JPH0430809B2 - - Google Patents

Description

[Detailed description of the invention]

<Industrial Application Field> The present invention is directed to the cultivation of parts of medicinal ginseng (panax ginseng, ginseng, American ginseng, ginseng, etc.) plants (e.g., growing points, leaves, stems, roots, seeds) through tissue culture. This invention relates to a method for mass-producing medicinal ginseng by regenerating a plant body from a callus derived from ginseng, etc., through the various steps of somatic embryo, stem and leaf differentiation, and rooting. <Background of the invention and conventional technology> Although there have been remarkable developments in biotechnology in recent years, there are only a limited number of regenerated plants that can be stably obtained using seed propagation techniques, and even among these, there are The results can vary greatly depending on the plant, site, and slight differences in culture conditions, and mass production of plants by tissue culture is still extremely difficult. Edible carrots are included in the stably obtained regenerated plants, and edible carrots belong to the Umbelliferae family, and the medicinal ginsengs mentioned above belong to the Araliaceae family. Therefore, these two are completely different types of plants, and medicinal ginseng has not yet been stably mass-produced by tissue culture. Also,
Typical methods for obtaining regenerated plants from plant tissue fragments using tissue culture methods include (a) regeneration, which involves the steps of callus induction, somatic embryo formation, shoot and leaf differentiation, and then rooting; (b) shoot apex or callus production; A method of propagating shoots derived from the same species by multiple shooting (multiple shoot method) has been proposed. The former (a) regeneration method is a method of propagating plant seedlings by propagating "somatic embryos" which can be said to be the "seeds" of the plant, while the latter (b)
The multiple shoot method is similar to the propagation of seedlings using cuttings, etc. in that shoots are divided and propagated. Medicinal ginseng (panax ginseng, ginseng ginseng, American ginseng, ginseng ginseng, etc., hereinafter the same) has been prized as a versatile noble medicine mainly for tonicity and tonicity since ancient times. Conventional methods for culturing medicinal ginseng tissue culture include JP-A-59-169486, JP-A-59-169487, and JP-A-59-169488, all of which involve culturing specific cells of medicinal ginseng into tissues. This relates to culturing and propagating. As recent literature, there is a method for producing regenerated plants by somatic embryo differentiation of Panax ginseng in Japanese Patent Application Laid-Open No. 151117/1983, and a method for propagating shoots derived from mature somatic embryos of Panax ginseng by multiple shooting is published in Japanese Patent Application Laid-Open No. 151117. There is No. 133922 (Sho 63-133). By the way, cells and/or
In the production process of regenerated seedlings, the selection, combination, and amount of plant growth regulators (plant hormones) are extremely important.・Determination of combinations and amounts of each compound is often the essential part of the research and invention. <Problems to be solved by the invention> The components contained in cultured cells obtained by tissue culturing a part of a medicinal ginseng plant are often different from the components contained in the cultivated plant. . Even if cultured cells contain active ingredients, the content is often small. Furthermore, even if the medicinal components contained in cultured cells were extracted and separated, they would not be recognized as drugs under the relevant laws and regulations of Japan. Additionally, it is generally said that many of the medicinal components are found in secondary metabolites. However, tissue culture methods
Although it can be an excellent means for obtaining primary metabolites, there is a problem in that it is inappropriate for obtaining secondary metabolites such as medicinal ingredients. By the way, medicinal ginseng takes five to six years from sowing seeds to harvesting. The period for sowing medicinal ginseng seeds is limited and is short throughout the four seasons, and there is a problem in that it is difficult to create and cultivate conditions for the seeds to germinate and grow. Furthermore, cultivated medicinal ginseng has large individual differences, and there are large qualitative and quantitative variations in active ingredients such as saponins due to individual differences in addition to cultivation conditions, and there is a problem that quality is highly heterogeneous depending on the production area. There is. Therefore, it is extremely difficult to obtain a plant body of medicinal ginseng with a stable composition and content of active ingredients. Furthermore, in the conventional production method of regenerated Panax ginseng plants, there is a problem that it is not suitable for mass production, especially when producing somatic embryos, because they are obtained using only a solid medium. There was a problem in that the quality of the active ingredients of medicinal ginseng obtained from these products varied due to individual differences in the body. This invention was completed as a result of intensive research to solve the above-mentioned problems, and was developed by regenerating a plant of similar quality from callus derived from a part of the plant of an excellent strain of medicinal ginseng. We aim to establish and provide a method for stably supplying large amounts of medicinal ginseng with stable quality, such as having good properties, all year round, regardless of the four seasons, and to foster and propagate superior varieties of medicinal ginseng. The purpose is to secure resources. <Means for Solving the Problems> In order to achieve the above object, the present invention provides a process for inducing callus from a piece of plant tissue by tissue culture;
