JPS63116691A - Tissue cultivation method for plant - Google Patents

Abstract

PURPOSE:To enhance productivity of secondary metabolites and efficiently produce an alkaloid, etc., by cultivating tissues of plants using a liquid culture medium containing an adsorbent and adsorbing the secondary metabolites of the above-mentioned plants released to the outside of cells onto the adsorbent. CONSTITUTION:Tissues of adventitious roots, etc., of a plant of the genus Duboisia in cultivated cells or cultivated tissues, e.g. roots, leaves, stems, seeds, flower buds, etc., are cultivated in a culture medium, containing preferably about 0.1muM-100mM inorganic components, about 1-100g/l carbon source, about 0.01-100muM phytohormones, e.g. auxin and cytokinins, etc., about 0.1-150mg/l vitamins and 0-100mg/l amino acids and added synthetic high polymer absorbent, e.g. Amberlite XAD-2, having giant network structure in an amount of 10-300g/l and prepared by using Nitsch.Nitsch, Linsmaier.Skoog or Murashige.Skoog culture medium to afford the aimed tropan based alkaloid, e.g. scopolamine, hyoscyamine, etc.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention aims to efficiently produce secondary metabolites by culturing am of a plant that produces secondary metabolites such as alkaloids using a specific medium. Concerning how to produce well.

[Conventional technology]

For example, scopolamine, which is one of the secondary metabolites, is used as an antispasmodic and analgesic, and hyoscyamine is used as a new parasympathetic nerve suppressant.These compounds are extracted from natural plants and manufactured. There is one. However, because they are made from natural materials, their production is dependent on the weather, and the harvest season is limited. Therefore, many studies have been conducted to produce these compounds by culturing plants [for example, Plant Cell Reports]
l-1101-103 (1982), etc.].

[Problem that the invention seeks to solve]

When aiming at industrial production of secondary metabolites by tissue culture, increasing productivity is an important issue. The present inventors believe that as a method to increase the productivity of secondary metabolites, if it is possible to efficiently release secondary metabolites produced inside the cells to the outside of the cells while culturing, it will be possible to increase the productivity of secondary metabolites compared to the conventional method. We considered this method to increase the productivity of metabolites and enable efficient cultivation.

[Summary of the invention]

As a result, the inventors discovered that the above object could be achieved by employing the following method and completed the present invention.

That is, according to the method of the present invention, plant tissues are cultured using a liquid medium containing an adsorbent, and the secondary metabolites of the plant released outside the cells are adsorbed to the adsorbent. A method for culturing plant tissue is provided.

[Detailed description of the invention]

As plants that can be used in the tissue culture of the present invention, the culture method of the present invention can be applied to any plant that can release secondary metabolites into cells. Whether the cell is capable of releasing secondary metabolites or not depends on the environment in which the cell is placed and the history of the cell.Specifically, the plant to which the method of the present invention is applied is Duboisia myoboroides (Duboisia m.
yoporoides), Zboisia Reichhardi (DuL+oisialeichhardi)
), plants of the genus Zboisia, such as Datura
a tal, ula), Datula arborea (Dat
, ura arborea), Datura stramonium (Datura sl, ramo+iium), Scobolia japonica (Scopolia japonica)
plants of the genus Scoboria such as A.
opa belladonna) and other plants of the genus Atropa;
onicaMakino), Seriba Ouren (C,,
1aponika Makin.

var, dussecta Nakai), Kikupaouren (C.

japonika Makino var, japon
ika), C. japonika
Makino var, ma, ior 5atake
), C. quinquefolia
Miq, ) and C. trifn.
plants of the genus Copteis such as lia 5alisb,), and Japanese larch (Thalictrum m1nus14.).
Examples thereof include plants of the genus Salictorum such as var hypolcucum Miq, ), plants of the genus Hydrastis, and plants of the genus Hydrastis.

In the present invention, a liquid medium containing an adsorbent is used to culture the plant tissue (including cells).

