JPH0626500B2 - Cultivation method of plants belonging to the genus Panatsux - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for culturing a plant belonging to the genus Panax. More specifically, the present invention relates to a culture method for supplying a somatic embryo of a plant belonging to the genus Panax by liquid culture to obtain a large amount of regenerated plants.

Prior art Medicinal carrots are perennial herbs that live in China, Korea, etc., and are one of the herbal medicines that have been used as herbal medicines or folk medicines as nutritional tonic or digestive system therapeutic agents.

Usually, medicinal carrots used as raw materials for crude drugs are
Mostly, the roots of 4th to 5th graders are dried, but it takes too long to grow, and diseases (tachigare disease, ragweed disease, hunten disease, etc.) and pests (Pleurotus spp. It is said to be much more valuable than the damage caused by the above (Tosho Ohsumi Tea, Agricultural Cooperative (1980)).

After that, in order to solve the above-mentioned problems, studies on medicinal carrots by callus culture were extensively conducted, but none of them could surely obtain regenerated plants. Also, 1
In 980, Hsing and Chang et al. Discovered that regenerated plants could be induced from somatic embryos [Theor. Appl. Genet., 57 , 133-135 (1980) and Natur.
e, 284 , 27 (1980))], all of them are methods of directly inducing regenerated plants by culturing somatic embryos isolated from the plant in a solid medium, and therefore, a large amount of regenerated plants can be passaged. I couldn't get it.

[Outline of Invention]

SUMMARY OF THE INVENTION The present invention aims to provide a solution to the above-mentioned problems.Adventitious embryos derived from tissue pieces of a plant belonging to the genus Panax are not directly cultured in a solid medium but are cultured in a liquid medium to give somatic embryos. A cell mass obtained by obtaining a cell mass that is proliferated and enlarged, and dividing this cell mass and then subjecting it to liquid culture to obtain the resulting cell mass containing the secondary somatic embryo, and carrying out this step at least once Alternatively, the objective is to achieve this object by culturing a cell mass containing a secondary somatic embryo on a solid medium to obtain a regenerated plant.

That is, the method for culturing a plant belonging to the genus Panax according to the present invention is characterized by comprising the following steps.

(A) To obtain an adventitious embryo by culturing a tissue piece of a plant belonging to the genus Panax in a medium.

(B) Obtaining a cell mass in which the somatic embryo proliferates and grows by subjecting the somatic embryo to culture in a liquid medium.

(C) Cell clusters containing secondary adventitious embryos obtained by dividing the cell clusters in which the somatic embryos obtained in step (b) have grown and enlarged and culturing these divided fragments in at least one liquid medium, if necessary. Carrying out the step consisting of obtaining at least once (in the second and subsequent passages, the cell mass containing the secondary somatic embryo obtained in the previous culture was divided into Subject to further culture).

(D) Obtaining regenerated plants by entrusting the cell mass obtained in step (b) or (c) or the cell mass containing secondary somatic embryos to a solid medium.

Effect The method for culturing a plant belonging to the genus Panax in the present invention is
Instead of separating the somatic embryo from the plant body each time, the once collected somatic embryo is subjected to at least one culture in a liquid medium to obtain a desired amount of somatic embryo, which is, so to speak, a so-called somatic embryo bank. ", The regenerated plant body can be arbitrarily induced from a large amount of somatic embryos supplied therefrom.

That is, by carrying out the acquisition of the somatic embryo from the plant body and the regeneration of the plant body from the somatic embryo as separate steps that do not depend on each other, the above-mentioned or other possible problems caused by the interdependence of both steps Does not exist. And, although the somatic embryo is originally collected from the plant body, after that, since it is obtained from the somatic embryo obtained by culturing in the liquid medium regardless of the plant body, the somatic embryo is cultured in the liquid medium. There is an inherent advantage, namely the possibility of being “manufactured” with increasing quantity due to somatic embryo growth.

Such an effect of the present invention is realized by culturing an adventitious embryo in a liquid medium, but it was unexpected that an adventitious embryo of a specific plant called Panax genus can be cultured in a liquid medium. It should be said. This is because the number of plants that can be cultivated in a liquid medium is extremely small, and no general knowledge has yet been known as to which plants give what somatic embryos. It is known that edible carrots (carrots) can be cultivated in a liquid medium of somatic embryos. However, even if the name "carrot" is given, edible carrots are a plant of the Sermaceae family Since it is different from the Panax genus plant which is a plant in terms of plant classification, it should not be possible to infer the nature of the somatic embryo of the Panax genus plant from the example of edible carrot.

[Specific Description of the Invention]

Plants belonging to the genus Panax The plants belonging to the genus Panax as used in the present invention refer to plants belonging to the phylum Seed plant, Apiaceae, Araliaceae, and specifically, for example, Panax ginseng (Panaxginseng), Panax japonicus (Panax japonicus) , American carrot (Panax quinquefolium), Radix Notoginseng (Panax noto)
ginseng) and so on.

Adventitious embryo The adventitious embryo referred to in the present invention refers to an embryo produced from somatic cells of a plant that undergoes a morphological change similar to a fertilized egg and differentiates into a plant body, "Iwanami Shoten," Biology Point of Time, "3rd Edition, page 1123 (1983 Year))), both are capable of differentiating into regenerated plants.

Regenerated plant The regenerated plant in the present invention refers to a state in which organs such as buds and roots are formed from plant tissue pieces through somatic embryos.

Cultivation of adventitious embryo Cultivation can be performed in any manner for the purpose of a joint. One preferred embodiment consists of the following unit steps.

(B) Induction of somatic embryo A part of the tissue of a plant belonging to the genus Panax is sterilized using any sterilizing agent such as sodium hypozincate, benzalkonium chloride, and ethylene alcohol, which has no linear effect on plant cells, After thoroughly washing with sterilized water, an appropriate amount of plant tissue (a part of roots, stems, leaves, etc., preferably roots) is excised with a sterilized scalpel, tweezers, etc. under a stereomicroscope to obtain a tissue piece.

As the basal medium, any liquid or solid medium usually used for plant tissue culture can be used. A preferred medium is Murashige-Sukuk medium or Gambouk B5 medium (both are liquid medium). Although saccharides can be mainly used as the carbon source of the medium, monosaccharides are preferable among them. A specific example of a particularly preferable carbon source is sucrose, as shown in Examples below. A plant growth regulator is usually used in this medium, and specific examples of such a substance include auxins, cytokinins, gibberellins and the like. In the present invention, auxins (eg, indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid, 1
-Naphthalene acetic acid and the like) are preferred, and particularly preferred specific examples thereof include 2,4-dichlorophenoxyacetic acid as shown in Examples below. The composition ratio of the medium is 1 to 6 times, preferably 2 to 4 times, the basic medium diluted with the carbon source and the plant growth regulator, 1 to 6% by weight, preferably 2 to 4% by weight, preferably adjusted to be contained.

The medium prepared by the above method is sterilized by an arbitrary sterilization method (preferably, it can be sterilized by a high-pressure sterilization method which is the simplest in operation), and then the tissue piece previously extracted as described above is removed. Transfer and culture.

Cultivation conditions are slightly different depending on the plant body, but usually 1 at room temperature (preferably 20 to 25 ° C.) under illumination of 500 to 500 lux (preferably 1000 to 3000 lux).
By culturing for ~ 3 months, a spherical or oval adventitious embryo can be obtained.

(B) Cultivation of adventitious embryo in liquid medium The adventitious embryo obtained in the step (a) is transplanted to a basal medium (liquid medium) containing a plant growth regulator and a carbon source, for example, 80 to 100 rpm, as described above. By culturing for 20 days to 2 months in a rotary shaking culture machine with a stroke of about 10 minutes, a cell mass in which an adventitious embryo proliferates and grows can be obtained.

A particularly preferred specific example of the plant growth regulator used here is a combination of naphthalene acetic acid (auxins) and benzyladenine (cytokinins) as shown in Examples below (the ratio of the two is almost equal). Amount is preferred).

The cell mass in which the adventitious embryo proliferates and grows thus obtained is divided or cut as it is or, if necessary, into an appropriate size, and at least one of them is transferred to a solid medium and cultured to regenerate a plant body. be able to. However, a preferred embodiment of the invention is to subject the cell mass thus obtained to a further culture in liquid medium.

This is the reason why the step (c) of the present invention is defined as the desired step as described above. In the definition of step (c), the fact that the cell mass of the somatic embryo obtained in step (b) in which the somatic embryo proliferates and expands is cultivated in a liquid medium "at least once in a passage" means the second time. The following is not the culture of the somatic embryo itself from the step (b) but the culture of the cell mass containing the secondary somatic embryo described later (here, in the second and subsequent cultures, it was obtained in the previous culture). This means that the cell mass containing the secondary somatic embryo is divided and then subjected to the following further culture).

