JPH01317397A - Production of phytoecdysteroid, induced root and callus containing phytoecdysteroid - Google Patents

Abstract

PURPOSE:To obtain the present compound useful for controlling harmful organisms and regulating growth of useful organisms industrially and advantageously by infecting a phytoecdysteroid-containing plant with a bacterium belonging to the genus Agrobacterium, subjecting the plant to tissue culture and extracting a product from the culture mixture. CONSTITUTION:A leaf of a phytoecdysteroid-containing plant such as Ajuga reptans var. atropurpurea cultivated in a greenhouse is cut, immersed in a culture solution of Agrobaccterium rhizogenes MAFF-03-01724 strain) of the genus Agrobaccterium for 5 minutes, infected with the bacterium, placed on agar, cultivated at 25 deg.C under illumination for 3 days, an induced root is treated by ultrasonic wave in methanol for 2 hours to give the aimed phytoecdysteroid.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention provides a method for producing phytoecdysteroids useful for controlling pests and controlling the growth of useful organisms, and a method for producing phytoecdysteroids containing induced roots and calluses containing phytoecdysteroids. Regarding.

[Prior Art and Problems to be Solved by the Invention] Phytoecdysteroids are insect molting metamorphosis hormones, are contained in all arthropods, and are physiologically active substances that control molting metamorphosis. Therefore, phytoecdysteroids can be used as insecticides by inducing excessive molting, abnormal metamorphosis, or inhibition of molting by insect pests, by inducing silk-spinning by administering to silkworms, and by inducing silk-spinning by administering phytoecdysteroids to crustaceans such as shrimp and crabs. In addition to being used as a drug to control shedding in the marine aquaculture industry, it is also expected to be used as an analgesic for neuralgia and rheumatism. In these fields of application, large amounts of phytoecdysteroids are required.

Conventionally, as a method for producing phytoecdysteroids typified by β-ecdysone, there has been known a method of extracting β-ecdysone from insects such as silkworms (Bonbyxlori). However, insect β
-Since the ecdysone content is extremely small, only a very small amount of β-ecdysone can be extracted from a large amount of insects. On the other hand, since 1968, β-ecdysone contained in insects and F-dysteroids having similar structures and activities have been discovered one after another in plants. These are collectively called phytoecdysteroids. The phytoecdysteroid content in these plants is high compared to that in insects and is now exclusively found in let, e.g.
n vulgare). However, since many of the plants containing phytoecdysteroids are ferns, woody weeds, or plants from tropical regions, it is difficult to collect or cultivate the required amount domestically and use them as extraction materials for phytoecdysteroids. be.

In addition, not only is the appropriate cultivation method unclear, but it is also difficult to constantly supply a constant amount because it is greatly influenced by natural conditions such as soil, soil quality, disease damage, and insect damage, or because it grows slowly. Furthermore, there is also the problem that the phytoecdysteroid content is not constant, resulting in variations in quality. Therefore, the above method is not suitable for mass production of phytoecdysteroids.

On the other hand, attempts have been made to mass-produce plant raw materials and useful substances of a certain quality by applying plant tissue culture technology.For example, cell masses (callus) obtained by dedifferentiating phytoecdysteroid-containing plants A method for culturing and extracting phytoecdysteroids has been reported. However, Inokozuchi (Achyranth)
eS) genus Shinatainokozuchi (eight, fauriei)
The content of phytoecdysteroids is 0.002% or less based on fresh weight [Chemical and Pharmaceutical Balletin (Cheiic
al and Pharmaceutical Bul
letin L 19.438-439. (1971
), and Pharmaceutical Journal, 95. (5), 581-
589. (1975)]. In this way, even if induced roots and callus are induced using plant tissue culture technology, the content of phytoecdysteroids is extremely small compared to the source plant, and it is difficult to efficiently and abundantly produce phytoecdysteroids. difficult to obtain. Furthermore, these methods require plant hormones to induce induced roots and callus.

Therefore, an object of the present invention is to obtain cultured cells with a high content of phytoecdysteroids and to produce phytoecdysteroids by the cells, thereby producing phytoecdysteroids of constant quality without being influenced by natural conditions. The objective is to establish a method for producing phytoecdysteroids that provides a stable supply of steroids.

An object of the present invention is to provide a method for producing phytoecdysteroids that can efficiently obtain large amounts of phytoecdysteroids by tissue culture.

Still another object of the present invention is a method for producing phytoecdysteroids that can proliferate cells with a high content of phytoecdysteroids and efficiently obtain a large amount of phytoecdysteroids even in the absence of 4a hormones. Our goal is to provide the following.

A further object of the present invention is to provide phytoecdysteroid-containing induced roots and callus that are highly capable of producing phytoecdysteroids.

