and the like. The medium is used in a liquid state or solidified with a solidifying agent depending on the purpose of use, the latter being called a solid medium.

Solidifying agents used in a solid medium are collectively referred to as a medium support, and several types of polysaccharides are generally used depending on the type of target cell / tissue, medium components and pH. The most frequently used medium support is red algae-derived agar, purified agarose, or microorganism-derived gellan gum.

It has long been known that the type of media support has a significant effect on the growth, differentiation and growth of cultures. For example, in inducing the differentiation of somatic organs in plant tissue culture, the induction rate is low when agar is used as a medium support, and when gellan gum is used, somatic organs that are approximately twice as large as agar are formed (Non-Patent Documents). 1).

Since this tendency may be reversed depending on the kind of plant, tissue / cell, it is necessary to examine a medium support suitable for a subject to be cultured in advance. In addition, when culturing for a relatively long period of several weeks to several months, agar contains a large amount of unbound water, so that it has a lot of water separation and weak water retention (see Non-Patent Document 2). Cracking occurs due to growth and drying of the medium, and gellan gum is gelated by crosslinking with divalent cations in the medium, so the cationization is reduced due to the decrease in the effective cation concentration in the medium and the absorption of cations in the culture. This has been a problem (see Non-Patent Document 3).
Atsushi Tanimoto, Hiroshi Harada (1991) “Introduction to Seminars on Plant Tissue / Cell Culture 4 Medium Solidifying Agent / pH of Medium” Shujunsha, Plant Cell Engineering 3 (3) P235-239 Yuji Buruhashi (1997) "Characteristics of agar gel and its application to food" Science Forum Inc., Gel Technology pp 332-337 Koichiro Shimomura (1997) “Use of gels in plants” Science Forum, Inc. Gel Technology pp162-170

The above-described conventional medium support mainly composed of agar or gellan gum is not suitable for a wide range of cultures and limits the culture target.

  The present invention is intended to solve the problems of such a conventional medium support, and provides a medium support that does not inhibit the growth, proliferation and differentiation of a wide range of cultures. It is an object.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found konjac, a taro family plant, scientific name Amorphophallus rivieri var. Konjac.
It has been found that the problem can be solved by using the derived polysaccharide konjac glucomannan as a main component of the medium support.

Konjac glucomannan does not gel and exists in a highly viscous sol state in acidic to neutral solutions. Therefore, unlike agar or gellan gum, when used alone, a commonly used acidic to neutral medium cannot be solidified.

For this reason, konjac glucomannan was considered inappropriate as a medium support. However, as a result of repeated studies by the inventor, it can be used as a medium support capable of forming a solid medium having sufficient strength by mixing agar, agarose or gellan gum and konjac glucomannan at an appropriate ratio. It became.

This novel medium support reduces the relative amount of agar, agarose or gellan gum added to the medium by incorporating konjac glucomannan, and is a culture found when these polysaccharides are used alone. There is almost no inhibitory effect on growth, differentiation, and proliferation.

In addition, as described above, konjac glucomannan does not form a strong gel in the medium that is normally used, so in addition to being able to hold the culture appropriately by flexibly deforming according to the growth of the culture, No cracking occurs even if the medium is dried by long-term culture. Furthermore, since light is transmitted more than agar, it is possible to confirm the state of growth / differentiation / proliferation of the culture in the medium, such as growth of the root of the culture. Thus, the culture medium support of the present invention solves the problems of the conventional culture medium support.

The culture medium support of the present invention can be used in the same manner as a conventional culture medium support. That is, it is provided in the form of a liquid, powder, granule, syrup-like culture composition mixed with medium components such as a medium support or nutrients, and after diluting with water or the like to an appropriate concentration, A solid medium can be obtained by heat sterilization with an autoclave or the like, and then cooling to solidify.

A medium support can be obtained with konjac glucomannan. In addition, a composition for culturing microorganisms / plants having an action of promoting proliferation / growth / differentiation of microorganisms / plants using konjac glucomannan can be obtained.

Hereinafter, the present invention will be described in detail. The konjac koji used as a raw material for the medium support of the present invention is Amorphophallus rivieri var. Konjac of the taro family that is a raw material for konjac used in oden and the like. There are native varieties of konjac varieties (red stalks, white birch, flat, Japanese and large varieties), Chinese varieties and binaka varieties (blue stalks, black leopards, long balls, stone balls, etc.) Harunuroro (Konnyaku Norin 1), Akagi Odama (Konnyaku No. 2), Myo Yutaka (Konnyaku No. 3) and Miyama Sari (Konnyaku No. 4) are known. Any varieties of konjac can be used. Also, one or more of the above varieties are used in combination. The parts that can be used are corms and pups. In addition, cultivated or natural konjac can be used in appropriate combination.

