JPH0315326A - Production of young seedling of plant of genus pinellia - Google Patents

Abstract

PURPOSE:To effectively produce a young seedling in a short time independent of the climate, soil, season, etc., by inducing an indefinite embryo by tissue- culture, preparing a secondary indefinite embryo from the indefinite embryo and differentiating the secondary indefinite embryo under irradiation of light. CONSTITUTION:A part of a plant tissue such as root, stalk or leaf of a plant belonging to family Araceae, order Arales, Monocotyledonaeae (e.g. Pinellia ternata or Pinellia tripartia) is incised, transplanted e.g. to Murashige-Skoog medium containing a plant hormone such as auxins and cultured in the medium. The obtained indefinite embryo is cultured on an agar medium and an effective indefinite embryo is selected from the resultant secondary indefinite embryo. The selected indefinite embryo is transplanted to a medium for redifferentiation and cultured at 15-35% under the illumination of light of 500-10,000lux to effect the sprouting of the indefinite embryo. A young seedling having leaf, stalk and root is produced by continuing the cultivation. A valuable plant such as Pinellia ternata or Pinellia tripartia containing large amounts of active components of Chinese herb drugs can be produced at a low cost.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention is directed to the use of a herbaceous herb that is used as a Chinese herbal medicine. The present invention relates to a method for producing a large amount of young seedlings of plants of the genus Pinellia, such as P. aeruginosa.

(Prior art) Pinellia ternata
) and Pinellia (Red Hill) are plants belonging to the genus Pinellia, and are perennial plants that grow naturally in Japan, mainland China, the Korean Peninsula, etc. These plants are. A corm with a diameter of 1 to 3 cm is attached as an underground stem. The outer skin of this corm is removed and dried, which is called hange, and has been used in Chinese medicine since ancient times.

This half summer. In Chinese medicine, it is used to treat nausea, vomiting, and morning sickness. Stomach hydration.
Heartache, cough e. Thunder in a. It is used as a prescription medicine for sore throats. The active ingredient of Hanka is not clear, but it is homogentisic acid. β-sitosterol,
Triterpenes, various glycosides. Gluconic acid derivatives are known.

Herbal medicines using Hanka include Hanka Kobokuto, Hankashashinto, Hanka Hakugyumato, and Hagireinka Hanka. Kohankashagireito, Hankasanryo. Half-summer bath. There are Osaiko-to, Shosaiko-to, Shiwa-yu, Saikokaryu-kotsu-yu-yu, Saiko-kei-waza-to, and Sairei-yu.

Among plants belonging to the genus Pinellia, the nutrient-storing parts that exist underground, such as corms, are used as herbal medicines. For example, it is relatively easy to cultivate the mid-summer mother plant, Karasubishaku, but the corms are difficult to cultivate. Usually only 3 to 6 pieces are obtained per plant per year. If you raise the plant and collect the corm, the plant will wither. To collect corms each year, you must save them and sow them. Therefore, it is not possible to obtain large quantities of corms. Furthermore, there are restrictions on the season and region in which this plant can be cultivated, and the effective proportion of the component contained in the corm of the plant varies depending on the region and climate where it is cultivated.
The disadvantage is that the quality is not constant. In order to obtain a large quantity of crude drugs of a predetermined quality, a method for effectively producing young seedlings of plants of the genus Pinellia in a short period of time is desired.

(Problem B to be solved by the invention) The present invention solves the above-mentioned conventional problems. What is its purpose? An object of the present invention is to provide a method for effectively obtaining young seedlings of plants of the genus Pinellia in a short time regardless of climate, soil, season, etc.

(Means for Solving the Problems) The method for producing young seedlings of plants of the genus Pinellia of the present invention is as follows.
A step of inducing a somatic embryo by culturing the U weave of a plant of the genus Pinellia using a medium for inducing somatic embryos containing plant hormones; Transferring the secondary somatic embryo to a somatic embryo propagation medium to obtain a secondary somatic embryo produced from the somatic embryo; and Transferring the secondary somatic embryo to a redifferentiation medium and irradiating it with light to differentiate it into seedlings. By doing so, the above purpose will be achieved.

What is a plant belonging to the genus Pinellia in the present invention? Monocots can refer to plants belonging to the order Araceae and the family Araceae.
lia ternata), Pinel
lia), etc.

The somatic embryo induction medium used in the present invention includes: Murashigeskoog medium commonly used for plant tissue culture,
White medium. Linsmeyer-Skoog's medium, Gauss-Lesotho's medium, Heller's medium. and these modified media can be utilized.

