JPH0740930B2 - Asunaro plant tissue culture method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for tissue culture of an Asunaro plant, which is a raw material for an antimicrobial agent, an aromatic agent, and the like.

[Conventional technology]

The evergreen Takagi coniferous cypress family Asunaro plant contains a tropolone compound such as hinokitiol having an antimicrobial effect, a phenol compound such as carvacrol, and tsuyopsene having an aroma. Therefore, if the callus tissue of a plant of the genus Asunaro can be cultured, the callus tissue can be used as a raw material such as an antimicrobial agent and an aromatic agent.

However, many tissue culture techniques for herbaceous plants such as rice and carrots and some woody plants such as poplar have been reported up to now. Until recently, no technology or medium composition was reported.

Under these circumstances, the present inventors have examined the method of culturing callus tissue of Asunaro plants, and recently, Asunaro plants on a medium containing a carbon source, an inorganic component and a plant hormone as essential components. It has been found that the callus tissue can be efficiently cultivated or subcultured, and a patent application has already been filed (Japanese Patent Application No. 63-327087).

[Problems to be Solved by the Invention]

However, in the above method, when the initial cell density was lowered,
Since the proliferation efficiency is very poor, further improvement has been required.

In view of the above circumstances, the present invention aims to provide a method capable of efficiently culturing callus tissue of a plant of the genus Asuna, more efficiently than the conventional method, and culturing the tissue even when the initial cell density is low. And

[Means for Solving the Problems]

In order to solve the above problems, the present invention is a method of culturing tissue of an Asunaro plant on a medium containing a carbon source, an inorganic component and a plant hormone as essential components, wherein the carbon source in the medium is 10% by weight. The above is characterized by glucose.

As a plant of the genus Asunaro applied to the tissue culture method for a plant of the genus Asunaro according to the present invention, cypress asunaro is preferable, but the plant is not particularly limited thereto.

The carbon source of the medium (hereinafter, simply referred to as “medium”) used in the method for tissue culture of Asunaro plant according to the present invention is not particularly limited, but is not limited to carbohydrates such as sucrose and its derivatives, and organic matter such as fatty acids. Examples thereof include acids and primary alcohols such as ethanol. However, in the method for culturing tissue of a plant of the genus Asunaro according to the present invention, it is necessary that 10% by weight or more of these carbon sources is glucose, and particularly 30% by weight or more is glucose.

The inorganic component of the medium is not particularly limited, for example, nitrogen, phosphorus, potassium, sodium, calcium,
Magnesium, iron, manganese, zinc, molybdenum, copper,
Inorganic salts containing elements such as cobalt, sulfur, boron, chlorine and iodine can be mentioned. Specific examples thereof include potassium nitrate, sodium nitrate, ammonium nitrate, potassium chloride, calcium chloride, potassium dihydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, ammonium sulfate, sodium sulfate, ferrous sulfate, ferric sulfate, and sulfuric acid. Examples thereof include compounds such as manganese, copper sulfate, zinc nitrate, sodium molybdate, potassium iodide, boric acid, and cobalt chloride.

In the tissue culture method of the plant of the genus Asunaro according to the present invention, it is necessary that the medium contains at least an auxin among auxins and cytokinins as a plant hormone. And, the concentration of auxins is in the range of 10 ⁻⁶ to 10 ⁻⁴ mol /, and if necessary, the concentration of cytokinins is 10 ⁻⁵ mol / or less, preferably 10 ⁵ or less.
^-6 mol / mol or less so that it is contained in the medium.
Tissue culture can be performed particularly efficiently by using a medium having an auxin concentration of 10 ⁻⁵ mol / and a cytokinin concentration of 10 ⁻⁷ mol /, but is not particularly limited thereto.

Examples of the auxins include α-naphthalene acetic acid (NAA), indole acetic acid (IAA), 2,4-dichlorophenoxyacetic acid (2,4-D), indole butyric acid (IBA) and their derivatives, and the like. Examples of the cytokinins include kinetin, benzyladenine (BA), zeatin, etc., but are not particularly limited thereto. Of these, when the auxin is α-naphthalene acetic acid or 2,4-dichlorophenoxyacetic acid and the cytokinin is kinetin or benzyladenine, the tissue culture of Asunaro can be performed particularly efficiently.

The medium may contain vitamins, amine acids, and the like, if necessary, in addition to the carbon source, the inorganic component, and the essential components of the plant hormone.

Examples of the vitamins include biotin, thiamine, pyridoxine, pyridoxal, pyridoxamine, calcium pantothenate, ascorbic acid, inositol, nicotinic acid, and nicotinic acid amide, but are not particularly limited thereto.

The amino acids include, for example, glycine,
Examples thereof include, but are not limited to, alanine, glutamic acid, cysteine, and phenylalanine.

The concentration of each component contained in the medium is usually 10 carbon sources.
To 50 g /, inorganic components 10 ^-6 to 0.1 mol /, plant hormones at the above-mentioned concentration, vitamins 0.1 to 150
It is desirable that the amount of mg / amino acids is about 0 to 1 g /, but the concentrations of components other than the carbon source and plant hormones are not particularly limited to these.