Specifically, the adsorbents include inorganic adsorbents such as activated carbon and activated alumina, Amberly 1 to XΔD-2 (a product of Rohm and Haas), Diaion HP-10 (a product of Mitsubishi Chemical), and Revaded QC1031 (a product of Rohm and Haas). Synthetic polymer adsorbents with a giant network structure such as Bayer's product), Amberlite IRC-50 (Rohm &Haas' product), Diaion WKIO (Mitsubishi Chemical's product), Revachit GNP (Bayer's product), etc. Examples include ion exchange resins. Among these, synthetic polymer absorbents with a giant network structure are preferred, and among them, Amberlite χΔ
D-2 is preferred. The ratio of the adsorbent used is usually 1 to 500 g, preferably 10 to 300 g, per 1β of the liquid medium, and culture is performed.

The liquid medium used in the present invention is a medium containing inorganic components and carbon sources as essential components, to which are added plant hormones and vitamins, and further added with amino acids as necessary.

Inorganic components include nitrogen, phosphorus, potassium, sodium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, molybdenum, chlorine, iodine, and cobal.
Examples include inorganic salts containing elements such as potassium nitrate, sodium nitrate, ammonium nitrate,
Ammonium chloride, potassium chloride, calcium chloride, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, magnesium chloride, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, copper sulfate, sodium molybdate, Examples include compounds such as molybdenum trioxide, 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.

Examples of plant hormones in the medium include naphthalene acetic acid (NAA), indole acetic acid (IAA), p-
Examples include auxins such as chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), indolebutyric acid (IBA), and derivatives thereof, and zytokinins such as benzyladenine (BA), kinetin, and zeatin. . In the present invention, it is generally desirable not to add cytokinins to the culture medium, but if they are added as necessary, the concentration of cytokinins is usually 10-''M (
It is preferable to use it at a low concentration of ( ), 02 mg/#') or less.

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 ribobravin (vitamin B2).

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

The medium of the present invention usually contains about 0.1 of the inorganic components.
! M to about 100 mM, the carbon source about 1 g/Il to about 100 g/β, and the plant hormones about 0.01
! M to about 100μM, and about 0.1M of the above vitamins.
g/7! 50 mg/R and O to about 100 mg/6 of the amino group.

Specifically, the medium used for the tissue culture of plants of the present invention may be a conventionally known culture medium used for tissue culture of plants, such as Murashige Skoog ('62
) CMurashige & Skoog) medium, Linsmeyer-Skoog (RM-1,965) (
Linsmaier &Skoog's medium, White's medium, Gamborg's B-5 medium, Mitsui's To9 medium,
An example is a medium prepared by adding the carbon source and plant hormones described above to a niche/niche (Nj, tsch old 1, sch) medium, etc., and further adding the vitamins and amino acids described above as necessary. However, in the present invention, among these, a medium prepared using an H.H.H., Linsmeyer-Skoog, or Murashige-Skoog medium is particularly preferred. The composition of the above-mentioned conventionally known culture medium is described, for example, in "New Plant Tissue Culture JP 386-P39, Asakura Shoten, 979," written by Yakuin, Nakajima, and Furuya.

The medium that can be used in the present invention is a liquid medium.

In the tissue culture of the present invention, a tissue piece of a plant that metabolizes a secondary metabolite is tissue cultured using the above medium to obtain cultured cells or cultured tissue containing the secondary metabolite.

In the present invention, when tissue of a plant of the genus Zboisia is used as the cultured tissue, adventitious roots are particularly preferred.

Specifically, the Mi tissue of the plant used in the tissue culture of the present invention includes roots, leaves, stems, seeds, flower buds, etc. of the plant, as well as tissue culture according to the present invention or other conventional tissues.
Examples include cultured cells or tissue of the plant obtained by the f5 cultivation method. In the present invention, in the case of plants belonging to the genus Zboisia, it is particularly preferable to use adventitious roots obtained by previously tissue culturing plant Jiff tissue and culture them in KJI culture using the medium according to the present invention.
In this case, the raw material adventitious roots are propagated and cultured using the medium of the present invention to obtain adventitious roots containing large amounts of tropane alkaloids such as scopolamine and hyoscyamine as secondary metabolites.