The konjac meal produces fine powder as a raw material of konjac and flying powder as a by-product. In this study, the fine powder can be used as konjac glucomannan. In addition, the purified konjac glucomannan can be used as konjac glucomannan by removing impurities by washing it with an organic solvent such as ethanol or a weak acid, weak alkaline solution, or surfactant. .

The culture medium support of the present invention contains 0.05 to 96%, preferably 0.09 to 84%, more preferably 50 to 84% of the konjac glucomannan. When the content of konjac glucomannan is less than 0.05%, no promoting action on proliferation, growth and differentiation of the culture is observed. Further, when the konjac glucomannan content is more than 84%, gelation of the medium support is not recognized. The component that forms the medium support together with konjac glucomannan may be a polysaccharide that gels at room temperature, such as agar and gellan gum, and agar or agarose is preferred.

Next, the composition for cultivating microorganisms and plants and the composition for plant cultivation obtained by blending the culture medium support of the present invention will be described. The preparation comprising the medium support of the present invention is used as it is or as a composition for culturing microorganisms / plants together with conventional medium nutrients, growth regulators, and inorganic salts, and used for culturing microorganisms or plants. be able to. The dosage form of these compositions is not particularly limited and may be appropriately selected as necessary. Examples thereof include solid agents such as tablets, capsules, granules, fine granules, and powders. It is done. In addition to the medium support of the present invention, surfactants, fluidity promoters and the like can be appropriately used for this type of preparation. The composition for cultivating microorganisms and plants containing the above-mentioned medium support may be supplied in a state divided into an aqueous solution containing a water-soluble nutrient component and a solid preparation containing a medium support, or as a suspension.

The medium support of the present invention is suspended in a liquid medium or water so that the final concentration in the medium becomes 0.01 to 3%, then melted by heating, and then cooled to form a gel. Can be used. The medium support of the present invention can be applied without any limitation on the use of the medium support so long as it is used for the purpose of inducing the proliferation, growth and differentiation of microorganisms and plants.

Next, the present invention will be described in more detail with reference to examples and comparative examples.

<Experimental example 1>
Example of preparation of solid medium: 1.96 g of mixed salt for Murashige-Skoog medium (manufactured by Nippon Pharmaceutical Co., Ltd.), 12 g of sucrose (manufactured by Wako Pure Chemical Industries, Ltd.), Murashige & Sukug vitamin solution (manufactured by Sigma Aldrich Co., Ltd.) ) Add 0.4 mL to 280 mL of water and stir. After dissolution, adjust the pH to 5.6 using an aqueous solution of sodium hydroxide (manufactured by Wako Pure Chemical Industries, Ltd.), add water to adjust the volume to 400 mL. Murashige-Skoog medium was prepared.

This was dispensed into four glass beakers of 100 mL each, and designated as treatment sections A, B, C, and D. While stirring the culture medium of each treatment group using a magnetic stirrer, 0.2 g of plant culture agar (manufactured by Wako Pure Chemical Industries, Ltd.) is added to treatment zone A, 0.5 g is the same to treatment zone B, and treatment zone C Was added 0.1 g of vegetable medium gellan gum (manufactured by Wako Pure Chemical Industries, Ltd.), and 0.2 g of the same was added to the treated area D, which was heated and completely melted. Furthermore, 1 g of glucomannan manufactured by Wako Pure Chemical Industries, Ltd. was added to each treatment zone as glucomannan, and the mixture was heated to the boil and melted as it was. The culture medium of each treatment group was dispensed 10 mL each into a test tube for plant tissue culture (Asahi Techno Glass Co., Ltd., diameter 20 mm, length 150 mm) and covered with a plastic cap for the test tube (Asahi Techno Glass Co., Ltd.), 120 Autoclaved at 20 ° C. for 20 minutes. After lightly stirring with a test tube mixer, it was cooled at room temperature and solidified.

<Experimental example 2>
Murashige-Skoog medium was prepared in the same manner as in Experimental Example 1, and 2,4-dichlorophenoxyacetic acid (manufactured by Wako Pure Chemical Industries, Ltd.) was added to a concentration of 0.5 mg / L. Dispense 100 mL of this liquid culture medium into a 200 mL glass Erlenmeyer flask, add 0.5 g of agar for plant culture medium (manufactured by Wako Pure Chemical Industries, Ltd.) and 1 g of glucomannan (manufactured by the same company), and cover with aluminum foil. And then autoclaved at 120 ° C. for 20 minutes. After lightly stirring, 20 mL each was dispensed into a plastic sterilization petri dish (manufactured by Nipro Corporation, diameter 90 mm, height 15 mm) and solidified at room temperature.