As a medium. Both solid and liquid media, such as agar media, can be used. Usually used as a medium for somatic embryo induction. To promote the induction of somatic embryos? Contains plant hormones (auxins, cytokinins, gibbelins, etc.). As auxins. Examples include 2,4-dichlorophenoxyacetic acid (2.4-D), indoleacetic acid (IAA), and naphthaleneacetic acid (NAA). For cytokinins. for example. These include kinetin and penzyladenine. Gibberellins include, for example,
Free gibberellin, bound gibberellin, etc. are used.
As iHI type gibberellin. for example. Gibberellin GA
, ~GA6■, etc., but examples of conjugated gibberellins include dihereryl glycosyl ethers (e.g., dihererin I'l3, A6+ AZ& + 8zt. At
* r Ass glycoside, etc.), gibberellin glycosyl ester (e.g. gibberellin An. As, A
I, AI7. A311. Aaa. glycosyl esters, etc.). These plant hormones are usually
They are contained in the medium at such a ratio that the amount of each of them is 0.01 to 10 ρρm.

The somatic embryo growth medium used in the present invention is as follows. A medium commonly used for plant tissue culture (a solid medium such as an agar medium or a liquid medium) can be used. As for the above-mentioned medium, somatic embryos can be grown more effectively when a liquid medium is used than when a solid medium is used. It is preferable that the plant hormone contained in the somatic embryo propagation medium has lower auxin activity than the above-mentioned somatic embryo induction medium, and the plant hormone may not be contained at all. Plant hormones are usually contained in the medium at a rate of 4 ppm or less.

When the concentration of plant hormones is too high. The growth rate of somatic embryos decreases.

As the redifferentiation medium, a medium commonly used for plant tissue culture can be used. Plant hormones. It is preferable to set the auxin activity lower than the above-mentioned medium for somatic embryo induction and medium for somatic embryo proliferation. It may not contain any plant hormones at all. The concentration of inorganic salts in the medium is preferably low in order to redifferentiate somatic embryos into seedlings. For example, a medium in which the inorganic salt concentration is reduced to 1/3 to 1/10 of the medium used for the above-mentioned somatic embryo induction medium or somatic embryo growth medium is preferably used.

To obtain young seedlings of plants of the genus Pinellia by the method of the present invention, first, the roots, stems,
Cut out a part of a plant tissue such as a leaf. Transfer to somatic embryo induction medium. The tissue pieces used are. Roots, stems. Any part, such as leaves, may be used, but in order to efficiently induce somatic embryos. Corms are preferred.

Cultivation conditions vary slightly depending on the plant, but usually 15 to 3
At 5°C and under illumination of 500~to,ooo lux. Culture for 1 to 3 months. This makes it possible to obtain spherical or oval somatic embryos.

Next, the following somatic embryos are transplanted into a somatic embryo growth medium and cultured. Cultivation conditions vary slightly depending on the plant, but usually 1
Preferably, the culture is carried out at 5 to 35° C. and under illumination of light of 500 to 10,000 lux for 20 to 60 days.

the result. An enlarged and grown somatic embryo is obtained. This somatic embryo is a spherical or oval somatic embryo. Or they are heart-shaped and torpedo-shaped somatic embryos that have further changed from these. for example. It can be an aggregate of many spherical somatic embryos or a cell mass containing somatic embryos. These somatic embryos are divided as appropriate and transferred to a new somatic embryo propagation medium as necessary to produce secondary somatic embryos (somatic embryos that are further produced secondary to somatic embryos derived from plants). ) is obtained. By performing subculture in this manner, somatic embryos that grow quickly and can form seedlings of plants with a high content of effective components are selected.

In this way, the somatic embryos that have been appropriately propagated and selected are transplanted into a redifferentiation medium and cultured. The type of somatic embryo to be transplanted is not particularly limited, but somatic embryos at the torpedo-shaped embryo or I'fcP stage are preferred. If only one somatic embryo is transplanted, it may take a long time for redifferentiation. usually,
An aggregate of somatic embryos or a cell mass containing somatic embryos is transplanted as is.

What are the culture conditions for redifferentiation? It varies slightly depending on the plant. It is usually carried out at 15-35° C. under lux night illumination of 9 times 500-100,000. If the amount of light is too low, differentiation is difficult to occur.

When cultured under these conditions, somatic embryos become globular embryos,
The embryo grows from a heart embryo and a torpedo embryo to a mature embryo, and budding occurs. moreover. As the culture continues, the tissue grows leaves and stems similar to those of the mother plant. Rooting then occurs from the contact surface with the medium. Usually, culture is carried out for 1 to 3 months. Seedlings with leaves, stems and roots are obtained.

(Effect) In the method of the present invention, somatic embryos are used to produce Pinel Iia
Young seedlings of plants of the genus are effectively produced. Although the growth of this plant is relatively slow, multiplication can occur relatively quickly in this somatic embryo form.

