JPS60985B2 - 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 tissue of a plant of the family Murasaceae containing a naphthoquinone pigment such as shikonin.

More specifically, by controlling the concentration of specific components in the liquid medium and tissue culturing the purple plant,
The present invention relates to a method for efficiently producing large quantities of naphthoquinone compounds and other useful ingredients. The roots of the purple root, which is a plant belonging to the Purple family, contain naphthoquinone-based compounds such as shikonin (R=-OH), which is represented by the following formula (R=-OH, -OCOCH3, etc.), and has traditionally been called "purple root". ” and is 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 Shiunbone 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 root are limited, and cultivating purple root takes time.
There are problems such as dependence on the natural environment and weather, and its stable supply is at risk.

In contrast, the use of tissue culture methods to propagate plants of the family Murasakiceae has been proposed by Mamoru Tabata, Hajime Mizukami, et al. 137
Page 6, “Phytochemistry”
It is reported in Volume 16, page 1183 of Mist Hi), Volume 17, page 95 of the same volume.

According to this method, regardless of the season or weather,
It is very advantageous because it can propagate plants of the family Murasakiceae. 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 of 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 a liquid medium that is suitable for tissue culture of plants of the family Murasaceae and that produces large amounts of naphthoquinone compounds such as shikonin, and as a result, the inventors have found that the concentration of specific components in the medium can be controlled. As a result, they found that the multiplication was rapid, ``a large amount of naphthoquinone compounds were produced, there was little variation in the amount produced, and stable production could be ensured.'' This led to the completion of this invention.

That is, the present invention relates to a method for culturing the tissue of a purple plant, which is characterized by using a liquid medium containing 5 rM or less of cytokinins.

As long as the concentration of cytokinins is within the above-mentioned range, the liquid medium used in this invention can have a wide range of types and concentrations of other medium components, and is similar to the medium composition normally used for plant tissue culture. The cytokinins of the present invention may be used by modifying their concentration.In addition, the cytokinins of the present invention include kinetin, benzyladenine, 6-(3-methyl-2-butenylamino)purine (2p), and zeatin. Examples include dihydrozeatin.

The liquid medium used in the present invention contains inorganic components and carbon sources as essential components, and at least one component selected from plant hormones including cytokinins, vitamins, amino acids, etc. is added thereto. A liquid medium can be exemplified.

Inorganic components include nitrogen, phosphorus, calcium, magnesium, sulfur, iron, manganese, zinc, boron, copper, molybdenum, chlorine, sodium, tocobalt iodine, etc. Specifically, potassium nitrate, sodium nitrate, calcium nitrate, phosphorus 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, etc.

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 other than cytokinins include auxins such as indoleacetic acid (IAA), naphthaleneacetic acid (NAA), P-chlorophenoxyisobutyric acid, and 2,4-dichlorophenoxyacetic acid (2,4-D); Examples of vitamins include biotin, thiamine (vitamin B), pyridoxine (pantothenic acid with vitamin B6, ascorbic acid (vitamin C), inositol, nicotinic acid, etc.).

Amino acids include glycine, alanine, glutamine, and cysteine.

The concentration of components other than cytokinins in the liquid medium can be varied within a wide range.

Usually, the inorganic content is about 0. lAM ~ about 100 M,
Add a carbon source of about 1 to 30 rM, plant hormones of about 0.01 to about 10 rM, and vitamins and amino acids of about 0.1 to 9/10 to about 10 rM each.
It is done that it is about 0 o/so. 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% 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 Murasakiceae, such as roots, growing points, leaves, stems, seeds, etc., it is hardened with agar or layered on a Ssmayer-Skoog solid column. After about 7 to 30 days at ~35°C, part of the tissue piece is turned into a callus. When the callus thus obtained is subcultured, the growth rate increases gradually and a stable callus is obtained. This callus are transferred to a liquid medium suitable for growth, such as a Linsmeyer-Skoog liquid medium, and grown.

There is a method of culturing callus in which the growth rate is further increased and stabilized in a liquid medium by adding it to the liquid medium of the present invention. In these methods, the initial concentration of callus in the liquid column can be varied within a wide range. Usually, it is desirable to add about 1 hour to about 200 hours (fresh weight) of callus per 1 volume of liquid medium. In the tissue culture of the present invention, light is not necessarily necessary, and culture in a stairwell is preferable for the growth of pigments such as shikonin, and the culture temperature is about 10 o'clock to about 35 o'clock, especially about 2 to about 28 o'clock.
is suitable.

If it is less than about 1000, the callus growth rate is low, and if it is about 35
Even if it exceeds 00, the growth rate of callus similarly decreases.
In order 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 a liquid soil is used, it is possible to perform mass culture using a tank, and furthermore, as a method for separating callus from a culture medium, simple operations such as decantation and furnace filtration can be adopted, which is industrially advantageous. It is.

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

Comparative example Lithospermum ebit
hrorhbonSecrying. et Zucc) root tissue pieces were layered on Linsmayer-Skoog agar solid medium,
Purple callus was obtained using the static culture method.

Furthermore, by culturing this callus in a Linsmeyer-Skoog liquid tower, the growth rate of the callus was increased.

On the other hand, in a 100' Erlenmeyer flask, a white liquid medium consisting of the composition shown in Table 1 (however, as plant hormones, indoleacetic acid was added to 1 ml and kinetin was added to 1 ml).
0 French M and sucrose as a carbon source (contains 20 mg/contains) 3
It was sterilized at 12,000 for 10 minutes.

To this white liquid medium, add 0.5 liters of the above-mentioned fresh purple callus with increased growth rate, and
Culture with rotation (amplitude 2) on a rotary shaker for 4 days.
5 days, 10 days pm).

After culturing, the purple callus was collected in an oven, dried at 35° C. for 24 hours, and its weight (dry weight) was measured to determine the growth dry weight of the cultured cells per night in the liquid column. In addition, shikonin was separated from the obtained callus by extraction, its weight was measured, and the total amount of shikonin produced per liquid medium was determined.

The results are shown in Table 2. Examples 1 to 3 In the comparative example, the same machine as the comparative example was used except that the concentration of kinetin among the culture medium components of the liquid tower was set to the value 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 in which the concentration of cytokinins is 5 μM or less. 2 The plant of the family Lithospermum is Lithospermum erythrorhiz.
on Sieb. et Zucc. ) The method according to claim 1, characterized in that: