JPS6232880A - Tissue culture of ranunculaceae family plant - Google Patents

Abstract

PURPOSE:To enable the mass production of isoquinoline-type alkaloid such as berberine in high efficiency, by the tissue culture of a plant of Ranunculacear family in a medium containing gibberellin. CONSTITUTION:The tissue or cell of a plant of Ranunculaceae family, e.g. Coptis japonica, Thalictrum thunbergii, etc., is cultured in a medium containing gibberellin at a concentration of usually >=10<-9>mol/l, preferably 10<-8>-10<-3>mol/l.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a tissue culture method for Ranunculaceae plants.

[Conventional technology]

BACKGROUND OF THE INVENTION The rhizomes of plants of the Ranunculaceae family, for example, Orientalis, contain isoquinoline alkaloids such as berberine, and these alkaloids are used, for example, as stomachic medicines and dyes, and are in great demand.

However, the method of directly collecting isoquinoline alkaloids such as berberi and nigra from naturally grown Ranunculaceae plants is difficult because the growth of the Ranunculaceae plants is affected by the natural environment and weather, and the collection of the plants is also time consuming. It is not an advantageous method as it is time consuming. Therefore, as an alternative method, for example,
1 (1981), and Phytochemistry Vol. 04, 1209-1.
As described on page 210 (1975), several tissue culture methods for Ranunculaceae plants have been proposed. However, even in these conventionally known tissue culture methods,
This method has a drawback in that the yield of the target isoquinoline alkaloid produced from the cultured spores is low.

[Problem that the invention seeks to solve]

Based on this background, the present inventors have conducted intensive studies on a method for tissue culturing Ranunculaceae plants to more efficiently produce isoquinoline alkaloids such as berberine than conventional methods, and have adopted the following method. Contains a lot of inquinoline alkaloids such as berberine.
We have discovered that this is possible, and have completed the present invention.

[Means for solving problems]

That is, according to the present invention, there is provided a tissue culture method for Ranunculaceae plants, which is characterized in that a medium containing dihererin is used for culturing tissues or cells of Ranunculaceae plants.

Examples of the Ranunculaceae plant used in the method of the present invention include Coptis japonicus
aMakino), Serihaouren (C, japon)
ica Makin.

var, dussecta Nakai), Gikuba ouren (C.

japonica Makino var, japon
ica ), C. japonica
Makino var, major 5atake
), C. quinquefolia
Miq, ) and Mitsuhaoren (C, trifol
ia 5alisb, ), and plants of the genus Copteis, such as L.
Examples include plants of the genus Thalictorum, plants of the genus Xanthorrhiza, and plants of the genus Hydrastis, such as Arhypoleucum Miq, ). In the present invention, among these plants, it is particularly preferable to use Seriba orensis or Japanese larch.

As the medium used for the tissue culture of the Ranunculaceae plant of the present invention, a medium characterized by containing a specific concentration of gibberellin among the conventionally known medium used for tissue culture of plants is used. be done. That is,
The medium used in the method of the present invention is a medium containing gibberellin usually at least 10 mol/E, preferably from 10 mol/1 to 10 mol/l. In the present invention, as long as the gibberellin concentration is maintained within the above range, other medium components other than gibberellin in the medium can be used by varying the concentration within the Hiroma concentration range as necessary.

Gibberellin, which is used as an essential component of the culture medium in the tissue culture of the present invention, is a general term for plant hormones having a diban nucleus represented by formula 11. Up to now, about 50 types of gibberellin [these are usually GAn ( n = an integer of 1 to 50)] However, in the present invention, any of these various gibberellins can be used.In the present invention, among these gibberellins, in particular, gibberellins expressed by the formula (2) It is preferable to use gibberellin A3 (GA3), which increases the content of isoquinoline alkaloids such as berberine.

The medium used in the present invention has gibberellin, an inorganic component, and a carbon source as essential components, and is a medium to which at least one component selected from plant hormones, vitamins, and amino acids other than gibberellin is added. Furthermore, other components may also be used in combination, if necessary.

Inorganic components of the medium include inorganic salts containing elements such as nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, copper, molybdenum, chlorine, sodium, iodine and cobalt. Specifically, potassium nitrate, sodium nitrate, ammonium nitrate, potassium monohydrogen phosphate, sodium dihydrogen phosphate, potassium chloride, calcium chloride, ammonium chloride, magnesium sulfate, sodium sulfate, ferrous sulfate, ferric sulfate, Examples include compounds such as manganese sulfate, copper sulfate, sodium molybdate, molybdenum dioxide, potassium iodide, zinc sulfate, boric acid, and cobalt chloride.

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

The plant hormone in the medium includes intole@acid (IAA
), naphthaleneacetic acid (NAA), P-chlorophenoxyisobutyric acid and 2,4-dichlorophenoxyacetic acid (2
, 4-D), and cytokinins such as kinetin, zeatin, and benzyladenine.

The vitamins in the medium include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pyridoxal, pyridoxamine, calcium pantothenate,
Examples include ascorbic acid (vitamin C), inositol, nicotinic acid, nicotinamide, and riboflavin (vitamin B2).

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

The medium of the present invention usually contains about 0.1 of the inorganic components.
μmol/ρ to about 100 mmol/l, and about 1 g/l to about 100 g/l of the carbon source! , about 0.01 μmol/l to about 20 μmol/l of the plant hormones other than gibberellin, and about 0.1 mg/j of each of the vitamins and amino acids! or about 150m
It is used containing about g/A.

