JPH02142429A - Method for culturing foliar body of plant belonging to genus schizandra - Google Patents

Abstract

PURPOSE:To obtain useful components, such as lignans, having improving action on hepatopathy, antitumor action, etc., in a large amount at a low cost by bedding and culturing a shoot apical part, etc., of a plant belonging to the genus Schizandra on a culture medium containing a specific phytohormone, such as cytokinin. CONSTITUTION:A shoot apical or auxiliary bud part of a plant belonging to the genus Schizandra is preferably cultured to provide a part of a foliar body containing the apex or auxiliary bud. The resultant part is then bedded on a culture medium containing at least one phytohormone selected from 0.005-150ppm cytokinin, 0.01-100ppm gibberellin and 0.001-30ppm auxin and cultured to afford the objective foliar body.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention involves culturing shoot tips or axillary buds obtained from the Schizandra humic plant in a glass vessel, etc. to obtain useful components such as lignans useful as pharmaceuticals. The present invention relates to a method for mass-cultivating shoots containing a high percentage of.

[Prior art and assignments] Schizophrenia belonging to the Schizandra genus
zandra chinensis Ba111. ) has been a small bone I since ancient times! It is used in Chinese herbal medicine formulations such as hot water, ninjinyoeito, seishoekkito, and seiryuto. Furthermore, among the lignans that contain Schizophrenia, Schizandrin (s
Chizandrin A=D, deoxyschizandrin, goaisin A A+D 5F-H%J, prego+5isin, etc. have liver disorder-improving effects and anti-ulcer effects, and are useful substances as pharmaceuticals. It is good (known) to be possible.

However, despite the fact that Schizandra plants contain useful ingredients as mentioned above, in order to supply them as medicines, methods are exclusively used to extract and purify natural Schizandra plants. However, no industrial synthesis method for these lignans has been reported yet.

However, with this method, the lignans contained in the plant are required, and the content is unstable, it takes a long time to cultivate the plant, and the amount of cultivation is small. For these reasons, it is difficult to say that this method is sufficient for mass production.

Therefore, as a method for mass production, a method can be considered in which the thallus derived from the shoot apex or axillary bud of a Schizandra-causing plant is cultured to a large size, and lignans are extracted from the thallus. There have been no reports of success in mass cultivation of the shoots of Schizandra plants, which are naturally growing.

[Means for Solving the Problems] As a result of careful study on a method for mass-cultivating shoots containing a large amount of lignans, the present inventors used the stem apex or axillary bud of a Schizandra-causing plant as a raw material, and found that The present invention was completed based on the discovery that a large amount of shoots containing lignans and H at a high rate can be obtained by culturing in a medium containing a high concentration of plant hormones.

That is, in the present invention, the shoot apex or axillary bud of a Schizandra plant is treated with 0.005 to 150 ppm of cytokinin, 0.01 to 10 ppm of gibberellin, and 0 ppm of auxin.
A method for culturing a stem of a plant of the genus Schizandra, which comprises placing the stem in a medium containing at least one plant hormone selected from 0.001 to 3 oppm, and culturing it to obtain a stem of a plant of the genus Schizandra, and a stem of a plant of the genus Schizandra. The part containing the apical axillary bud of the shoot body obtained by culturing the apical part or axillary bud part was treated with 50 pp of cytokinin O,005 to I.
The Schizandra plant is placed in a medium containing at least two plant hormones selected from m, gibberellin 001 to 100 ppm and auxin 0.001 to 30 ppm, and cultured to obtain a foliage of a Schizandra plant. This is a method for culturing plant stems.

The present invention will be explained in detail below.

In the present invention, the term "stem body" refers to a tissue obtained by culturing the shoot apex or axillary bud and multiplying it with multiple buds.

Examples of the Schizandra-causing plant in the present invention include plants of the genus Schizandra of the family Elegantaceae, specifically Schisandra schizandra, Schisandra schizandra, Schizandra safflower, and the like.

The stem apex or axillary bud, which is the raw material from the Schizandra-causing plant, can be sterilized using commonly used sterilization methods (e.g., methods using ethanol aqueous solution, hypochlorous acid aqueous solution, sterilized water, etc.). ) and then removing the tops of the sterile buds.

In addition, seeds of Schizandra-causing plants are treated with gibberellin etc. to break dormancy, and after the same sterilization treatment as above, they are placed in a culture medium and grown under aerobic conditions at a temperature of 10 to 35°C, preferably 25°C. Seedlings obtained by culturing for 2 to 8 weeks at 0.degree. C. and germination can also be used as the shoot apex or axillary bud as a raw material.

The shoot apex or axillary bud thus obtained is cultured in a medium containing a certain concentration of plant hormones.

