JPS60987B2 - Tissue culture method for plants of the family Murasakiceae - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for culturing the tissues of purple plants containing naphthoquinone pigments such as shikonin.

More specifically, the present invention relates to a method for efficiently producing a large amount of naphthoquinone compounds and other useful components by tissue culturing plants of the family Prunusaceae using a liquid medium with a specific composition. The root of Murasaki, a plant belonging to the Purple family, contains naphthoquinone compounds such as shikonin (R=-OH) shown by the following formula, and has traditionally been called "purple root" and used in Chinese medicine. .

In other words, the ointment obtained by extracting shikonin and other substances from Shikonin using oils such as sesame oil is called Shiyunlip and is used to treat various skin diseases, cuts, burns, hemorrhoids, etc., and has anti-inflammatory and granulation-forming effects. It is known that there is
However, the medicinal ingredients such as shikonin that can be extracted from the purple plate are only available in small quantities, and the cultivation of purple is time-consuming.
There are problems such as dependence on the natural environment and weather, and its stable supply is at risk.

On the other hand, Mamoru Tabata, Hajime Mizukami, and others proposed propagating plants of the Phytochemistry family using tissue culture methods.
Registration page 927, "Pharmaceutical magazine" No. 95 registration page 1376, "Phytochemistry"
(st) Volume 16, page 1183, Volume 17, page 95
Reported on the page.

According to this method, regardless of the season or weather,
It is very advantageous because it allows the cultivation of purple plants. However, the methods disclosed in these publications all use agar as a solid medium, and are unsuitable for mass production. Therefore, the present inventors investigated a method for growing callus in a similar manner using a liquid tower suitable for mass production, and first added agar to the medium used by Tabata et al. (Linsmeyer-Skoog medium). It was used for tissue culture of purple violet in the form of a liquid medium, but
Although the callus proliferated to some extent, the amount of pigments such as shikonin produced was small, and the amount produced also varied widely, making it impossible to secure a stable yield.

The present inventors have conducted further studies on liquid media that are suitable for tissue culture of plants of the family Murasaceae and that produce large amounts of naphthoquinone compounds such as shikonin. The present inventors have discovered that the growth is rapid, a large amount of naphthoquinone compounds are produced, and there is little variation in the amount produced, ensuring stable production, leading to the completion of this invention.

That is, the present invention relates to a method for culturing the tissue of a plant belonging to the family Murasaceae, which is characterized by using a liquid medium having a sulfate ion concentration of 0.1 M or more.

In the present invention, the sulfate ion concentration in the liquid medium is 0.1 m
Other medium components can be varied over a wide range as long as the medium is adjusted to M or higher, especially within the range of 0.2mM to 45mM, and the medium conventionally used for plant tissue culture can be used with various modifications. be able to. When the sulfate ion concentration is less than 0.1 mM, the amount of naphthoquinone compounds produced decreases, and even when it exceeds 45 M, no major change is observed, but a slight decrease in the amount produced is observed.

Conventional media used for plant tissue culture include those that have inorganic components and carbon sources as essential components, and to which are added at least one component selected from plant hormones, vitamins, and amino acids. , other components may be used in combination as necessary.

Inorganic components include nitrogen, phosphorus, potassium, lucium, magnesium, sulfur, iron, manganese, zinc, boron, copper, molybdenum, chlorine, sodium, iodine, cobalt, etc. Specifically, potassium nitrate, sodium nitrate, Calcium nitrate, monopotassium phosphate, disodium phosphate, potassium chloride, calcium chloride, magnesium sulfate, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, zinc sulfate, boric acid, steel sulfate, sodium molybdate , molybdenum trioxide, potassium iodide, cobalt chloride, and the like.

Examples of carbon sources include hydrocarbons such as sucrose, derivatives thereof, organic acids such as fatty acids, and primary alcohols such as ethanol. Plant hormones include indoleacetic acid (IAA), naphthaleneacetic acid (NAA), P-chlorophenoxyisobutyric acid, and 214-dichlorophenoxyacetic acid (
Examples include auxins such as 2.4-D), and cytokinins such as kinetin, zeatin, and dihydrozeatin.

Vitamins include biotin, thiamin (vitamin B),
Examples include pyridoxine (vitamin B6), patetothenic acid, ascorbic acid (vitamin C), inositol, and nicotinic acid.