A method of producing a regenerated plant through a somatic embryo formation process, a shoot and leaf differentiation process, and a rooting process, in which a part of the medicinal ginseng plant is tissue cultured using a predetermined reference medium to regenerate the plant. In the process of forming somatic embryos from callus of medicinal ginseng, after forming somatic embryos from callus, the somatic embryos of medicinal ginseng are shaken in a liquid medium to which no plant growth regulators such as cytokinin or auxin are added. A method for mass production of medicinal ginseng by tissue culture, which comprises propagating somatic embryos of medicinal ginseng by culturing. This invention was constructed based on the following. The reference medium that can be used in this invention includes:
Murashige & Skoog medium (hereinafter referred to as "MS medium"), Linsmaier & Skoog medium (hereinafter referred to as "LS medium"), White medium (hereinafter referred to as "W medium"), Gauslet (Gautheret medium (hereinafter referred to as "G medium"), Tulecke medium (hereinafter referred to as "T medium"), Morel medium (hereinafter referred to as "M medium")
), Hildebrandt medium (hereinafter referred to as "H medium"), and the like can be used. Among them, MS medium, LS medium, W medium, etc. are preferable, and MS medium, LS medium, etc. are particularly suitable. Furthermore, various auxins, cytokinins, and others can be used as plant growth regulators that can be used in the tissue culture according to the present invention. The concentration of this plant growth regulator is within the commonly used concentration range.
Available in 10 ^-6 mg/~10 ² mg/. The method for mass-producing medicinal ginseng by tissue culture according to the present invention will be described below. The outline of the method for mass production of medicinal ginseng by tissue culture according to the present invention is to induce callus derived from parts of medicinal ginseng plants (e.g., growing points, leaves, stems, roots, seeds, etc.) by a known method. This callus is subcultured by a known method or in a solid medium containing a predetermined plant hormone to form a somatic embryo, and the somatic embryo is further grown by shaking culture in a liquid medium containing a predetermined plant hormone. After that, this somatic embryo is differentiated into foliage on a solid medium containing a combination of predetermined plant hormones, if necessary, and the differentiated foliage solid is cultured on a solid medium containing a predetermined phytohormone, or a method known in the art. This will cause roots to develop and eventually regenerate the plant. First, medicinal ginseng [Panax ginseng]
ginseng CAMEYER), Panax japonicum CAMEYER, American ginseng (Panax quinquefolium L.), Panax notoginseng FHCHEN, etc., hereinafter referred to as "medicinal ginseng (panax ginseng, etc.)"]
plant parts (e.g., growing points, leaves, stems,
Calli derived from roots, seeds, etc.) are induced by known methods. That is, for the induction of callus, known auxins such as 2,4-dichlorophenoxyacetic acid (hereinafter referred to as "2,4-D"),
Naphthalene acetic acid (hereinafter referred to as ``NAA''), indolebutyric acid (hereinafter referred to as ``IBA''), indole acetic acid (hereinafter referred to as ``IAA''), etc. can be used, and at least one auxin is required.Among them, 2,4-D, NAA is particularly suitable. Also, as the cytokinin, kinetin, benzyladenine (hereinafter referred to as "BA"), zeatin (hereinafter referred to as "Z"), etc. can be used.The above-mentioned various auxins and cytokinin are used in appropriate combinations. This promotes callus induction in medicinal ginseng.For example, kinetin and 2,4-
Use in combination with D or IAA (combination use of 2), or use combination of BA and NAA or IBA (combination use of 2), Z and 2,4-D, IAA, IBA or NAA When used in combination (combination use of 4), callus induction of the medicinal ginseng (panax ginseng, etc.) is promoted. The concentration of the plant growth regulator used in this callus induction step is within the normally used concentration range.
10 ^-6 mg/~10 ² mg/ is appropriate. In this callus induction step, the best effect is obtained when 2,4-D and kinetin are added in a concentration range of 0.1 mg/~10 mg/. Also, kinetin, benzyladenine (BA), zeatin (Z)
Although these are not essential plant hormones, their additional addition promotes callus induction. In conclusion, 2,4-D, NAA,