As a medium, a medium based on various known inorganic synthetic media to which sugars, vitamins, natural substances, etc. are added, or a solid medium obtained by adding agar, noelan gum, etc. to the medium and solidifying it can be used. .

Typical synthetic media include White's medium and Murashige & Skoog.
Skoog's medium (hereinafter referred to as MS medium), Linsmeyer-Skoog's (Linsa + Eier & S
Koog) medium, Nitsch medium, Etch Niche (Nitsch & N1Lsch) medium,
Gamborg et al.'s medium, W P M
(Woody Plant Medium), Heller's medium, and other mediums based on these mediums with modified compositions.

Specific examples of inorganic components of the inorganic synthetic medium include copper, nitrogen,
Phosphorus, potassium, calcium, magnesium, sulfur,
Iron, manganese, zinc, boron, molybdenum, chlorine, sodium, iodine, cobalt, etc. More specifically, potassium nitrate, sodium nitrate, calcium nitrate, ammonium nitrate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, sodium edylenoaminetetraacetate, Potassium chloride, calcium chloride, magnesium sulfate, sodium sulfate, ammonium sulfate, ferrous sulfate 1. Examples include ferric sulfate, manganese sulfate, zinc sulfate, boric acid, copper sulfate, sodium molybdate, molybdenum dioxide, potassium iodide, and cobalt chloride.

Specific examples of sugars include carbohydrates such as sucrose, inositol, and glucose, and derivatives thereof.

Specific examples of vitamins include biotin, diamine (vitamin B1), pyridoxine (vitamin B8), pantothenic acid, ascorbic acid (vitamin C), nicotinic acid, etc., and specific examples of amino acids include glycine, alanine, etc. , glutamine, protein, etc.

Specific examples of natural substances include casein hydrolyzate, coconut milk, yeast extract, etc. In addition to the above-mentioned specific examples, substances necessary for culture may be added as appropriate.

At least one plant hormone selected from cytokinin, gibberellin, and auxin is added to the medium containing the above components. Specific examples of cytokinin include kinetin, zeatin, dihydrozeatin, benzyladenine (BA), and isopentenyladenine. etc. are mentioned,
A specific example of gibberellin is gibberellin A3, and a specific example of auxin is indole acetic acid (IAA).
), α-naphthaleneacetic acid (NAA), 2,4-dichlorophenoquinacetic acid (2,4-D), indolebutyric acid (IBA), and the like.

The concentration of cytokinin is 0.005 to 150.0 pp
m, preferably 0.01 to IO, Oppm.

More preferably 0.03 to 3.0 ppm is appropriate.

The concentration of gibberellin is 0.01 to 100.0 ppm.

Preferably 0. O1-1.0 ppm, more preferably 0.01-0.1 ppm is suitable.

The 6 degrees of auxin is 0.001-30, Oppm.

Preferably 0.001-1. Oppms more preferably 0.001 to 0. ippm is suitable.

In addition, these plant hormones can be added in appropriate combinations, and cytokinin 0.01-100.0
ppm, preferably 0.03-3, 0 ppm and auxin 0.001-1, oppls preferably 0
,01-0, I ppta, and cytokinin 0.01-io, Oppm, preferably 0.03-3.0 ppm and gibberellin 0.01-1
．． 01ta1 preferably 0,03~0,lpp-
A combination of these is appropriate.

Cultivation is carried out under conditions typical for plant growth. That is, under aerobic conditions, at a temperature of 20 to 30°C, preferably 25°C, they are grown into stems in 3 to 8 weeks.

In order to culture a larger amount of stems, the tops of the stems obtained or the tissues containing the tops are cultured in the same manner as described above, and by repeating this process, more stems can be obtained.

Next, whether lignans are contained in the stems of plants of the genus Schizandra that can be obtained according to the present invention will be explained below by giving experimental examples.

Experimental Example 1 The H content per dry weight of the lignans contained in the phyllodes 19 obtained in Examples 1-14 described later was measured by high performance liquid chromatography using Gomisin A as an index.

The results are shown in Table 1 below.

As shown in Table 1, the content of gomisin A in the shoots per dry weight was high.

EXAMPLES Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereto.

Example 1 After peeling 200 seeds of Schisandra japonica, 100 p.
Dormancy was broken by treatment with pm gibberellin for 24 hours. Then, treated with 70% ethanol for 1 minute, and then treated with 70% ethanol for 3 minutes.
% sodium hypochlorite for 20 minutes to sterilize. The germination of the obtained sterile seeds was allowed to stand for 3 weeks in a culture chamber (25° C.), thereby obtaining 100 seedlings.