(C-1) Induction of secondary somatic embryo The cell mass in which the somatic embryo proliferates and expands is divided into appropriate sizes (usually 10 to 20 cells), and cultured under the same conditions as in step (b). After 1 to 2 months, the small fragment (divided fragment) becomes a cell mass (including a secondary somatic embryo) that has grown and enlarged, and a large number of secondary somatic embryos can be obtained.

(C-2) Propagation of secondary somatic embryo The cell mass containing the secondary somatic embryo obtained in step (C-1) is further divided and cut into an appropriate size (usually 10 to 20),
By culturing for 1-2 months under the same conditions as in step (b), the small fragments (divided fragments) again become enlarged and enlarged cell clusters (including secondary somatic embryos), and many secondary somatic embryos are generated. Obtainable.

(C-3) Continuous Cultivation of Secondary Somatic Embryos By repeating the step (C-2) as necessary, a large amount of somatic embryos can be continuously supplied.

(D) Acquisition of regenerated plant Transfer the secondary somatic embryo or the cell cluster containing the secondary somatic embryo obtained in the above step to a basal medium (solid medium) containing a plant growth regulator and a carbon source in the same manner as above. Thus, a regenerated plant can be obtained.

Example 5th grade medicinal carrots (Panax ginseng CAMe after fruit removal)
yer) About 50 flower bud specimens were extracted from the rhizome. 3 in test tube
Add 25 ml of 1/4 diluted Murashige-Sukuk medium containing 1% by weight of sucrose and 1 ppm of 2,4-dichlorophenoxyacetic acid, sterilize by autoclave, and put the extracted flower buds into the medium one by one. , Cultured. Cultivation was performed in a rotary incubator rotating at 3 revolutions per minute under irradiation with a fluorescent lamp of 1000 lux. After 8 weeks, 5 to 10 somatic embryos become visible in one culture sample. This adventitious embryo is sucrose 3% by weight, naphthalene acetic acid 4 pp
m, benzyladenine 4 ppm in a Murashige-Skoose medium (30 ml), and the cells were cultivated at 25 ° C. in a dark room with shaking for 1 month to grow them. The cell mass in which the adventitious embryo proliferated and enlarged was divided and cut with a scalpel into 10 to 20 pieces per specimen to obtain a small fragment (divided fragment), which was subjected to culture for 2 months under the same conditions. Secondary somatic embryos develop from the divided sections.
3% by weight sucrose was added to the cell mass containing the secondary somatic embryo.
When transplanted to Murashige-Sukuk agar medium containing 1 ppm of benzyladenine and 1 ppm of gibberellin, and cultured under illumination, shoots were obtained. Further, the cell mass containing the secondary somatic embryo was further cut into 10 to 20 pieces and cultured under the same conditions. After 2 months, a cell mass containing a secondary somatic embryo was obtained in the same manner. In this way, subculture is continued about every 2 months, and after 26 months from the start of culture, when a cell mass containing an adventitious embryo is transplanted to the above-mentioned seedling medium, a shoot is obtained in the same manner. If the culture is continued for 2 months, the height is 2 to 5 cm and the leaf height is 1 to 1.
A shoot having a shape of 2 cm and a leaf width of 0.5 to 1 cm was obtained. Also, when this shoot was transplanted to Murashige-Sukuk agar medium containing 3% by weight of sucrose, 1 ppm of 2,4-dichlorophenoxyacetic acid and 1 ppm of gibberellin, root development was observed from the shoot portion after about 1 month, and after 2 months. 2-3
Grow into cm roots.

Claims (1)

[Claims] 1. A method for culturing a plant belonging to the genus Panax, which comprises the following steps. (A) To obtain an adventitious embryo by culturing a tissue piece of a plant belonging to the genus Panax in a medium. (B) Obtaining a cell mass in which the somatic embryo proliferates and grows by subjecting the somatic embryo to culture in a liquid medium. (C) If necessary, dividing the cell mass in which the adventitious embryo obtained in step (b) is hypertrophied and culturing the cell mass containing the secondary somatic embryo by culturing these divided fragments in at least one liquid medium. Carrying out the step consisting of acquisition at least once in succession (Here, in the second and subsequent passages, the cell mass containing the secondary somatic embryo obtained in the previous culture is divided into Subject to further culture). (D) Transplanting the cell mass obtained in step (b) or (c) or the cell mass containing a secondary somatic embryo into a solid medium to obtain a regenerated plant.