If at least one of IAA and IBA is present, callus derived from a part of a medicinal ginseng (panax ginseng, etc.) plant can be effectively induced. The basic procedure for callus induction is to aseptically remove parts of the plant body (e.g., growing points, leaves, stems, roots, seeds, etc.) of medicinal ginseng (panax ginseng, American ginseng, Chikusetsu ginseng, Sanchi ginseng, etc.). The explants were cut into appropriate sizes with a scalpel and treated with the plant growth regulator [2,4-D, NAA,
Place the plate on a standard agar medium supplemented with IBA or IAA (the above four are essential plant growth regulators) and kinetin, benzyladenine, or zeatin as necessary), and place it at 15-30℃ in a light or dark place. Callus can be induced by culturing for several weeks (usually about 3 to 6 weeks). Next, the process of forming somatic embryos from the induced callus will be explained. That is, the callus derived from a part of a plant of medicinal ginseng (panax ginseng, etc.) is prepared in the standard medium without cytokinin, but with essential plant growth regulators such as 2,4-D, IBA, IAA, NAA, etc. Addition of at least one of the following auxins (concentration range: 10 ^-6
15 to 20 mg/~10mg/) of the drug in a solid medium
℃, subculturing in the light at regular intervals (about every 4 weeks to about 8 weeks), repeating the subculturing for a fixed period of time (about 1 year, 48 weeks to 54 weeks), and indeterminate from the induced callus. Allow the embryo to form. Thereafter, in order to propagate the final product, the regenerated plant, this somatic embryo was transferred to a liquid reference medium without the addition of plant growth regulators such as cytokinin and auxin.
The somatic embryos are grown by culturing with shaking (rotation or reciprocation) for about 20 days at 20-25°C in the light. In this case, when the somatic embryo is cultured with shaking in the liquid medium, its proliferation can be promoted incomparably more efficiently than when the somatic embryo is cultured in a solid medium, and moreover, the somatic embryo can be homogeneously cultured. can be obtained. Next, the process of causing this somatic embryo to differentiate into shoots and leaves will be explained. By the way, in general, when redifferentiating somatic embryos of plant tissue pieces, that is, when somatic embryos are differentiated into shoots and roots, it has been reported that good results can be obtained by culturing in a medium with a reduced concentration of inorganic salts. Therefore, a solid medium (standard medium or modified medium) in which a plant growth regulator is added to a standard medium or a modified medium with a reduced content of inorganic salts (less than 1/1 to 1/20 or more) is used for growth. The somatic embryos (that is, the somatic embryos cultured and grown in a liquid medium) are transplanted and incubated under certain conditions (approximately 15 to
The somatic embryos are cultured at 20° C. in the light for about 3 to 6 weeks) to differentiate into stems and leaves. Plant growth regulators that can be used in this shoot and leaf differentiation process include IBA and
A combination with BA or a combination of IAA and BA is preferred. In addition, the amount of the plant growth regulators (IBA, IAA, BA) used in this shoot and leaf differentiation step is as follows:
The amount of each addition is preferably about 10 ^-2 mg/~10 mg/. Next, the process of rooting the stem-and-leaf-differentiated solid will be explained. That is, the above-mentioned foliage-differentiated solid is transplanted to a standard solid medium supplemented with a plant growth regulator and cultured for a certain period of time (about 4 to 10 weeks) at about 15 to 20° C. in the light. It has been found that gibberellic acid (GA), which is neither auxin nor cytokinin, can be used as a plant growth regulator used during the rooting process of this foliage-differentiated solid. Further, the amount of the plant growth regulator added to the reference medium is preferably within the range commonly used, that is, 10 ^-3 mg/10 mg/. <Example> (1) In the method for mass culturing medicinal ginseng according to the present invention, an example will first be described in which Murashige & Skoog (MS) medium is used as the reference medium. (a) Example of Callus Induction Callus was induced by a known method.
Namely, medicinal ginseng (Panax ginseng, Panax
An example of inducing callus from the roots of C. ginseng CAMEYER is as follows. Medicinal ginseng (Panax ginseng, Panax)
ginseng CAMEYER) roots are aseptically punched out with a cork borer and cut into approximately 2-3 mm thick pieces with a scalpel to prepare disc-shaped explants. On the other hand, 2,4-D as a plant growth regulator
and the concentration of kinetin,
A total of 16 types of MS solid media are prepared by adding combinations of 0.0, 0.1, 1.0, and 10 mg/4 types each. Then, the disk-shaped explant of the medicinal ginseng was placed on this solid medium (the first
(Figure), culture is performed for 6 weeks at 25°C in the light to induce callus derived from the roots of Panax ginseng.
(Figure 2) The results of this callus induction are shown in Table 1. As is clear from Table 1, 2,
4-D and kinetin each 1.0mg/
This suggests that callus formation in the added area is optimal.