50 shoot tips obtained from these seedlings were placed on MS medium (3%
A medium containing 0.3 ppm of I'3A as cytokinin in a medium (containing nuclose, 0.2% gellan gum)
pH 5,6) at 25°C and 2,000 lux.
After culturing for several months, 100 shoots were obtained. The top part of the obtained shoot was collected, and 5 pieces of the nuchal part of the shoot were obtained by repeating subculturing for 1 month 5 times.
As a result of culturing for one month at 25° C., 3,000 lux, and l 00 rpm, the shoots grew elongated.

Example 2 Collected 200 wild buds of the wild Schistemrum, washed with water, treated with 70% ethanol for 30 seconds, and then treated with 70% ethanol for 30 seconds.
It was sterilized by treatment with sodium hypochlorite for 15 minutes.

The tops of the resulting sterile buds were taken out and added to MS medium (containing 3% sucrose) as cytokinins such as BAo and 3p.
Using a liquid medium (pl-15, 6) supplemented with pm,
By paper wick method using filter paper bed, 25℃, 2.0
00 Rutus for one month, and 200 shoots were obtained. 100 of the obtained shoots were grown at 25°C and 20.00 lux using MS medium (containing 3% sucrose and 0.2% gellan gum) containing 3 ppm of BAo as cytokinin (pH 5, 6). As a result of culturing for one month,
200 shoots were obtained. The tops of the resulting shoots were collected, and the tops of the shoots were further subcultured for 1 month 5 times, and the resulting 3 tops of the shoots were cultured using a liquid medium 60-, which was obtained by removing gellan gum from the medium listed above. As a result of culturing for one month at 25° C., 3,000 lux, and IOOrp, the shoots grew elongated (34 times).

Example 3 BA was used as cytokinin at 0. The process was carried out in the same manner as in Example 2 except that 3 ppm of O was added to obtain shoots with an elongation growth rate of 19 times.

Example 4 The same treatment as in Example 2 was performed except that O and l ppm of BA were added as cytokinin to obtain shoots with an elongation growth rate of 14 times.

Example 5 The same treatment as in Example 2 was performed except that 1.0 ppffi of BA was added as cytokinin, and the elongation growth rate was 30.
Twice as many shoots were obtained.

Example 6 The same treatment as in Example 2 was carried out except that 3.0 ppm of BA was added as cytokinin to obtain shoots with an elongation growth rate of 28 times.

Example 7 The same treatment as in Example 2 was carried out except that 0.3 ppm of kinetin was added as cytokinin to obtain shoots with an elongation growth rate of 11 times.

Example 8 Gibberellin was used instead of cytokinin at 0, i.p.
The process was carried out in the same manner as in Example 2 except that pm was added to obtain shoots with an elongation growth rate of 11 times.

Example 9 The same treatment as in Example 2 was performed except that 0 and 3 ppm of BA and 0 and I ppm of gibberellin were added as cytokinins to obtain shoots with an elongation growth rate of 20 times.

Example 1O The same treatment as in Example 2 was performed except that 0 and 3 ppm of BA were added as cytokinin, and 0 and 1 ppm of NAA were added as auxin to obtain shoots with an elongation growth rate of 30 times.

Example 11 Example 2 except for using white medium as the basic medium
By treating in the same manner as above, shoots with an elongation growth rate of 4 times were obtained.

Example 12 The same treatment as in Example 2 was performed except that a niche medium was used as the basic medium to obtain a shoot with an elongation growth rate of 8 times.

Example 13 The same treatment as in Example 2 was carried out except that WPM medium was used as the basic medium to obtain shoots with an elongation growth rate of 11 times.

Example 14 Except for using Gamborg B-5 medium as the basic medium,
The same treatment as in Example 2 was carried out to obtain shoots with an elongation growth rate of 18 times.

[Effects of the Invention] According to the present invention, it is possible to obtain a large amount of stems containing a high percentage of useful components such as lignans that can be used in pharmaceuticals at low cost.

Claims (2)

[Claims] (1) The stem apex or axillary bud of a Schizandra plant was treated with 0.005 to 150 ppm of cytokinin and 0.0 gibberellin.
1-100ppm and auxin 0.001-30p
1. A method for culturing a foliage of a plant of the genus Schizandra, which comprises placing it on a medium containing at least one plant hormone selected from pm and culturing it to obtain a thallus of a plant of the genus Schizandra. (2) Cytokinin-0
．． 005-150ppm, gibberellin 0.01-100
1. A method for culturing a foliage of a plant of the genus Schizandra, which comprises placing the foliage in a medium containing at least one plant hormone selected from 0.001 to 30 ppm of auxin and 0.001 to 30 ppm of auxin, and culturing the thallus of a plant of the genus Schizandra.