As a method for preparing a medium, a medium that has been conventionally used for tissue culture of plants, for example, Murashige-Skoog medium,
Rinsmeier-Skoog medium, White medium, Gamborg B-5 medium, niche-niche medium, etc. are used as basic medium, and in addition to carbon sources and plant hormones, if necessary, vitamins, amino acids, etc. A method of adding and preparing can be exemplified, but the method is not particularly limited. The basal medium originally contains the inorganic component, but the inorganic component may be appropriately added if necessary. Of the basal mediums, particularly desirable in the present invention are Murashige-Skoog medium and Gamborg B-5 medium.

The medium thus prepared may be used as a liquid medium or a solid medium containing a polysaccharide such as agar. The polysaccharide is usually contained in the medium in an amount of about 0.1 to 1% by weight, but is not particularly limited thereto.

Tissue culture is performed by culturing or subculturing explants cut from Asunaro plants, particularly callus pieces well induced from petiole sites and lateral buds, on the medium prepared as described above. On the above medium, callus tissue can be stably grown and grown without necrosis. The same applies to subculture.

[Action]

In the tissue culture method of Asunaro plant according to the present invention, carbon source, in culturing the callus tissue of an Asunaro plant on a medium with an inorganic substance and a plant hormone essential component, 10% by weight or more of the carbon source is glucose. According to this method, according to this method, the callus tissue of the plant of the genus Asunaro can be cultured more efficiently as compared with the conventional method, and the low cell density, which was very poor in the proliferation efficiency in the conventional method. Can be efficiently cultured.

〔Example〕

Specific examples and comparative examples of the present invention will be shown below, but the present invention is not limited to the following examples.

Culturing in the following Examples and Comparative Examples was performed as follows. That is, the concentration of the carbon source is 3% by weight, α
-Natural acetic acid concentration of 10 ^-5 mol / each was added to Murashige-Skoog's medium to prepare 20 ml of liquid medium and hinoki asunaro callus pieces, 100 ml volume triangle containing finely crushed callus pieces The flask was placed on a rotary shaker and cultivated under the conditions of 100 rpm, 25 ° C. and dark.

-Examples 1 to 4 In a medium containing glucose and sucrose in an amount shown in Table 1 below as a carbon source, about 0.4 g of callus pieces of Hinoki Asunaro.
Was added and cultured for 7 days to examine the growth rate of callus tissue.

-Comparative Example 1-As a carbon source, about 0.4 g of callus pieces of Hinoki Asunaro was added to a medium containing only sucrose in the amount shown in Table 1 below,
The cells were cultured for one day and the growth rate of callus tissue was examined.

-Examples 5 to 8-About 0.4 g of callus pieces of Hinoki Asunaro in a medium containing glucose and sucrose in the amounts shown in Table 1 below as a carbon source.
Was added and cultured for 14 days to examine the growth rate of callus tissue.

-Comparative Example 2-As a carbon source, about 0.4 g of callus pieces of Hinoki Asunaro was placed in a medium containing only sucrose in an amount shown in Table 1 below, and 14
The cells were cultured for one day and the growth rate of callus tissue was examined.

-Examples 9 to 12-About 0.2 g of callus pieces of Hinoki Asunaro in a medium containing glucose and sucrose in the amounts shown in Table 1 below as a carbon source.
Was added and cultured for 7 days to examine the growth rate of callus tissue.

-Comparative Example 3-As a carbon source, about 0.2 g of callus pieces of Hinoki Asunaro was added to a medium containing only the amount of sucrose shown in Table 1 below,
The cells were cultured for one day and the growth rate of callus tissue was examined.

The respective results are shown in Table 1.

As shown in Table 1, callus tissue of Hinoki Asunaro cultivated on a medium containing 10% by weight or more of glucose is a carbon source, cultivated on a medium containing a carbon source of 100% sucrose. Proliferate more efficiently than what was done. In addition, when the initial cell density of callus tissue is low (10 g /),
Callus tissue of Hinoki Asunaro cultivated on a medium containing a carbon source containing 10% by weight or more of glucose is significantly higher than that on a medium containing a carbon source containing 100% sucrose. Proliferate efficiently.

〔The invention's effect〕

According to the method of culturing a tissue of the genus Asuna of the present invention, the callus tissue of the plant of the genus Asuna, which is used as a raw material for an antimicrobial agent, a fragrance, etc., can be cultured more efficiently than in the conventional method. In addition, it is possible to efficiently cultivate callus tissue of a plant of the genus Asunaro from a low cell density, which has a very poor culture efficiency in the conventional method.

Claims (2)

[Claims] 1. A method for culturing a tissue of an Asunaro plant on a medium containing a carbon source, an inorganic component and a plant hormone as essential components, wherein 10% by weight or more of the carbon source in the medium is glucose. A method for tissue culture of a plant of the genus Asunaro, which comprises: 2. The method for tissue culture of a plant of the genus Asunaro according to claim 1, wherein the plant of the genus Asunaro is cypress asunaro.