[Table] Yes, -: Indicates callus induction (e.g., callus induction not recognized)
The callus induction results described above show almost the same tendency when each combination of IAA, NAA, or IBA and kinetin is used as a plant growth regulator. The callus induction results in this case are as shown in Table 2.

[Table] Yes, -: Indicates callus induction (e.g., callus induction not recognized)
(b) Example of somatic embryo formation from the induced callus. To the callus induced in (a) above, 1.0 mg/2,4-D and 0.0 mg/kinetin were added as plant growth regulators (that is, no kinetin was added).
The cells are transplanted into MS medium (solid medium), and subculturing is repeated every 6 weeks for about 1 year (about 48 to 54 weeks) at 20°C in the light. At the beginning of induction, the callus is pale white and soft, but after about one year of subculturing, the yellow part of the somatic embryo gradually becomes visible. (Figure 3) Table 3 shows plant growth regulators such as 2,4-D, IAA, IBA,
The results of somatic embryo formation when using NAA are shown in comparison. The somatic embryos thus obtained are prepared without the addition of a plant growth regulator (2,4-D) or with the addition of a low concentration (approximately 10 ^-2 mg/~10 ^-3 mg/).
Transplant into MS medium (liquid medium) and culture under rotating shaking culture (110-115 rpm) or reciprocating shaking culture (110-130 cycles/min) at 25°C in the light.
Culture for 20 days to propagate somatic embryos.

[Table] Yes, -: Indicates the formation results of somatic embryos, such as no formation of somatic embryos)
(c) Example of stem and leaf differentiation of the somatic embryo. The somatic embryos obtained in (b) above were placed in MS medium or
The concentration of inorganic salts contained in MS medium should be less than 1/1/1/20.
Indole acetic acid (IAA) or indole butyric acid (IBA) as a plant growth regulator was added to the MS-amended medium, which had been reduced in concentration to 0.0 mg/, 1.0 mg/, 5.0 mg/, or 10.0 mg/, respectively.
mg/ and each concentration of benzyladenine (BA)
0.0mg/, 0.1mg/, 1.0mg/, 10.0mg/
A total of 16 types of each additive amount for the above combinations (i.e., two sets of IAA and BA, IBA and BA) (attached to each combination)
Transplant onto each solid medium and incubate at 20℃ under light for 30 minutes.
The somatic embryos obtained in (b) above are cultured for 1 day and differentiated into stems and leaves. (Figure 4) Table 4 shows somatic embryos for each test concentration of the plant growth regulator (combination of IAA and BA, and combination of IBA and BA), with the inorganic salt concentration of the MS medium set to 1/2. The results of stem and leaf differentiation are shown.

