JPS619227A - Tissue culture of buttercup plant - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] [Field of invention] The present invention is a method for tissue culture of Ranunculaceae plants.

Regarding.

[Industrial application field]

The rhizomes of plants belonging to the Ranunculaceae family, for example, the rhizomes of the Ranunculaceae family, contain inquinoline alkaloids such as berberine, and these alkaloids are used, for example, as stomachic agents and dyes. The method of directly collecting isoquinoline alkaloids cannot be said to be an advantageous method because the growth of the Ranunculaceae plants depends on the natural environment and weather, and it takes time and effort to collect the plants. Therefore, as an alternative method, for example, the Journal of Crude Pharmacology, Vol. 65, pp. 15-21 (1981) and Phytochemistry
As described in Vol. 14, pp. 1209-1210 (1975), several tissue culture methods for Ranunculaceae plants have been proposed. However, these conventional tissue culture methods also have the drawback that the yield of the target isoquinoline alkaloid produced from the cultured cells obtained by the method is low. Based on this background, the present inventors improved the conventionally known method of tissue culturing Ranunculaceae plants using a liquid medium, and conducted intensive studies on methods for efficiently producing inquinoline alkaloids such as berberine. As a result, it was discovered that cultured cells containing a large amount of inquinoline alkaloids such as berberine could be obtained by employing the following method. A method for tissue culture of Ranunculaceae plants, characterized in that the concentration of copper ions contained in is 0.2 μmol/13 or more,
is provided.

As the Ranunculaceae plant used in the method of the present invention, for example, Coptis coponic
a Makino), Seriba ouren (C, japon)
ika Makino var, dussect,
aNakai), Kikubaoun (G, japoni)
kaMakino var, japoniKa),
Kose IJ Ba,t Uren (C0japonixa M
aKino Var.

major 5ataKe Papaikaou V (C.

quinquefolia Miq, ) and trifolia 5alisb (c, trifolia 5alisb, ), etc.
ctrumminus L, var hypoleuc
um Miq, ), plants of the genus Salictorum, plants of the genus Xanthorrhiza, and plants of the genus Hydrastis.

In the present invention, among these plants, it is particularly preferable to use Seriba orensis.

The liquid medium used for the tissue culture of Ranunculaceae plants of the present invention is the same as the liquid medium used for conventionally known plant tissue culture, but is made by adding a specific concentration of copper ions. A characterized liquid medium is used.

That is, when preparing the liquid culture medium of the present invention, it is necessary to set the concentration of copper ions to 0.2 μmol/l or more, and in particular, the concentration of copper ions must be set between 0.2 μmol/l and 50.0 μmol/l. It is preferable for the method of the present invention to adjust the amount to a range of mol/l. In the method of the present invention, as long as the total copper ion concentration is maintained at 0.2 μmol/1 or more, medium components other than copper ions in the liquid medium can be varied over a wide range of concentrations as necessary. can be used. Here, regarding the effects of copper ions on tissue culture of Ranunculaceae plants, 7.66 and 1. Kayutsu. When the concentration is less than 0.2 μmol/1, the cultured cell mass obtained by the tissue culture (hereinafter referred to as callus)
The production amount of inquinoline alkaloids such as berberine decreased, and the concentration of copper ions was reduced to 50.0 μmol/
Even if it is increased to /1 or more, the amount of the alkaloid produced is slightly reduced. From this, it is preferable that the concentration of copper ions in the liquid medium be within the above concentration range of the present invention.

The liquid medium used in the present invention has an inorganic component such as copper ions and a carbon source as essential components, and a medium containing at least one component selected from plant hormones, vitamins, and amino acids. Furthermore, other components may be used in combination as necessary.

Inorganic components of the liquid medium (c) In addition to copper, nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, iron,
Mention may be made of inorganic salts containing elements such as manganese, zinc, boron, molybdenum, chlorine, sodium, iodine and cobalt, specifically potassium nitrate, sodium nitrate, ammonium nitrate, monopotassium phosphate, disodium phosphate, chloride. Compounds such as potassium, calcium chloride, magnesium sulfate, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, copper sulfate, sodium molybdate, molybdenum trioxide, potassium iodide, zinc sulfate, boric acid, cobalt chloride, etc. can be exemplified.

Examples of carbon sources for the liquid medium include carbohydrates such as sucrose and their derivatives, M organic acids such as fatty acids, and primary alcohols such as ethanol.

The liquid medium also contains intoluacetic acid (I) as a plant hormone.
AA), naphthaleneacetic acid (NAA), P-chlorophenoxyisobutyric acid and 2,4-dichlorophenoxyacetic acid (
Examples include auxins such as 2,4-D) and cytokinins such as kinetin, zeatin and benzyladenine.

The vitamins in the liquid medium include biotin, thiamine (vitamin B1), pyridoxine (vitamin B-), pyridoxal, pyridoxamine, calcium pantothenate, ascorbic acid (vitamin C), inositol, nicotinic acid, nicotinamide, and riboflavin ( Vitamin B.

), etc.

Examples of amino acids in the liquid medium include glycine, alanine, glutamic acid, cis9ine, and lysine.

The liquid medium of the present invention usually contains about 0 of the inorganic components.
．． About 1 μmol/l to about 100 mmol/β, the carbon source is about 'Ig/1. to about 1OO&H, the plant hormones about 0.01 μmol/l to about 20 μmol/A, and the vitamins and amino acids about 0.1 μmol/b to about 150 my/β, respectively. used.