<Example 1> Examination of plant shoot growth-promoting action Preparation of culture: Komatsuna "Ajisai" (manufactured by Tohoku Mating Co., Ltd.) Seeds are sequentially washed with water, ethanol, 1% hypochlorous acid solution and sterilized , Seeded on an elementary agar medium consisting of 0.8% plant medium agar (manufactured by Wako Pure Chemical Industries, Ltd.) and 0.5% sucrose (manufactured by the same company) . Using a sterilized scalpel, a 1 cm long section was cut from the shoot apex, planted vertically on the solid medium of Experimental Example 1, cultured at 25 ° C. under continuous light for 2 weeks, and then the fresh weight of the plant body was measured. . The results are shown in Table 1.

<Comparative Example 1> A 200 mL Murashige-Skoog medium was prepared in the same procedure as in Experimental Example 1 and dispensed into two glass beakers, each of which was 1 g of plant medium agar (manufactured by Wako Pure Chemical Industries, Ltd.) ) Or 0.2 g of gellan gum for plant medium, and after melting, dispense 10 mL each into a test tube for plant tissue culture (Asahi Techno Glass Co., Ltd., diameter 20 mm, length 150 mm). (Asahi Techno Glass Co., Ltd.) was applied and autoclaved at 120 ° C. for 20 minutes. After lightly stirring with a test tube mixer, it was cooled at room temperature and solidified. A section of radish was planted in this solid medium in the same manner as in Example 1, and after culturing at 25 ° C. under continuous light for 2 weeks, the fresh weight of the plant was measured. The results are shown in Table 1.

<Example 2> Examination of plant cell growth promoting action Preparation of plant section: Carrot “Tokinashi 5 inch ginseng red” (manufactured by Tohoku Co., Ltd.) seeds were sterilized and sown in the same manner as the radish seeds of Example 1. . After culturing at 25 ° C. under continuous light for 1 week, 1 cm was cut off from the shoot apex, and the remaining hypocotyl was cut to a length of 1 cm to prepare a section.
Preparation of callus induction medium: 2,4-dichlorophenoxyacetic acid was added to the Murashige-Skoog medium prepared in the same manner as in Experimental Example 2 to a concentration of 0.5 mg / mL. To 100 mL of this medium, 0.8 g of agar for plant medium (manufactured by Wako Pure Chemical Industries, Ltd.) was added, dispensed into a plastic sterilized petri dish after autoclaving, and solidified at room temperature.
Induction of callus formation: Carrot sections were planted horizontally on the solid medium prepared by the above procedure and cultured at 25 ° C. under continuous light for 4 weeks.

Preparation of callus: 30 ml of Murashige-Skoog medium containing 0.5 mg / mL 2,4-dichlorophenoxyacetic acid was dispensed into a 100 mL flask, capped with aluminum foil, autoclaved, and cooled to room temperature. About 100 μL of undifferentiated cells (callus) formed on the sections cultured by the above procedure were taken, transferred to this medium, and cultured at 70 rpm, 25 ° C. in the dark for 2 weeks.

Planting in glucomannan medium: 1 mL of carrot callus grown in the above procedure was planted in the solid medium prepared in Experimental Example 2 and cultured at 25 ° C. in the dark for 1 month. Callus was collected and fresh weight was measured. The results are shown in Table 2.

<Comparative example 2>
1 g of plant medium agar was added to 100 mL of 2,4-D-containing Murashige-Skoog medium prepared in the same procedure as in Experimental Example 2, autoclaved, and dispensed into a plastic sterilized petri dish. 1 mL of carrot callus prepared by the same procedure as in Example 2 was planted in this medium, and cultured at 25 ° C. in the dark for 1 month. Callus was collected and fresh weight was measured. The results are shown in Table 2.

Claims (5)

A medium support comprising konjac glucomannan as a main component. The medium support according to claim 1, wherein the raw material of konjac glucomannan is Amorphophallus rivieri var.   The medium support according to claim 1 or 2, characterized by containing 0.05 to 96% by weight of konjac glucomannan. 4. The medium support according to claim 1, which has an action of inducing plant indefinite organs or promoting growth and differentiation of cultures. A composition for cultivating microorganisms and plants, comprising the medium support according to claim 1 as an active ingredient.