[Table] Yes, -: Shows the results of stem and leaf differentiation of indeterminate embryos, such as no shoot and leaf differentiation of somatic embryos)
From the results in Table 4, IAA or IBA is 1.0
mg/~5.0mg/, BA 0.1mg/~1.0
It has been suggested that mg/each added is suitable. Note that the results of stem and leaf differentiation of somatic embryos related to the combination of IAA and BA and the combination of IBA and BA according to the present invention are different from those of NAA and IAA, which have been conventionally used as examples of good combinations of plant growth regulators. Or, when each combination of the three types of auxin in IBA and BA (that is, the three combinations of NAA-BA, IAA-BA, and IBA-BA) was used, almost similar trends were shown. Table 5 shows an example of test results regarding the influence of the inorganic salt concentration in the medium on the shoot and leaf differentiation of somatic embryos. The results of stem and leaf differentiation of somatic embryos for each composition are shown when the composition is 1/1 to 1/30 of the composition. However, the amount of plant growth regulator added is determined by IAA.
An example of the results when BA is 1.0mg/ and BA is 0.1mg/ is shown. The results in Table 5 suggest that a standard medium or modified medium in which the inorganic salt concentration of the standard medium is reduced to 1/1 or 1/20 of the standard medium yields favorable results, which is consistent with previous reports. do.

[Table] Shows the results of stem and leaf differentiation, such as Yes and -: No effect observed. )
In addition, some of the solids that have differentiated into stems and leaves include
Some plants root at the same time as stem and leaf differentiation, but many remain in stem and leaf differentiation. (d) Example of rooting of a leaf-differentiated solid. An example of the step of rooting the stem-and-leaf differentiated solid in (c) above will be described. In MS medium (1) As a comparison with known usage examples, auxin [indolebutyric acid (IBA),
A solid medium supplemented with naphthalic acid (NAA) and indole acetic acid (IAA); (2) plants other than auxin and cytokinin as an example for searching for plant growth regulators that can be used according to the present invention; The solids differentiated in (c) above were transplanted to a solid medium supplemented with the hormone gibberellic acid (GA), and cultured for 8 weeks at 20°C in the light to develop the solids differentiated into leaves. Let it take root. (Figure 5)
Thereafter, the rooted plant is transplanted into soil as necessary. (FIG. 6) Table 6 shows a comparative example of the rooting results of solids with shoot and leaf differentiation in the GA-added medium.

[Table] Yes, -: Negative, *: Examples of implementation results according to this invention are shown respectively)
(2) Regarding the reference medium that can be used in this invention, the test results of each step of the method for mass production of medicinal ginseng from (a) induction of callus to (b) rooting of differentiated solids in (1) above are as follows: As shown in Table 7. In addition to the above-mentioned MS medium, the reference medium used for the test is Linsmaier & Skoog medium (Linsmaier & Skoog medium / LS medium), White medium (White medium / W medium), Gautheret medium (Gautheret medium / G medium), Churetzke's medium. Culture media (Tulecke medium/T medium), Morel medium (Morel medium/M medium), and Hildebrandt medium (Hildebrandt medium/H medium) are tested. Furthermore, the concentration of inorganic salt in the standard medium used for shoot and leaf differentiation is 1/2 that of each standard medium.