[Structure of the Invention] As a result of repeated research aimed at solving the above problems, the present inventors have found that plants containing phytoecdysteroids are infected with bacteria of the genus Agrobacterium, and that certain plants are infected with Agrobacterium spp. It has been found that cultures containing large amounts of phytoecdysteroids can be obtained by tissue culture without infection with bacteria of the genus. That is,
The present invention involves infecting a phytoecdysteroid-containing plant with bacteria of the genus Agrobacterium and culturing the tissue, extracting phytoecdysteroids from the resulting culture, or culturing a plant of the genus Vitex and culturing the resulting culture. The above problem is solved by a method for producing phytoecdysteroids that extracts phytoecdysteroids from substances. In addition, as each of the above-mentioned cultures, a culture of induced roots, a callus, or a plant regenerated from the same is preferable.

Furthermore, the present invention solves the above problems by using phytoecdysteroid-containing induced roots and callus obtained by infecting a phytoecdysteroid-containing plant with bacteria of the genus Agrobacterium.

In the present invention, a plant containing a phytoecdysteroid represented by β-ecdysone, which is an insect molting hormone, is used. As a phytoecdysteroid, β-
In addition to ecdysone, nearly 50 other types are known, including α-ecdysone, cidasterone, bonasterone A-C, inocosterone, butyrosterone, adjugasterone, maxisterone, thiasterone, and cengosterone.

Plants containing these phytoecdysteroids include Os11unda, such as Os11unda, Blec japonica, etc.
For example, Shishigashira (B, n1ponic)
Chei 1anthes, such as Chei argentea (C, argentea) and Chei 1anthes (Chei 1anthes).
C, brandtii), etc.; the genus Ivy is ennaph
For example, 70 ivy (L, n1c
rophyllus);
), for example, Podocarpus, the + ypteris), etc.: Podocarpus, for example, dog? 4(
P. 11acrophyllui) and Nagi (P. n.
agi) etc.; Polypodiun such as P. veitchii; Pteridiun such as Bracken bracken (P.
aquilinulm) and Oshagujidenda (p, j
aponicun), etc.: of the genus Taxus, such as T. cusprtiata; of the genus Serratu la, such as S.
, coronata) etc.: Dacrydium sp.
diuI), such as Dacridium intermedium (
C, 1nH3rlediul) etc.;
For example, a) Abta velutina (^, velutin)
a) etc.; Sesuvium fsesubiun) such as Sesbium porcellacastrum (S, portul
acastrun) etc.; Baffia spp. (ptarria)
For example, Brazilian ginseng (P, 1resinoid
es), etc. ;; Inocyranthes
Examples include A. japonica and A. reptans (A. fauriel et al.; Juga CI').
var, atropurpurea), atropurpurea (
8, decumbens) and A. decunbens (A, p.
yHaea) etc.: members of the genus Vitex (ν1tex), such as Vitex serena (ν, 5ereti), Vitex lemanni (ν, rehlanni),
Vitex madiensis (V, Iadiens)
is), vitex thyrsifofV, thyr
siflora), Vitex gelabrata (V,
glabrata), Vitex micranta (V
.

1icrantha), Vitex doniana (V,
doniana), Vitex strickeri (V
, 5trickeri), Vitex mombasae (V, nombasae), Vitex-boo V
buchananii (V, buchananii), Vitex ferruginea (V, ferruginea), etc.
There are over 250 species in 33 families [Journal of Scientific and Industrial
Research (Journal of 5cientifi)
c and 1industrial research
), 38.632-647 (1979)].

Any of the above phytoecdysteroid-containing glues can be used.

Among the above-mentioned phytoecdysteroid-containing plants, plants of the genus Vitex produce large amounts of phytoecdysteroids by tissue culture without being infected with bacteria of the genus Agrobacterium.

When not infected with bacteria of the genus Agrobacterium, the culture produced by tissue culture of plants of the genus Vitex may be induced roots, callus, or N objects regenerated from these, but callus is preferable. Plants, especially Vitex strigeri, Vitex
Doniana, Vitex madiensis, Vitex micranta, etc. contain large amounts of phytoecdysteroids. In particular, Vitex strickeri is a high-content plant containing about 5% by weight of β-ecdysone on a dry weight basis. These plants of the genus Vitex are
It is a woody herb that grows wild in eastern Africa, and its fruit is
It is also used medicinally in Africa and other countries.

The organ to be subjected to tissue culture of the phytoecdysteroid-containing plant may be any of leaves, stems, roots, etc. Particularly from the viewpoint of surface sterilization, leaves and stems are preferable, and the organs of the Vitex genus plants are treated with disinfectants such as 1 to 10% by weight sodium hypochlorite solution, mercury chloride, etc. After soaking in liquid 8 or bleaching powder solution, wash thoroughly with sterilized water and cut into appropriate sizes.

For leaves, for example, about 1■×1α, for stems, for example, ICl11
It is preferable to cut it to a certain extent.

In the case of plants belonging to the genus Vitex, the above-mentioned sections may be tissue cultured as they are, but in order to efficiently produce phytoecdysteroids, it is preferable to infect them with bacteria belonging to the genus Agrobacterium.