Proliferated somatic embryos are relatively easy to redifferentiate, and seedlings can be easily obtained through redifferentiation. In this way, a large amount of P in a short period of time
Young seedlings of plants of the genus Inellia are obtained. If you select somatic embryos that multiply rapidly during somatic embryo multiplication. Young seedlings of fast-growing plants can be obtained.

(Example) The invention will be described with reference to the following examples.

(A) Induction of somatic embryos of Pinellia ternata
) After washing the corms with 70% ethanol, the surface was sterilized with an aqueous sodium hypochlorite solution (CI concentration 2%).

After removing the flower bud from this corm. It was cut into pieces of about 5 to 15 mm and cultured in a somatic embryo induction medium. As a medium for somatic embryo induction, 1.0% of 2.4-D is used as a plant hormone.
Murashigeskoog's liquid medium was used, containing the mineral salts in ppm and the inorganic salt concentration as %. 10 at 25°C
Under 00 lux fluorescent lighting. After 49 days of low-speed rotational culture (3 times/min), somatic embryos were formed.

(B) Propagation of somatic embryos The somatic embryos formed in section (8) were transplanted into a somatic embryo propagation medium. As a medium for somatic embryo propagation, 1.0 ppm of NAA and 1.0 ppm of BA are used as plant hormones.
Murashige Skoog's liquid medium containing the following ratio was used. 28 at 25°C under 3000 lux fluorescent lighting
Orally cultured. Somatic embryos were grown. Small fragments obtained by dividing and cutting the cell mass of a proliferated and enlarged somatic embryo. 28 under the same conditions
The cells were cultured with low speed rotation for days. Secondary somatic embryos were developed from the sections divided above.

(C) Redifferentiation of somatic embryos (Cell mass containing secondary somatic embryos) obtained in section (B)
were transplanted into regeneration medium. No plant hormones were added to the medium. Murashige Skoog agar solid medium with an inorganic salt concentration of 1/5 was used.

At 25℃, 16 hours/day. Culture was performed under illumination of 2000 lux. Approximately 50 days after transplantation, the somatic embryo underwent the process of ripening, budded, and differentiated into cotyledons. Rooting was observed after cotyledon differentiation.

The seedlings obtained as described above were complete plants similar to seedlings, and were transplanted into soil for cultivation.

Small fragments were obtained by dividing and cutting the cell mass containing the secondary somatic embryo obtained in Section (B) of Example 1. Low speed rotation culture was carried out for 28 days under the same conditions as in Example 1 (B). Further somatic embryos were developed in the same manner from the sections divided above.

In this way, subculturing was continued approximately every month, and the culture was carried out for 24 months. Next, when the proliferated somatic embryos were transplanted into the same regeneration medium as in Example 1, the same seedlings as in Example 1 were obtained. Actually JIJL (A) Induction of somatic embryos Instead of Pinelli
The only exception was that the corms of A. a. Example 1 Same as section (A). Callus and somatic embryos were observed in the corm sections.

(B) Growth of somatic embryos Except that no plant hormones were added and Murashigeskoog agar medium with an inorganic salt concentration of % was used as the medium for somatic embryo growth. This is the same as Section (B) of Example 1.

(C) Regeneration of somatic embryos Redifferentiation of somatic embryos was carried out in the same manner as in Example 1 (C) except that Murashigeskoog's agar medium with an inorganic salt concentration of 1/10 was used. Ta. After going through almost the same process as in Example 1, a large number of young seedlings of Ohange were obtained.

Small fragments obtained by dividing and cutting the cell mass containing the secondary somatic embryo obtained in Example 2 (B) were cultured at low speed rotation for 28 days under the same conditions as in Example 2 (B). did. Further somatic embryos were developed in the same manner from the sections divided above.

In this way, subculturing was continued approximately every month, and the culture was carried out for 24 months. Next, when the proliferated somatic embryos were transplanted into the same redifferentiation medium as in Example 2, the same seedlings as in Example 2 were obtained. (Effects of the Invention) According to the present invention, as described above, young seedlings of plants of the genus Pinellia are grown under the climate. It can be obtained effectively in a short period of time without being affected by soil or season. By appropriately selecting somatic embryos when propagating somatic embryos, seedlings capable of forming plants containing a large amount of the active ingredient can be obtained. Using this method, plants useful as Chinese herbal medicines, such as crow's birch and chinensis, can be produced at low cost.

that's all

Claims (1)

[Scope of Claims] 1. A step of inducing somatic embryos by culturing tissues of plants of the genus Pinellia using a somatic embryo induction medium containing plant hormones; and a step of transferring the secondary somatic embryo to a redifferentiation medium and irradiating it with light to differentiate it into young seedlings. Method for producing plant seedlings.