【table】

[Table] Approved, -: Indicates negative or poor, respectively)
<Effects of the Invention> (a) We have succeeded in regenerating a large amount of plants from callus derived from parts of medicinal ginseng (panax ginseng, ginseng, American ginseng, ginseng, etc.), so that it can be used in different seasons. It is possible to obtain a large quantity of medicinal ginseng of excellent variety with stable quality. (b) Furthermore, since the method for producing medicinal ginseng according to the present invention allows a large number of plants to be regenerated from one plant, a large amount of medicinal ginseng with stable quality can be obtained with almost no individual differences among the regenerated plants. It is possible to secure resources by cultivating and multiplying superior varieties of medicinal ginseng. (c) In addition, somatic embryos were formed from the induced callus according to the present invention, and the somatic embryos could be multiplied in large quantities by liquid shaking culture, which is different from conventional methods.
The multiplication rate of somatic embryos has been dramatically increased compared to the conventional method of increasing somatic embryos on a solid medium, or obtaining multiple shoots from shoots and attempting to propagate seedlings on a solid medium. It becomes possible to increase the (d) The method for mass production of medicinal ginseng according to the present invention can be applied to the mass production of various types of medicinal ginseng, such as Panax ginseng, Panax ginseng, American ginseng, Panax ginseng, etc. It grows wild in shady areas, and its cultivation is said to be difficult. According to the production method according to the present invention, valuable ginseng can be grown in the required amount at the required time by tissue culture by simply setting light and temperature conditions appropriately.
Moreover, it is possible to mass-produce excellent varieties with uniform quality. By repeating subculturing at 20°C to 25°C, simple cell proliferation was suppressed and somatic embryos were gradually formed.
Since the somatic embryos were propagated by shaking culture at 0.degree. C., the formation of somatic embryos was reliably carried out with an extremely high probability, and the propagation was achieved rapidly by shaking culture. (f) Furthermore, the foliage differentiation process and rooting process of this invention are carried out at a low temperature of 15°C to 20°C, so the conditions are close to the climate of the habitat of medicinal ginseng, ensuring that foliage differentiation and rooting occur. carried out, etc., it has a remarkable effect of achieving the purpose of the invention.

[Brief explanation of drawings]

●Figure 1 is a photograph showing the biological morphology of a Panax ginseng root section placed on a standard (solid) medium.
●Figure 2 is a photograph showing the biological form of a callus derived from the roots of Panax ginseng.●Figure 3 is a photograph showing the biological form of a somatic embryo formed from the root callus of Panax ginseng.●Figure 4 is a photograph showing the biological form of a somatic embryo formed from a somatic embryo of Panax ginseng. ●Figure 5 is a photo showing the biological morphology of differentiated stems and leaves of Panax ginseng. ●Figure 6 is a seedling obtained from the callus derived from the roots of Panax ginseng. This is a photograph showing the biological form of a plant transplanted into soil.

Claims (1)

[Scope of Claims] 1. A method for producing a regenerated plant from a plant tissue piece through tissue culture through a callus induction step, a somatic embryo formation step, a shoot and leaf differentiation step, and a rooting step, wherein a predetermined reference medium is used to produce a medicinal plant. A method for regenerating a plant by tissue culturing a part of a ginseng plant, which involves forming somatic embryos from medicinal ginseng callus.
A large amount of medicinal ginseng is produced by tissue culture, which is characterized in that the somatic embryos of medicinal ginseng are grown by shaking culture in a liquid medium to which no plant growth regulators such as cytokinin or auxin are added. Production method. 2 The medicinal ginseng is Panax ginseng (Panax
ginseng CAMEYER) or Panax Japonicum CAMEYER or American ginseng (Panax quinquefolium L.) or Panax notoginseng FH
CHEN) A method for mass producing medicinal ginseng by tissue culture according to claim 1. 3. Claim 1, wherein the reference medium is any medium selected from Murashige and Skoog medium, Linsmeyer and Skoog medium, Gauslett medium, Churetzke medium, Morrell medium, and Hildebrand medium. A method for mass production of medicinal ginseng by tissue culture as described in . 4. In the shoot and leaf differentiation step, a solid medium is used as the reference medium, and the plant growth regulator added is either a combination of indolebutyric acid and benzyladenine, or a combination of indoleacetic acid and benzyladenine. 2. The method for mass producing medicinal ginseng by tissue culture according to claim 1, wherein the ginseng is cultured at a temperature of about 15°C to 20°C. 5. In the rooting step, a solid medium is used as the reference medium, and the plant growth regulator added is:
Claim 1, characterized in that the culture is performed using gibberellic acid at a temperature of about 15°C to 20°C.
A method for mass production of medicinal ginseng by tissue culture as described in . 6 In the somatic embryo formation step, a solid medium is used as the reference medium, the induced callus is repeatedly subcultured at a temperature of 15°C to 20°C to form a somatic embryo, and this somatic embryo is transferred to the liquid medium. About 20 years after transplanting
2. The method for mass-producing medicinal ginseng by tissue culture according to claim 1, wherein the shaking culture is carried out at a temperature of 25°C to 25°C.