Specifically, the secondary metabolites according to the present invention include various alkaloids such as tropane alkaloids (scopolamine, hyoscyamine, etc.), isoquinoline alkaloids (berberine, etc.), pigments such as shikonin, and various glycosides such as arbutin. For example, it is a metabolic product of plants.

In the present invention, as a method for separating secondary metabolites from cultured cells containing secondary metabolites, for example, conventional methods described in the Pharmacy Act and the like can be employed.

〔Effect of the invention〕

According to the method of tissue culture in which an adsorbent is present in a liquid medium of the present invention, secondary metabolites released to the outside of the cells are adsorbed by the adsorbent, thereby lowering the concentration of secondary metabolites in the liquid medium. The amount of secondary metabolites released outside the cells increases compared to when culturing is performed without an adsorbent, so the amount of secondary metabolites produced increases and alkaloids are efficiently produced compared to conventional methods. etc. can be produced.

〔Example〕

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

Example 1 2Qm of liquid medium of Nirachae in a 100cc Erlenmeyer flask! After 2 weeks of culture, Amberly I-XΔD-2 (Rho 1, a product of And Haas Co., Ltd.), a commercially available synthetic resin adsorbent, was added. 2. (After adding 1 g of the culture solution and culturing for another 3 weeks, Amberly 1-XAD-2 was collected by filtration. Next, this Amberlyte XAD-2 was packed into a column, and 100 mff was The alkaloids scopolamine and atrobin adsorbed on Amberlite XAD-2 are desorbed and eluted by flowing methanol to form an eluate (a
It was recovered as l. On the other hand, after crushing the cultured adventitious roots obtained by filtering the culture mixture, they were mixed with 170 polm of basic glue.
Extracted with methanol solution. IN sulfuric acid was added to this to transfer the alkaloids to the sulfuric acid layer, and aqueous ammonia and chloroform were added to transfer the alkaloids to the chloroform layer, which was then concentrated under reduced pressure to obtain a cultured adventitious root extract (bl).The alkaloids were quantified using 5ilicone [1V- 17 (1%)
Culture solution (C) obtained by filtering and removing the previous eluate (al, cultured adventitious root extract (bl, and cultured adventitious roots and amber rye) from the culture mixture) by gas chromatography using an onChromosorb W column. The amount of alkaloids in adventitious roots was determined by quantifying the amount of scopolamine and atrobin contained in this.
Among the alkaloids released to the outside of the cells, the amount of alkaloids adsorbed to the adsorbent and the amount of unadsorbed alkaloids were determined.

The results are shown in Table 1.

Examples 2 to 3 Results carried out in the same manner as in Example 1 except that the adsorbent was changed from Amberly I-XAD-2 to Levachitsu) CNP (Example 2) or activated carbon (Example 3). are shown in Table 1.

Comparative Example 1 Zboisia adventitious roots were cultured in the same manner as in Example 1, except that the culture was carried out without adding the adsorbents Amberly 1 to XAD-2.

In other words, when seeds of Zboisia adventitious roots were cultured under the same conditions for 5 weeks after transplanting, the amounts of atlobin and scopolamine released into the medium were small as shown in Table 1, and they remained in the adventitious roots without being released. The total amount of alkaloids produced by the culture, including alkaloids, was 932 μg for scopolamine and 2 μg for athrobin.
It was 64 μg, which was lower than Examples 1 to 3.

Japanese Patent Application Publication No. 111'163-116691 (5) Corridor

Claims (1)

[Claims] (1) Plant tissues are cultured using a liquid medium containing an adsorbent, and the secondary metabolites of the plant released outside the cells are adsorbed to the adsorbent. Tissue culture methods.