In the method of the present invention, the amount of inquinoline alkaloids such as berberine produced in callus is controlled by maintaining copper ions in the liquid medium within the above concentration range and adjusting the concentrations of the other components in the medium. It is possible to further increase the For example, the concentration of manganese ions 'rIU
By controlling the concentration to 0 μmol/l or less, the alkaloid can be cultured in tissue using the liquid medium. The tissue culture method in this case will be described in detail below.

First, the plant body of a plant belonging to the Ranunculaceae family, for example, the roots,
Tissue pieces collected from growing points, leaves, violets, fruits, seeds, etc., are subjected to, for example, New Plant Tissue Culture (Breakfast Shoten 1979 Edition), 2
Place the plate on Rinsmeyer Skoog's medium (RM-1965) hardened with agar described on page 1,
A part of the tissue piece is formed into a callus by culturing at 35° C. for about 7 to 60 days. When the thus obtained callus of the Ranunculaceae plant is subcultured by a commonly known method, the growth rate of the callus gradually increases.

Next, this callus is grown in a liquid medium suitable for propagation, such as the Linsmeyer-Skoog liquid medium (hereinafter referred to as liquid medium A) described in New Plant Tissue Culture (Breakfast Shoten 1979 edition), page 21. When the cultured cells are transferred to a certain type of culture medium and further propagated, the growth rate of the cultured cells is further increased and stabilized cultured cells can be obtained.

In the method of the present invention, the thus obtained stabilized cultured cells are added to the liquid medium of the present invention (hereinafter sometimes referred to as liquid medium B) and further culture is performed.

In the method of the present invention, when culturing the stabilized cultured cells in the liquid medium B, the initial concentration of the cultured cells can be varied within a wide range; About 1 to about 200 g of the cultured cells expressed as fresh weight for 11 of the liquid medium B of
It is desirable to add about 10 to about 40 g, preferably about 10 to about 40 g.

゛The culture temperature in the tissue culture of the present invention is usually about 10 to about 35°C, particularly preferably about 23 to about 28°C, and when the temperature is less than about 10°C, the growth rate of the cultured cells increases. is small, and when the temperature is raised to 55°C or higher, the growth rate of cultured cells similarly becomes small.

Although light is not necessarily necessary for performing the tissue culture of the present invention, irradiation with light does not interfere with the production of alkaloids such as berberine.

In the method of the present invention, after the culture is completed, the cultured cells are separated from the liquid medium B by a method such as decantation or passing, and then the target isoquinoline alkaloid such as berberine is extracted from the cultured cells in a conventional manner. It can be separated by a method such as extraction, which is applied to known natural products such as Oren and Oriental. The purity of the alkaloid thus obtained can be further increased by a method such as recrystallization, if necessary.

〔Effect of the invention〕

Since the method of the present invention uses a liquid medium, it is possible to perform mass culture using a tank or the like, and furthermore, the method of the present invention is industrially advantageous because the cultured cells can rapidly proliferate and alkaloids such as berberine can be reliably mass-produced. It's a method.

[Embodiments of the invention]

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Examples 1 to 4 A Linsmeyer-Skoog liquid medium (corresponding to the above-mentioned liquid medium A) whose tissue culture medium components have the composition shown in Table 1 was prepared using a solid medium solidified with agar (1 layer of agar 11) Ooptis sterilized with 2% antiformin solution or 70% ethanol solution, etc.
japonica Makinnvar, disse
cta Nakai) leaves were placed on a bed at 25°C.
A callus of Seriba aurifolia was obtained by static culture in a dark place. Next, under the same conditions as above, C. chinensis callus was planted every 14 days in a Linsmeyer-Skoog liquid medium (liquid medium A above), and cultured with rotation on a rotary shaker (amplitude 25 mm, 1100 rpm). As a result, the growth rate of the cultured cells of Seriba ouren was increased and stabilized cultured cells of Seriba ouren were obtained.

On the other hand, separately from this, in the liquid medium A, the concentration of copper ions = 0.5 μmol/β, 1. Oμmol/#, 5.0
Four types of liquid media (corresponding to the opaque liquid medium B with part J6) having the same composition as liquid medium A except that μmol/β, 1 U, and 0 μmol/6 were respectively prepared were prepared. Next, 20mA1 of these four types of liquid media,
The contents of each sample were placed in a 41,100 ml Erlenmeyer flask, added to each liquid medium that had been sterilized by holding at 120°C for 10 minutes, and incubated at 25°C for 1.4 days. Incubate with rotation on a low creep kneeker (amplitude 25 mm, 1tJOrpm) at 40°C.
After air-drying at night, its weight (dry lid) was measured, extracted using 2μ of culture medium per liquid medium, and quantitatively determined by comparing with a standard product using high-performance liquid chromatography.

The results are shown in Table 2.

Comparative Example 1 In the culture medium components of liquid medium B used in Examples 1 to 3, the total concentration of copper ions was set to 0.1 mol/l. The rest of the components listed are in the water surface. M indicates mol/l. Examples 5 to 7.
halictrum m1nus L, 'Var,
), and the copper ion concentration of liquid medium B of this example was 0.1 μmol/L0.5.
Mol/l.

1. Tissue culture was carried out in the same manner as in the example except that Qμmol/l was used to obtain a culture mixture containing cultured Japanese larch cells and a culture solution.

After culturing, the cultured Japanese larch cells were collected by p-filtration.

In addition, some berberine was released from the cultured cells and dissolved in the culture solution, but this berberine was separated and collected by passing the culture solution through an ion exchange resin (AmberliteXAD-2).

Claims (1)

[Claims] When tissue culturing Ranunculaceae plants using a liquid medium, the concentration of copper ions contained in the liquid medium is set to 0.2μ.
A method for culturing tissue of Ranunculaceae plants, characterized in that the concentration is mol/l or more.