The medium that can be used in the present invention is a liquid medium or a solid medium containing usually 0.5 to 1% agar, but it is preferable to use a liquid medium in the present invention.

In the method of the present invention, it is possible to further increase the amount of isoquinoline alkaloids such as berberine produced in the medium while maintaining the concentration of gibberellin in the medium within the above range. For example, the present applicant
If the method proposed in No. 27467, in which the concentration of ions in the medium is increased to 0.2 mol/β or more, is applied to the method of the present invention as necessary, the amount of the alkaloid produced can be increased. Therefore, it is preferable.

Specifically, the medium used for tissue culture of a Ranunculaceae plant in the present invention includes a culture medium conventionally used for tissue culture of a predatory plant, for example, Murashige Skoog ('62) [: Murashige & Sk
oog) medium, Linsmeyer-Skoog (RM
1965) CLinsmaier & Skoog)
medium, White ('63)
medium, Gamborg's B-5 medium, three pieces of M-9 medium, Etch Niche's medium (N1ts
For example, a medium prepared by adding a medium such as Niche-Hetch, Linsmeyer-Skoog, or Murashige-Skoog is particularly preferred in the present invention. Regarding the composition of the above-mentioned conventionally known culture medium, for example,
[New Plant Tissue Culture JP P386~] by Nakajima and Furuya
P391, Asakura Shoten, 1979.

In the method of the present invention, Ranunculaceae plants are tissue cultured using the medium. The tissue culture method in this case will be described in detail below. First, tissue pieces collected from the plant body of a plant belonging to the Ranunculaceae family, such as roots, growing points, leaves, violets, fruits, seeds, etc., are placed in, for example, New Plant Tissue Culture # (Asakura Shoten 1979 edition), 10-35. A part of the tissue piece is formed into a callus by culturing at °C for about 7 to 30 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 New Plant Tissue Culture (Asakura Shoten 1).
When the callus is transferred to a liquid medium (medium A) of Linsmeyer Skoog (medium A) described in 1979 edition), p. 21, and further grown, the growth rate of callus is further increased and stable callus can be obtained. In the method of the present invention, the stabilized callus thus obtained is added to the medium of the present invention (liquid medium B) and further culture is performed.

In the method of the present invention, when culturing the stabilized callus in the medium B, the initial concentration of the callus can be varied within a wide range; It is desirable to add about 1 to about 200 g of the callus, preferably about 10 to about 40 g, based on the fresh weight of the callus.

The culture temperature in the tissue culture of the present invention is usually as follows:
About 10 to about 35°C, particularly about 23 to about 28°C, is preferred; when the temperature is less than about 10°C, the growth rate of callus is low, and when the temperature is above 35°C, the growth rate of callus is also low. The callus growth rate decreases. Although light is not necessarily required for 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 completion of culture, the callus is separated from the medium B by a method such as decantation or filtration, and then the desired isoquinoline alkaloid such as berberine is extracted from the callus using a conventionally known natural product. It can be separated by methods such as extraction, which are applied to Aurena and Aurena. The purity of the alkaloid thus obtained can be further increased by a method such as recrystallization, if necessary.

Since the method of the present invention can also use a liquid medium, it is possible to perform mass culture using a tank or the like, and furthermore, the callus can be rapidly multiplied, and alkaloids such as berherine can be reliably mass-produced. This is an industrially advantageous method.

〔Effect of the invention〕

By employing the method of the present invention, isoquinoline alkaloids such as berberine can be produced efficiently in large quantities compared to conventional methods.

〔Example〕

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

Examples 1 to 7 A Linsmeyer-Skoog liquid medium whose tissue culture medium components have the composition shown in Table 1 was added to a solid medium (1% by weight of agar) solidified with agar and preliminarily mixed with 2% antiformin solution or Coptis japonica Maki sterilized with 70% ethanol solution etc.
A part of the leaves of ``no var, dissecta Nakai'' was placed on a bed and cultured stationary in the dark at 25°C to obtain each callus. These calli were then cultured in a Linsmeyer-Skoog liquid medium under the same conditions as above.
The callus was transplanted every 14 days and then cultured in a rotational manner on a rotary shaker (amplitude 25 degrees, 1100 rp) to accelerate the growth rate of the callus and obtain stabilized calli.

Separately, 20mZ of the above liquid medium was placed in separate Erlenmeyer flasks with an internal volume of room/room, and after sterilizing the flasks by holding them at 120"C for 10 minutes, they were filtered through a membrane filter. A fixed amount of gibberellin A3, which had been sterilized using
1.10 mol/l, 10-' mol/1107 l/l
and 10 mol/l.

Next, 0.20 g of the previously obtained fresh and stabilized Seriba callus with increased growth rate was added to each liquid medium, and the mixture was cultured with rotation on a rotary shaker at 25°C for 14 days (amplitude 25 mm). , 1100rp
) did.

The callus after culture was collected by filtration, air-dried at 40°C for one day and night, and its weight (dry weight) was measured.1. The weight of the cultured cells grown per 11 parts of the liquid medium was determined.

I11 of alkaloids such as berberine was determined by extracting the obtained dry callus using methanol or the like, and comparing it with a standard product using high performance liquid chromatography.

The results are shown in Table 0.

Comparative Example 1 In the medium components of the liquid medium used in Examples 1 to 7,
Examples 1 to 7, except that gibberellin is not included.
I did it in the same way.

The results are shown in Table 2.

Claims (1)

[Scope of Claims] A method for culturing a tissue of a Ranunculaceae plant, which comprises using a medium containing gibberellin in culturing tissues or cells of a Ranunculaceae plant.