As the microorganism used in the present invention, any strain of Agrobacterium that produces a culture with a high content of phytoecdysteroids can be used. Such bacteria of the genus Agrobacterium include Agrobacterium tumefaciens (A, tulefaciens).
) and Agrobacterium rhizogenes (A, rhiz
ogenes) is preferred. Among these bacteria, the former has a large plasmid called Ti plasmid and the latter has a large plasmid called Ri plasmid, and a part of it has T-DN.
Transformation occurs when A is integrated into the plant chromosome. At that time, since the T-DNA contains genes involved in the synthesis of auxins and cytokinins,
The transformed plant cells can grow on a plant hormone-free medium and have a high ability to produce phytoecdysteroids.

Among the strains of bacteria belonging to the genus Agrobacterium, the strain of Agrobacterium tumefacinus is C58,737.
Strains with tuberin-type Ti plasmids such as A6, B
Strains with octobin-type Ti plasmids such as 6.15955 and Ac7h5, and agrobin-type Ti plasmids such as Bo342.
Examples include strains having Grasmid. In addition, Agrobacterium rhizogenes strains include Agrobacterium rhizogenes ATCC15834 and A4.819.
6, NCPPB2659, MAFF-03-01,
724, MAFF-03-01725, MAFF-03
Examples include strains having Ri plasmids such as agrobin type, mannopine type, and cucumopine type, such as -01726 and MAFF-03-01727. These Agrobacterium bacteria are generally difficult to control, many of them are foreign strains, and moreover, they are harmful plant pathogens that cause serious damage to agriculture. Therefore, when used in Japan, permission to import harmful microorganisms is required based on the Plant Protection Law, and the use of harmful microorganisms must be handled with sufficient care to avoid dispersal inside and outside of licensed experimental facilities. It is subject to various restrictions.

On the other hand, Agrobacterium rhizogenes M AFF
03-01724, MAFFO3-01725, MAF
FO301726, MAFFO3-01727, etc. are strains called melon hair root disease fungi discovered in Japan (Shiomi et al., Journal of the Japanese Society of Plant Pathology, 454-45).
9. (1987) ), there are no restrictions on its use and it is preferable from the viewpoint of plant protection. Although the melon hairy root disease fungus has a plasmid that induces roots, it is also possible to use a melon hairy root disease fungus that has an artificially modified plasmid. At least one type of the above Agrobacterium bacteria can be used.

The bacterial suspension of Agrobacterium bacteria is prepared using a conventional culture medium such as potato dextrose medium, Nutriendo broth medium, Luria
It can be prepared by culturing in broth medium, YEB medium, AB medium, YM medium, etc. Among the above-mentioned media, Nutriendo Broth medium, Luria Broth medium, etc. are preferred; solid media and liquid media can be used as the medium. More specifically, the bacterial solution is prepared by subculturing Agrobacterium bacteria at predetermined intervals, such as once every month, on a Luria broth medium solidified with 1.5% by weight agar, and then subculturing the bacteria in an appropriate amount. Liquid medium, e.g. 20ml
It can be prepared by transplanting the cells into a liquid Luria broth medium and culturing them under appropriate conditions, for example, overnight at a temperature of 30°C.

The method of infecting plants belonging to the genus Vitex with these bacteria is not particularly limited as long as the T-DNA of the bacteria is integrated into the plant chromosome for transformation. A simple inoculation method is, for example, by puncturing a needle with bacteria attached to a seedling that has sprouted from a seed of a phytoecdysteroid-containing plant, or a stem of a seedling obtained by cutting off the shoot tip and planting it on a medium. Plant inoculation methods, such as applying a fungal solution to the stem when damaging it, applying a fungal solution to the cut surface of the stem, and soaking cut pieces of leaves and stems in the fungus solution; Leaf disk method Examples include a method of co-cultivating bacteria and suspension cells, callus or protoplasts of plants containing phytoecdysteroids. There is also a method in which protoplasts from plants containing phytoecdysteroids and spheroplasts from Agrobacterium genus bacteria are mixed under fusion conditions, and protoplasts are mixed with plasmid T from Agrobacterium in a medium containing polyethylene glycol and calcium ions. - Cells transformed with Agrobacterium bacteria may be transformed by mixing with DNA, etc. As mentioned above, cells of the genus Agrobacterium do not require plant hormones during proliferation, so they can be transformed without hormones. It can be cultured in a medium and only those that have grown can be transplanted and subcultured. In this case, as the six antibiotics that are preferably added to eradicate bacteria, various antibiotics such as carbenicillin, vancomycin, and cefotaxime can be used singly or in combination of two or more. The antibiotic is added at an appropriate concentration, e.g.
A concentration of about 0 μg/Tl1 is sufficient.

In addition, whether or not a phytoecdysteroid-containing plant is infected and transformed by Agrobacterium bacteria depends on the non-protein amino acids specifically synthesized by plant cells transformed with Ti, Ri plasmid T-DNA. This can be done by detecting opine, which is a generic term for. That is, a part of the culture is sonicated or triturated,
Detection can be performed by spotting the centrifuged supernatant onto a piece of paper, performing RIIF; electrophoresis, and then coloring with a coloring reagent.

The culture medium used for induced roots from cells transformed with Agrobacterium bacteria and for callus induction and culture is used for plant tissue culture and contains inorganic components,
Various media containing organic components etc. can be used. For example, the commonly used Murashige Skoog
e-3koog, 83) medium, Linsnaier-3koog (13) medium, White 1 old te, W) medium, Heller,
[1) Medium, Niche (Nitsch-Nits)
ch, NN) medium, Gamborg B5 (B5) medium, etc., or a medium prepared by making various modifications using these as a basic medium may be used. The medium can be used in the form of either a liquid medium or a solid medium. The solid medium may be solidified using a gelling agent such as agar, agarose, carrageenan, gellan gum, etc. The solid medium may be solidified using an appropriate concentration of agar, such as about 1% by weight agar. However, it is preferable to solidify with gellan gum, particularly about 0.2% by weight of gellan gum.

Plants of the genus Vitex transformed by bacteria of the genus Agrobacterium do not necessarily require plant hormones, but plants of the genus Vitex that have not been transformed require plant hormones. V1'! Conventional hormones can be used as IA hormones, and as plant hormone auxins, natural and synthetic auxins, such as 2,4-dichlorophenoxyacetic acid (2,4-D), indole #acid (
IAA), indolebutyric acid (IBA), β-naphthaleneacetic acid (NAA), 2,3.4-triiodobenzoic acid, phenyl′# acid, and the like. Among these auxins, 2.4-D, IAA, IBA, NAA, etc. are preferable.The auxins can be used at an appropriate concentration, for example, 0.01 to 1
It is preferable to use it at a concentration of about 0 .mu./J, especially about 0.05 to 5 .mu./J.

Examples of cytokinins include kinetin, benzylaminopurine (BAP), zeatin, and 2-isopentenyladenine. Among the cytokinins, kinetin, BAP, etc. are preferred.

Cytokinins can be used at appropriate concentrations, for example from 0.001 to
It is preferable to use it at a concentration of about 10 .mu./J, particularly about 0.001 to 5 .mu./J. As gibberellins, gibberellins Al to A13, abscisic acid, etc. may be used. Among gibberellins, gibberellic acid (GA3) is preferred. Gibberellins are usually used at a concentration of about 0.001 to 5 μ/J. The above plant hormones may be used alone or in combination.

When cultured in a medium supplemented with these plant hormones, regardless of the presence or absence of transformation by Agrobacterium bacteria, not only the growth of induced roots and callus is improved, but also the proliferation is increased, and Phi 1 to Ecdaily The amount of steroid production increases.

Furthermore, an energy source, a carbon source, vitamins, amino acids, etc. may be added to the medium. Examples of the energy source and carbon source include glucose, fructose, and sucrose, with sucrose being particularly preferred.

The energy source and carbon source can be used at an appropriate concentration, for example, 1 to 5% by weight, preferably about Bffl%.

Examples of vitamins include inositol, thiamine hydrochloride, pyridoxine, nicotinamide, ascorbic acid, calcium pantothenate, choline chloride, folic acid, ρ-aminobenzoic acid, vitamin A, and vitamin 812, and examples of amino acids include, for example. Examples include glycine and alanine.

Furthermore, coconut milk, casamino acids, organic acids such as sodium pyruvate, citric acid, and maleic acid, yeast extract, casein hydrolyzate, yeast extract, malt extract, and tomato and potato extracts are added. Good too,
In addition, in a medium containing about 0.5 to 10% by weight of coconut milk, callus etc. grow well even in plants of the genus Vitex that have not been transformed with bacteria of the genus Agrobacterium, and phytoecdai The amount of steroids produced also increases.

The liquid medium is, for example, an aqueous solution of the components excluding the gelling agent, and the pH is adjusted to 5.7 to 5.0 using sodium hydroxide, hydrochloric acid, or the like.
It can be produced by adjusting the temperature to about 8 and sterilizing it by high-pressure steam sterilization using an autoclave or the like or filtration sterilization using a membrane filter or the like. In addition, although the pH may decrease with high-pressure steam sterilization, there is no particular problem if it is used as is.Also, a solid medium can be prepared by adding a gelling agent to the sterilization liquid, and this solid medium can be prepared by adding a gelling agent to the sterilization solution. It may also be a medium.

The induced roots generated from the above sections and cells may be cultured under conditions that do not adversely affect cell growth or metabolic activity, including antibiotics, and under appropriate conditions according to the cell proliferation rate, metabolic activity, etc. The method is to subculture an appropriate number of times in a suitable medium, and finally to grow sterile induced roots by culturing in an antibiotic-free medium, and to cultivate induced roots that have many branches and a fast growth rate. This can be done by More specifically, approximately 5 to 10 rrllT+ of induced roots are cut from the tip and placed on a solid medium containing the antibiotic. Next, under appropriate conditions, for example, under illumination at a temperature of 25 to 28°C and illuminance of about 500 to 3000 lux, or in darkness,
When cultured for an appropriate period of time, for example, about 2 weeks, induced roots that have elongated by 5C11 or more are generated, so cut off the elongated induced roots by about 5 = 10 mm from the tip and transplant them into another fresh solid medium. Approximately 2 weeks later, the induced roots that have elongated 5 cm or more are again cut by about 5 to 10 mm from the tip and transplanted into another fresh solid medium.

Two weeks later, the tips of the roots that have grown to 5 cm or more are transplanted onto a solid medium that does not contain antibiotics, so that Agrobacterium bacteria are completely removed and only sterile induced roots grow. These induced roots are cultured for about a month under the same conditions as above, and those with a fast growth rate and many branches are selected and grown on another fresh solid medium. The fast and highly proliferative induced roots are cultured as follows. That is, approximately 20 to 40 square roots with high growth potential are cultured in a liquid medium with shaking. To protect the morphology of the roots, the rotation speed was set at 150. It is preferable to rotate and shake at a temperature below rpn. In this case, culturing may be carried out under light illumination. If cultured in this way for about 2 to 4 weeks, induced roots with a high content of phytoecdysteroids can be obtained. . At that time, it is preferable to extract phytoecdysteroids from each of the obtained induced roots and select induced roots with high phytoecdysteroid-producing ability.Incidentally, a substance that induces phytoecdysteroids is added to the liquid medium. For example, the phytoecdysteroid content can be increased by applying stress by adding the above-mentioned sugars, yeast extracts, etc., or changing conditions such as light and temperature. When these highly proliferative phytoecdysteroid-producing roots are extensively cultured, a large amount of phytoecdysteroids can be efficiently obtained.

In addition, callus can be induced from sections and cells by, for example, placing the sections on a solid medium or suspending them in a liquid medium and culturing them under the same conditions as above for an appropriate period of time, for example, about one month. can be done. The induced callus can be subcultured an appropriate number of times in the same medium as used for callus induction, or in an appropriate medium depending on the cell proliferation rate, metabolic activity, etc.

This subculture can be carried out under conditions that do not adversely affect cell growth or metabolic activity, such as the same conditions as above.

Furthermore, the plant body regenerated from the induced roots and callus also has a high content of phytoecdysteroids.

Therefore, induced roots such as highly proliferative phytoecdysteroid-producing induced roots or callus may be organ differentiated, grown in soil or in a culture vessel, and phytoecdysteroids may be extracted from the grown plants.

Regeneration of plants can be carried out by various conventional methods. For example, a regenerated plant can be obtained by culturing induced roots or callus in a medium containing plant hormones to differentiate adventitious buds, and then rooting the adventitious buds and transplanting them into soil or the like. More specifically, the induced roots and callus are treated with plant hormones, such as NAAO~5■/J, BAPI~10■/
The cells are placed on a solid medium containing J or the like, for example, an MS agar medium.

If this is cultured for more than two weeks in a cycle of 12 hours under light illumination of about 5,000 to 20,000 lux and 12 hours in the dark, adventitious buds will differentiate.

Incidentally, adventitious buds may also differentiate by transplanting onto a solid medium such as MS agar medium that does not contain plant hormones and culturing in the dark for 2 weeks or more.

Adventitious buds may also differentiate by transplanting to a solid medium containing antibiotics, such as MS agar medium, and culturing for 2 to 3 days under light illumination of about 20,000 lux. When the adventitious buds thus obtained are cut and planted in a medium containing no plant hormones, such as an MS agar medium, they will root in a few days. When the obtained regenerated plants are transplanted into soil and cultivated, the regenerated plants grow more rapidly than the original plants, producing large amounts of phytoecdysteroids,
The time to produce a certain amount of phytoecdysteroids is shortened. In particular, the regenerated plants derived from the highly proliferative phytoecdysteroid-induced roots have a faster growth rate than the original plants, and the growth of regenerated plants in culture vessels is generally inferior to that in soil. The growth rate is increased by culturing with shaking in a liquid medium such as an MS liquid medium under light illumination, for example under light illumination of about 5000 lux.

Note that the culture is not limited to the above-mentioned induced roots, callus, or plant bodies, but may also be organ differentiated bodies obtained by culturing callus or the like in a shoot differentiation medium or the like.

Phytoecdysteroids can be obtained by extracting them from the culture obtained as described above. Extraction solvents include, for example, alcohols such as methanol, ethanol, propatool, inpropatol, n-butanol, halogenated hydrocarbons such as chloroform, carbon tetrachloride, dichloroethane, methyl acetate, ethyl acetate, butyl acetate, etc. Esters, ketones such as acetone and methyl ethyl getone, ethers such as dioxane, diethyl ether, and tetrahydrofuran, and mixed solvents thereof can be used. For extraction of phytoecdysteroids, highly pure phytoecdysteroids can be obtained by grinding the culture, repeating extraction with the above extraction solvent, and separating by separation and purification means such as column chromatography.

[Effects of the Invention] As described above, according to the present invention, a phytoecdysteroid-containing plant is infected with Agrobacterium bacteria, tissue culture is performed, and a phytoecdysteroid is extracted from the resulting culture, Since plants of the genus Vitex are tissue cultured and phytoecdysteroids are extracted from the resulting culture, a large amount of phytoecdysteroids can be efficiently obtained from the culture without being influenced by natural conditions.

In addition, in the method of infecting phytoecdysteroid-containing plants with Agrobacterium bacteria, culturing the tissues, and extracting phytoecdysteroids from the resulting culture, the ability to produce phytoecdysteroids can be improved even in the absence of plant hormones. Cells can be grown at a high rate, and even larger amounts of phytoecdysteroids can be efficiently obtained. In addition, when the culture is an induced root, especially a high-proliferative induced root that grows rapidly, an induced root having a content of callus from a phytoecdysteroid-containing plant that is 10 to 100 times that of a callus derived from a phytoecdysteroid-containing plant. can be selected whose phytoecdysteroid content is comparable to or exceeds that of the source plant.

By culturing these highly proliferative roots and extracting phytoecdysteroids from the culture, phytoecdysteroids can be produced in large quantities and within a short period of time. Furthermore, when the culture is callus, a large amount of phytoecdysteroids can be efficiently produced compared to callus produced by conventional methods. Furthermore, phytoecdysteroids can be produced in large amounts by extracting them from plants regenerated using induced roots and callus derived from transformed cells.

Furthermore, the induced roots and callus obtained by the present invention have a significantly high ability to produce phytoecdysteroids.

[Examples] The present invention will be described in more detail below based on Examples.

Example 1 Melon hair root disease fungus of Agrobacterium genus bacteria (Agrobacterium rhizogenes strain MAFFO3-01724)
25 g of Nutriendo broth, 15 g of agar and 1 part of water, pH
7,5). Also, the night before using this bacteria, tryptone 10g, yeast extract 5g and sodium chloride 5g
Luria-Brose liquid medium (pH 7) adjusted to 1.1 with water
, 2) Using 20 to 30 cal, shaking culture was performed overnight at a temperature of 25°C.

Ajuga reptans var atropuabrea grown in a greenhouse
After washing the leaves of atropurpurea), the chlorine concentration is 0.
．． It was immersed in a 5% sodium hypochlorite solution for 7 minutes and washed with plenty of sterile water. The leaves were cut into 1011 x 1 am squares, immersed in melon hairy root disease fungus solution for 5 minutes, and inoculated.
Excess bacterial liquid was absorbed with sterilized P paper, and the cells were placed on 1% by weight agar and cultured at a temperature of 25° C. under 1 light for 3 days.

Next, the cells were transplanted onto MS medium containing 500 μg/ml carbenicillin and 500 μg/0111 vancomycin as antibiotics and solidified with 0.2% gellan gum, and cultured at a temperature of 25° C. under illumination.

After about 10 days, the tips of the induced roots that appeared @ 10 m were cut off, transplanted to the above medium, and cultured at a temperature of 25°C and under 1 light. After repeating this operation twice, only induced roots with especially good growth and many branching were selected, transplanted onto MS medium containing no antibiotics and solidified with 0.2% by weight gellan gum, and cultured. Fast-growing induced roots grow about 5 m in a day.
m offshore elongated.

Cut off 20 to 40 inches of the tips of the induced roots that were particularly fast elongated, and place them in 250 ml of MS liquid medium at a temperature of 25°C.
Rotary shaking culture was performed under illumination with stirring at a rotation speed of 150 rpm. After culturing for 4 weeks, the proliferation rate of induced roots was calculated from the fresh weight, and some were found to have a proliferation rate of 500 times.

Phytoecdysteroids were extracted from these induced roots. The extraction procedure is as follows.

That is, 100 to 200 induced roots cultured with shaking were placed in a 5 m
Sonicate for 2 hours in l of methanol.

After evaporating the methanol fraction to dryness and dissolving in 5 ml of water, two-layer partitioning is performed twice with equal amounts of butyl acetate, and the aqueous layer is extracted twice with butanol. The butanol layer was evaporated to dryness and
After dissolving in 0μ of methanol, this 10μ was subjected to high performance liquid chromatography (HPLC) to quantify β-ecdysone.

The quantitative results are shown in Table 1.

In addition, the production of ajugalactone, thiasterone, and norsengosterone as phytoecdysteroids other than β-ecdysone was confirmed.

Table 1 Ne1: Content% based on fresh weight Ne2: Clone No. of induced roots.

From Table 1, it was found that when infected and cultured with Agrobacterium bacteria, the induced roots contained a large amount of phytoecdysteroids.

Example 2 Chyrant cultivated in a greenhouse
Hes fauriei) leaves were sterilized in the same manner as in Example 1 and cut into 1 an x 1 an square.
The samples were inoculated by immersion into a melon hair root disease fungus solution containing Agrobacterium bacteria.

Next, the roots were cultured in the same manner as in Example 1, and among the induced roots that appeared, those with a fast growth rate were selected and subjected to liquid culture in the same manner as in Example 1. After 4 weeks of culture, the induced root proliferation rate was calculated from the fresh weight, and some were found to be 180 times as large.

Example 3 A bacterial solution of Agrobacterium rhizogenes ATCC15834 strain was prepared in the same manner as in Example 1.

Leaves of H. chinensis grown in a greenhouse were sterilized using the same method as in Example 1, and cut into 13×1α squares. Next, the above bacterial solution was inoculated onto a leaf disk and cultured in the same manner as in Example 1, and the induced roots that appeared were cultured in the same manner as in Example 1. After culturing for one month, the induced root proliferation rate was calculated from the fresh weight, and the induced root proliferation was approximately 170 times.

Example 4 The induced roots of Ajuga reptans bar atropoabrea from Example 1 were grown on MS medium containing 500 μg/not carbenicillin and 500 μg/[+11] vancomycin and solidified with 0.2 wt% gellan gum. 2
When cultured under illumination of 0,000 lux, adventitious buds often differentiated, which were cut out and grown on the above medium.
When cultured under 00 lux illumination, a complete plant was regenerated. β-ecdysone was extracted from this plant according to the procedure of Example 1 and quantified by HPLC. The quantitative results are shown in Table 2.

In addition, the production of ajugalactone, thiasterone, and norsengosterone as phytoecdysteroids other than β-ecdysone was also confirmed.

(Hereafter, margin) Table 2 *I: Content% relative to fresh weight Ne2: Regenerated plant clone No.

From Table 2, it was found that the regenerated plants also contained large amounts of phytoecdysteroids.

Example 5 Leaves of Ajuga reptans bar atropuabrea grown in a greenhouse were sterilized using the same method as in Example 1, and 1×
It was cut into 1 square piece. Next, the bacterial solution of Agrobacterium rhizogenes ATCC 15834 strain of Example 3 was inoculated into leaf discs, and the induced roots that appeared were cultured in the same manner as in Example 1, and they grew by about 1 n+m per day. A complete plant was regenerated from this induced root in the same manner as in Example 4. In addition, agropine and mannovin, which are ovines, were detected in the induced roots. Note that agropine and mannovin were identified by the method described below.

Example 6 Vitex strickeri (ν1t) grown in a greenhouse
Leaves of Vitex ex 5trickeri), Vitex doniana (V, 1oniana), Vitex micrantha (V, 11iCrantha), and Vitex madiensis (ν, nadiensis) were sterilized using the same method as in Example 1, and 1caX After cutting into 1 cx squares, the roots were immersed and inoculated in the Agrobacterium genus melon hair root disease fungus solution of Example 1.Next, they were cultured in the same manner as in Example 1, and among the induced roots that appeared, those with the fastest growth rate were inoculated. The cells were selected and subjected to liquid culture in the same manner as in Example 1. After culturing for 4 weeks, the proliferation rate of induced roots was calculated from the fresh weight, and it was observed that the proliferation rate reached 100 times. β-ecdysone was extracted from these induced roots according to the procedure of Example 1 and quantified by HPLC. The quantitative results are shown in Table 3.

(The following is a margin) Table 3 *3 = Content % based on dry weight When plants of the genus Hytex were infected with bacteria of the genus Agrobacterium and cultured, it was found that the induced roots contained a large amount of phytoecdysteroids.

Example 7 In the same manner as in Example 1, Acrobacterium rhizogenes MAFF-03-01725 strain, which is a melon hairy root disease fungus,
MAFF-03-01726 strain, MAFF-03-01
Bacterial liquids of 727 strains were prepared. Plants of the genus Vitex were infected and cultured in the same manner as in Example 6, except that the bacterial liquid of each melon hairy root disease fungus was used instead of the bacterial liquid of Example 6, and the same results as in Example 6 were obtained. Obtained.

Example 8 Cut the leaves of Vitex Strickeri grown in a greenhouse, remove surface contamination with a commercially available detergent, thoroughly wash with water, and then immerse in a 10% by weight sodium hypochlorite solution for 7 minutes to sterilize the surface. did. After thoroughly washing the sterilized leaves with a large amount of sterilized water, cut them into 1 x 1:11 squares, and cut them into 1
of IAA and 3 wt% sucrose, placed on MS medium solidified with 0.2 wt% gellan gum, and kept at a temperature of 25°C.
The cells were cultured for one month under illumination. One gram of calli that appeared was cut, transplanted onto fresh MS medium, and subcultured every month.

Finally, 1 g of callus was placed on MS medium containing no plant hormones, and MS containing 0.001 to 1 μ/J of IBA.
The callus was transplanted into a medium, an MS medium containing o, ooi ~ 1 ■/J of kinetin, and an MS medium containing 0.001 ~ 1 ■/J of G A 3, respectively, and cultured for one month. Ecdysone content was measured. The content of β-ecdysone in the callus was determined by extracting β-ecdysone in the callus with methanol, then sequentially extracting with butyl acetate and n-butanol, and then quantifying the content by column chroma 1 graphography. The results are shown in Table 4.

Table 4 shows that plants of the genus Vitex produce large amounts of β-ecdysone in the presence of plant hormones even without infection with bacteria of the genus Agrobacterium.

Example 9 1 g of callus obtained in Example 8 was finally transplanted to MS medium containing 0.1 to 10% by weight of coconut milk, and cultured for 1 month in the same manner as in Example 8 above. Fresh form and β-ecdysone content were measured. The results are shown in Table 5.

Table 5 shows that plants of the genus Vitex produce a large amount of β-ecdysone in the presence of coconut milk even without being infected with bacteria of the genus Agrobacterium.

(Hereinafter, blank space) Example 10 Leaves of Vitex Strigeri grown in a greenhouse were cut, surface sterilized in the same manner as in Example 8, and then 1■X
It was cut into lG1 corner. In addition, Agrobacterium rhizogenes MAFF-03-01724, which is a melon hair root disease fungus among bacteria belonging to the genus Agrobacterium, was cultured overnight at a temperature of 25°C in a Luria Broth medium to prepare a bacterial solution. After immersing cut pieces of leaves in each bacterial solution for 5 minutes, they were cultured on 1% by weight plain agar at a temperature of 25° C. under 1 light for 2 days to infect them with the bacteria. Then, 500 μg/ml carbenicillin and 500 μg/ml valecomycin were added, along with 3 wt.% sucrose and 0.2 wt.%
Transplant onto MS medium solidified with gellan gum and incubate at 25°C.
Transformed calli growing on hormone-free medium were selected by culturing in the dark. 1 g of the generated callus was placed in MS medium to which no plant hormones were added, B5 medium, H
After transplanting onto medium, NN medium, and W medium and culturing for one month, the fresh weight of the callus and the content of β-ecdysone were measured in the same manner as in Example 8. In addition, to identify whether or not a plant of the genus Vitex has been transformed with the above bacteria, Ti
This was done by detecting opine, which is a general term for non-protein amino acids that are specifically synthesized by plant cells transformed with Ri plasmid T-DNA. That is, after sufficiently grinding the callus, the callus is separated in a centrifugal separator at 10,000 rpm for 5 minutes, and the supernatant is collected. Next, 5 μj of the supernatant was spotted on P paper for chromatography and air-dried. In the case of octopine and tubarin, acetic acid/xylic acid/water = 15/
Using a migration buffer consisting of 5/80 (wt%), electrophoresis was performed for 1 to 2 hours at an applied voltage of sov,
After air drying, it was colored with phenanthraquine (80% ethanol solution containing 2% sodium hydroxide), which is a coloring reagent, and detected with short wavelength ultraviolet light. In the case of aglobin and mannovin, electrophoresis was performed in the same manner as above using the above-mentioned electrophoresis buffer, and after air-drying, a black spot was generated by spraying alkaline silver nitrate, which is a coloring reagent, to detect the electrophoresis. The results are shown in Table 6.

As is clear from Table 6, when transformed with Agrobacterium bacteria, it was found that the cells proliferated significantly even in the absence of plant hormones, and a large amount of β-ecdysone was produced.

Example 11 Instead of the bacterial solution of Example 10, Agrobacterium rhizogenes MAFF-03-01725 prepared in Example 7
strain, MAFF-03-01726 strain, MAFF-03-
Example 10 except that bacterial liquid of strain 01727 was used.
When a Pitenchus plant was infected and cultured in the same manner as in Example 10, the same results as in Example 10 were obtained.

Claims (1)

[Claims] 1. A phytoecdysteroid-containing plant, which is characterized by infecting a phytoecdysteroid-containing plant with bacteria belonging to the genus Agrobacterium, culturing the tissue, and extracting a phytoecdysteroid from the resulting culture. Manufacturing method for disteroids. 2. Agrobacterium bacteria are A. tumefaciens or A. rhizogenes.
The method for producing a phytoecdysteroid according to claim 1, wherein the phytoecdysteroid is 3. The method for producing a phytoecdysteroid according to claim 1 or 2, wherein the Agrobacterium bacterium is a melon hair root disease bacterium. 4. The method for producing a phytoecdysteroid according to claim 1, wherein the culture is a culture of induced roots. 5. The method for producing a phytoecdysteroid according to claim 1, wherein the culture is callus. 6. The method for producing a phytoecdysteroid according to claim 1, wherein the culture is a plant regenerated from induced roots or callus. 7. Tissue culture of plants of the genus Vitex,
A method for producing phytoecdysteroids, which comprises extracting phytoecdysteroids from the produced culture. 8. The method for producing a phytoecdysteroid according to claim 7, wherein the culture is callus. 9. The plant of the genus Vitex is V. strickeri, V. doniana, V. madiensis, or V. mcrantha. The method for producing a phytoecdysteroid according to claim 7. 10. A phytoecdysteroid-containing induced root obtained by infecting a phytoecdysteroid-containing plant with Agrobacterium bacteria. 11. A phytoecdysteroid-containing callus obtained by infecting a phytoecdysteroid-containing plant with Agrobacterium bacteria.