Examples of amino acids include glycine, alanine, glutamine, and cysteine.

The types and concentrations of components other than sulfate ions in the liquid medium can be varied within a wide range.

Usually, the inorganic component is about 0.1 French M to about 100 mM,
Add a carbon source to about 1 to 30 μM, plant hormones from about 0.01 to about 10 μM, and vitamins and amino acids from about 0.1 to about 9/2 to about 10
The value is set to about 9/h of 0. In the present invention, it is also possible to further increase the amount of naphthoquinone compounds produced by adjusting other components in the medium.

For example, if the ratio of ammonium ions to the total nitrogen source is about 10 mol % or less, the amount of naphthoquinone compounds produced will further increase. A preferred example of this invention is the following method.

That is, after sterilizing tissue pieces collected from the plant body of a plant belonging to the family Prunusaceae, such as roots, growing points, leaves, stems, seeds, etc., solidification with agar or layering on the ground of a Skoog solid tower is carried out for 10 minutes. After about 7 to 30 days at ~35°C, a part of the tissue piece is formed into a callus. When the callus thus obtained is subcultured, the growth rate gradually increases and stable callus is obtained. This callus is transferred to a liquid medium suitable for growth, such as a Linsmeyer-Skoog liquid medium, and grown.

There is a method of culturing callus whose growth rate is further increased and stabilized in a liquid column by adding it to the liquid medium of the present invention.

In these methods, the initial concentration of callus in the liquid medium can be varied within a wide range.

Usually, one liquid medium is used to grow callus for about 1 hour to about 20 minutes.
It is desirable to add about 0.0 kg (fresh weight). In the tissue culture of the present invention, light is not necessarily required, and culture in a stairwell is preferable for the production of pigments such as shikonin.

In addition, the culture temperature is about 10°C to about 35°C, especially about 23°C.
00 to about 28q○ is preferable; if it is less than about 1000, the callus growth rate is low, and if it exceeds about 35oo, the callus growth rate is similarly low. To separate and collect naphthoquinone compounds from callus and liquid medium, methods such as extraction that have been conventionally applied to the natural product "Shikon" can be employed.

According to the present invention, since liquid beach soil is used, it is possible to perform mass culture using a tank, and furthermore, as a method for separating callus from the culture medium, simple operations such as decantation and furnace filtration can be adopted, which is industrially advantageous. It is.

Furthermore, callus multiplies rapidly, and naphthoquinone compounds such as shikonin can be reliably produced in large quantities.

Comparative example Lithospermum erythr
orhbonSe rank. etZucc. ) root tissue pieces were plated on Linsmayer-Skoog agar solid medium,
Purple callus was obtained using the static culture method.

By culturing this callus in a Linsmayer-Skoog liquid column, the growth rate of the callus was increased.Meanwhile, a modified white liquid having the composition shown in Table 1 was added to a 100' Erlenmayer flask. Toji (contains 1rM of indoleacetic acid, 10rM of kinetin, and 20% of sucrose as a carbon source as plant hormones)
It was sterilized in a Z-box of 30°C at 120°C for 10 minutes.

To this liquid tower, 0.5 days of fresh purple callus with increased growth rate was added, and cultured with rotation (amplitude 25 bars, 1
0 ratio pm). After culturing, the purple callus was collected by filtering, dried at 35o0 for 2 hours, and its weight (
The growth rate of cultured cells per 1 volume of liquid medium was determined.

In addition, shikonin was separated from the obtained callus by extraction,
The weight was measured, and the total amount of shikonin produced per liquid column was determined.

The results are shown in Table 2.

Examples 1 to 3 Comparative Examples were conducted in the same manner as in Comparative Examples except that the sulfate ion concentration among the medium components of the liquid medium was set to the values shown in Table 2.

Table 1 Table 2

Claims (1)

[Scope of Claims] 1. A method for cultivating tissues of plants of the family Murasaceae, characterized by using a liquid medium having a sulfate ion concentration of 0.1 mM or more. 2. The method according to claim 1, characterized in that a liquid medium having a sulfate ion concentration of 0.2 mM to 45 mM is used. 3 Lithospermum erythrorhiz
on Sieb. et Zucc. ) The method according to claim 1, characterized in that: