JPH0622754A - Production method for substance by dedifferentiated initial cell line - Google Patents

Abstract

PURPOSE:To efficiently obtain temporally and quantitatively chemical substances such as alkaloids or pigments from plant cells by extracting plant cell lumps at the initial stage of dedifferentiation or by stimulating such cell lumps with an elicitor. CONSTITUTION:Plant cell lumps at the initial stage of dedifferentiation is either directly extracted or stimulated with an elicitor. The elicitor is e.g. a composition derived from an organism such as yeast extract, heat, temperature, rays of light etc., in a single form or a combination thereof. The stimulation with the elicitor is made, for example, by immersing the above-mentioned cell lumps in a yeast extract-contg. solution for 1 to 2 days.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a cell mass in the early stage of dedifferentiation and a method for producing a useful substance by stimulating a cell mass in the early stage of dedifferentiation with an elicitor. Since this method can produce substances having various uses, it is expected that the fields of industrial application will be extremely wide. In particular, natural organic compounds produced by plants (for example,
It is possible to efficiently obtain alkaloids, terpenes, phenolic compounds, pigment components, etc., and use them in the fields of medicine, agricultural chemicals, foods, fragrances and the like.

[0002]

2. Description of the Related Art Conventionally, methods for obtaining chemical substances using plants have been widely used. For example, as shown in the example of extraction / separation of crude drug components, there is a method of directly obtaining from the plant itself. However, with this method, it is difficult to produce a large amount of a substance originally produced only in a trace amount in a plant or a substance that has a high metabolic rate and could hardly be detected.

Further, as seen in the example of obtaining shikonin from shikon callus, there is a method of culturing and proliferating a dedifferentiated cell line, and then obtaining the same through cultured cells. However, when the dedifferentiation progresses to such an extent that the dedifferentiated cells grow stably, the productivity of the useful substance found in the mother plant generally decreases. Therefore, with such a method, it is difficult to efficiently produce a desired substance.

In order to deal with such problems of the conventional method, it has been studied by trial and error to select a plant species containing a relatively large amount of a useful substance and to improve a method for extracting a useful substance. It was However, the technology for controlling the type and amount of substances produced by plants has not yet been developed.

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for efficiently obtaining a desired chemical substance from a plant cell in time and quantity. Another object of the present invention is to provide a method for obtaining a substance that was difficult to obtain by the conventional method.

[0006]

[Means for Solving the Problems] The object is to provide a method for producing a substance, which comprises a step of stimulating a cell mass in the early stage of dedifferentiation derived from an arbitrary explant or a cell mass in the early stage of dedifferentiation with an elicitor. Achieved by developing.

According to the present invention, in the cell mass in the early stage of dedifferentiation, that is, in the cell mass of several generations before being derived from the explant and capable of stable growth, the secondary mass exceeding the amount originally present in the explant is present. It was discovered that metabolites accumulate, new secondary metabolites that are hard to find in explants accumulate in large amounts, and that susceptibility to elicitors greatly changes to efficiently produce and accumulate organic compounds. It was completed based on this. The present invention in which a useful substance is obtained from cells in the early stage of dedifferentiation or by allowing elicitor to act on the cells,
It is a new technology that has never been tried before.

The cell mass in the early stage of dedifferentiation used in the present invention is
It is not particularly limited by plant species, parts and organizational differences. For example, cells in the early stage of dedifferentiation from alfalfa cotyledons or pea hypocotyls are used. The cells to be dedifferentiated can be collected from various parts such as plant seeds, roots, leaves, stems and flowers.

The elicitor to be given to cells in the early stage of dedifferentiation is not particularly limited in kind as long as the cells can produce a useful compound by giving the stimulus. Therefore, a composition of biological origin, such as yeast extract,
Light, heat, temperature, humidity and the like can be used as the elicitor alone or in combination. The elicitor may be applied at once, divided into several times, or maintained for a certain period of time. For example, a method of immersing in a solution containing a composition of biological origin for 1 to 2 days can be used.

The cell mass itself in the early stage of dedifferentiation and useful substances produced by stimulation with elicitor can be isolated by separation / purification techniques well known to those skilled in the art. The useful substance may be accumulated inside the cell or may be released to the surroundings.In either case, the cells or the surrounding medium are extracted with an organic solvent such as acetone or ether, and then, by chromatography or the like. The target substance can be obtained by purification.

The present invention has various advantages over conventional methods.

[0012] First, in order to efficiently obtain a specific substance, the cell mass itself after a specific number of days after the start of dedifferentiation is directly extracted, or the cell mass in this state is stimulated by elicitor, A large amount of a specific substance accumulates inside the cell or is released outside the cell. Therefore, a large amount of substance can be efficiently obtained by performing the operation of the present invention under the condition that the concentration of the desired substance is the highest.

For example, when a partially purified yeast extract is stimulated to cells in the early stage of dedifferentiation of alfalfa, the amount of the substance released outside the cells changes as shown in FIG. 1 depending on the number of days after the start of dedifferentiation. (Example 1). For example, a substance with tR = 4.6 has about four times the release amount when cells are stimulated 5 days after the start of dedifferentiation, and is released when cells are stimulated 7 days after dedifferentiation. It becomes about 1.5 times.
The increase in the amount of release due to such stimulation was also confirmed in the case of tR = 8.0 (4 ′, 7-dihydroxyflavone) and tR = 14.8 (formononetin) released by cells on the 10th day after dedifferentiation. . In this way, confirm in advance the case where the release amount increases due to the stimulation of elicitor,
If the desired substance is produced and separated under the conditions, the acquisition efficiency can be improved. The alfalfa system shown in Example 1 remarkably induces several kinds of phenolic substances, and thus can be an effective means for promoting the separation and purification of new elicitors.

Stimulation by elicitors not only increases the amount of substance released, but may change the release pattern in some cases. For example, tR = shown in FIG.
The release amount of the substance of 8.0 is maximum in the cells 5 days after the start of dedifferentiation when untreated, but it is minimum in the cells 5 days after the start of dedifferentiation when stimulated by elicitor. The reverse phenomenon occurs. FIG. 2 showing changes in intracellular substance accumulation
According to the data, when stimulated by elicitor, tR = 8.0 is remarkably accumulated in the cells 5 days after the start of dedifferentiation. Therefore, if the present invention is applied under these conditions and the cells are extracted, tR = 8.0 is extremely efficient.
Can obtain the substance.

The present invention is characterized not only by such quantitative efficiency but also by high time efficiency. That is, the time required for the present invention using cells at the early stage of dedifferentiation is significantly shortened as compared with the conventional method in which a substance is obtained after culturing and proliferating a dedifferentiated cell line as cultured cells. Therefore, the present invention is extremely useful when it is desired to quickly obtain a desired substance.

Furthermore, according to the present invention, not only known substances but also new derivatives can be obtained. For example, when pea cells are stimulated with an aqueous solution of chitosan, 2-hydroxypisatin (tR = 1), which is a novel substance, is stimulated.
A large amount of 0.0) is accumulated in the cells. Therefore, according to the present invention, it is expected to be possible to discover a novel substance having physiological activity and to provide a new raw material for synthesizing a useful substance.

Further, according to the present invention, there is a great feature in that an intermediate, which has been considered difficult to obtain from plants by the conventional method, can be obtained efficiently. For example, assume a reaction system in which the substance A becomes an intermediate B by the action of the enzyme α and is further changed to the substance C by the enzyme β in the plant body. At this time, if the reaction rate from the intermediate B to the substance C is faster than the reaction rate from the substance A to the intermediate B, it becomes extremely difficult to obtain the intermediate B. Therefore, if an elicitor that weakens the activity of the enzyme β is allowed to act according to the present invention, the intermediate B is introduced into the plant cell.
Will be accumulated and can be acquired. In addition, the expression pattern of the enzymes involved in the synthesis of substances A, B, and C present in the mother plant greatly changes with the progress of dedifferentiation, and
Since the converting enzyme from the enzyme to the substance C does not express the activity, a large amount of the newly accumulated substance B can be efficiently obtained. Therefore, according to the present invention, it becomes possible to obtain an unstable substance or a substance having a short existing time, which has been difficult to obtain conventionally, and it is expected that its application range is extremely wide.

As described above, the present invention efficiently obtains various substances such as natural organic compounds produced by plants (for example, alkaloids, terpenes, phenolic compounds, pigment components) to obtain pharmaceuticals, It can be used in a wide range of fields such as agricultural chemicals, foods, and fragrances.

[0019]

EXAMPLES The present invention will be specifically described below with reference to examples, but these examples do not limit the scope of the present invention.

Example 1 To 200 g of yeast extract (Nacalai), 1 liter of deionized water and 4 liter of methanol were added and stirred, and the mixture was allowed to stand at room temperature for 2 hours.
It was left for 4 hours. The supernatant was removed, 1 liter of deionized water and 4 liter of methanol were added again to the remaining precipitate, and the mixture was stirred and allowed to stand at room temperature for 24 hours. The supernatant was removed, and 1 L of deionized water was added to the remaining precipitate, which was passed through an anion exchange resin (Diaion SA20A), and the eluate was placed in an eggplant-shaped flask and dried in a distiller (4.2 g). . Deionized water was added to this to make 500 ml, and a partially purified yeast extract was obtained.

[0021] Aseptically grown alfalfa cotyledons were aseptically excised and dedifferentiated in MS medium containing 2,4-D. On the 0th, 3rd, 5th, 7th, and 10th days after the start of dedifferentiation, the cells were taken out, and a part of them was immersed in an elicitor solution containing partially purified yeast extract to
Rotation culture was carried out at 8 ° C. for 48 hours in the dark. Further, the remaining cells were cultivated in the dark at 18 ° C. for 48 hours in the dark without immersing in the elicitor solution for control. After spin culture, the sample was filtered to separate the cell mass and the filtrate.

The filtrate was extracted with ethyl acetate and analyzed by HPLC [developing solution: methanol-water (7: 3), flow rate:
0.5 ml / min, detection wavelength: 254 nm]. tR = 4.6, 6.
For components of 3, 8.0, 14.8 and 16.4, 2
The relationship between the peak area at 54 nm and the number of days of dedifferentiation is shown in FIG.

The cell mass was extracted with acetone and analyzed by HPLC in the same manner. tR = 8.0, 11.6 and 1
The relationship between the amount of the 4.8 component and the number of days of dedifferentiation is shown in FIG. The components with tR = 8.0 and tR = 14.8 were identified as 4 ′, 7-dihydroxyflavone and formononetin which are known substances by instrumental analysis.

Example 2 The same operation as in Example 1 was performed using an unpurified yeast extract instead of the partially purified yeast extract. The cell mass was extracted with acetone and analyzed by HPLC. The results are shown in FIG.

Example 3 A plant piece was cut out from a stalk of pea seedlings grown aseptically, and the plant hormone composition NAA was 10.7 μM, 6BA.
The cells were dedifferentiated in the dark at 26 ° C. on 4.4 μM MS medium. On day 0, day 2, day 4, day 6, and day 8 after the start of dedifferentiation, the cells were taken out, and part of them was immersed in an aqueous chitosan solution (4 mg / ml) for 24 hours in the dark at 25 ° C. The cells were cultivated by rotation. The remaining cells were cultivated in the dark at 25 ° C. for 24 hours in the dark without immersing in the chitosan aqueous solution for control.

After the spin culture, the cell mass obtained by filtering the sample was extracted with acetone and analyzed by HPLC. tR = 10.
The relationship between the peak area at the detection wavelength of 285 nm and the number of days of dedifferentiation for the components 0 and 20.0 is shown in FIG. Also,
When the same analysis was carried out with the detection wavelengths of 254 nm, 285 nm and 310 nm, tR = 20.0 was tR = 10.
It was confirmed that the peak area was changed at almost the same ratio as 0.

The component with tR = 10.0 was identified as (+) pisatin by comparison with the standard, and the component with tR = 20.0 was the novel substance 2-not described in the literature from the following ¹ H NMR data. Identified as hydroxypisatine. ¹ H NMR: 2.33, 3.83, 3.95, 4.13,
5.21, 5.26, 5.88, 5.91, 6.38, 6.4
4,6.77,6.97

[0028]

Industrial Applicability According to the method for producing a substance by the dedifferentiation early stage cell line of the present invention, a desired chemical substance can be efficiently produced from plant cells in time and quantity. Further, it is possible to efficiently obtain a substance that has been conventionally difficult to obtain.

[Brief description of drawings]

FIG. 1 shows the relationship between the number of days after dedifferentiation and the amount of substance released from cells.

FIG. 2 shows the relationship between the number of days after dedifferentiation and the amount of substances accumulated in cell clusters.

FIG. 3 shows the relationship between the number of days after dedifferentiation and the amount of substances accumulated in cell mass.

FIG. 4 shows the relationship between the number of days after dedifferentiation and the amount of substance released from cells.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yasuki Akiyama 214-9 Kamaya, Mitsu-cho, Ibo-gun, Hyogo Prefecture (72) Inventor Akihiro Tai 7966-1 Danshicho, Takamatsu-shi, Kagawa Inventor Yasuko Nakamoto Hiroshima 4-5-3 Funakoshi, Aki-ku, Hiroshima Prefecture

Claims (1)

[Claims] 1. A method for producing a substance, which comprises a step of extracting from a cell mass in the early stage of dedifferentiation and a step of stimulating the cell mass in the early stage of dedifferentiation with an elicitor.