JP3191953B2 - Method for inducing somatic embryo of medicinal carrot and method of mass-producing medicinal carrot - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for obtaining a large amount of medicinal carrot seedlings by tissue culture.

[0002]

[Prior Art] Panax ginseng CA
Meyer), Panax japonicum CA Me
yer), American carrot (Panax quinquefolium)
L.), Panax notoginseng FH Chen
Are cultivated as important medicinal plants and are usually bred by seeds. In particular, Panax ginseng is a kind of crude drug that has been widely used as a herbal medicine and a folk medicine since ancient times, and has recently been used for health foods, and the demand is growing more and more.

[0003] However, medicinal carrots used as raw materials for crude drugs usually require four to seven years to harvest, their growth is easily affected by the weather, and diseases (eg, Negusare disease,
The cultivation requires a great deal of labor due to the large amount of damage caused by tatigare disease, hanten disease, etc.) and pests (such as weevil weevil, beetles). Therefore, stable preservation and supply of excellent varieties have been desired. However, since these Panax plants are self-reproductive and require three years to collect seeds, it is difficult to obtain a large number of excellent varieties of seedlings. And it was.

[0004] In order to solve the above problems, callus is induced from tissues such as roots, stems and leaves of Panax ginseng by using a callus induction medium containing auxins and cytokinins, and the callus is proliferated. A method of redifferentiating the callus under light irradiation (JP-A-61-216619) has been proposed.

[0005] This method requires a process of inducing roots after inducing the above-ground parts, which is troublesome, and has problems in rooting efficiency and survival of young seedlings in the field. Furthermore, the number of seedlings obtained by regeneration depends on the amount of callus, and the callus mutates at a considerable rate after repeated passage, so it is limited to the amount of normal cloned seedlings obtained from one plant. There is a problem that there is.

On the other hand, a number of techniques have been reported to induce somatic embryos from cultured cells by various methods and to regenerate seedlings in many plants such as edible carrots. Of calli from rice and cotyledons, somatic embryos and redifferentiation to obtain seedlings (WCChang, YI Hsing; THEORETIC
AL AND APPLIED GENETICS, 57, 133, (1980), JP 62
-151117 and JP-A-63-248321) have been proposed.

This method has a problem in the efficiency of inducing somatic embryos, and has a drawback that it takes a long time from the start of culture to the time when seedlings are obtained. Furthermore, since the callus is passed through the callus in the same manner as the method of redifferentiating from callus by light irradiation, the ability to form adventitious embryos and redifferentiation is lost during repeated subcultures,
In addition, since mutation occurs frequently, there is a problem that the amount of normal cloned seedlings obtained from one individual is limited.

To solve these problems, a method for inducing somatic embryos using flower buds as a material (Japanese Patent Laid-Open No. 63-133921)
), A method of growing somatic embryos using a liquid medium (Japanese Patent Laid-Open No. 62-201520), a method of inducing seedlings from somatic embryos, multiplying the seedlings and growing them (Japanese Patent Laid-Open No. 63-133922, Japanese Unexamined Patent Publication No. Hei 1-95772 and Japanese Unexamined Patent Publication No. Hei 1-98480) have been proposed.

However, all of these methods have a problem that the induction efficiency of the adventitious embryo is low and the amount and timing of the material are limited except when a specific tissue called flower bud is used. In addition, the method of growing in a liquid medium has a disadvantage that the adventitious embryo formation ability is reduced by continuing liquid culture for a long period of time, and the method of growing into a multibud is time-consuming in that it requires a rooting process, There is also a problem with survival in the field.

[0010]

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned problems, to obtain a large amount of medicinal carrot seedlings having the same trait in a short period of time, and to improve the habituation rate and field transplantation. An object of the present invention is to provide a seedling having a high survival rate in a later soil, and to provide a somatic embryo induction method suitable for the seedling.

[0011]

According to the present invention, a medicinal carrot tissue of Panax sp .
Processing time range of ~ 96 hours, processing temperature range of 30 ~ 50 ° C
Area and two straight lines (however, one straight line has a processing time of 3:00
And the processing temperature 35 ° C, the processing time 6 hours and the processing temperature
The straight line connecting the point at 30 ° C and the other straight line
Time 3 hours and processing temperature 50 ° C and processing time 96 hours
And a straight line connecting a point at a processing temperature of 35 ° C.)
Processing at the processing temperature and processing time of the area, then 25 ℃
A method for inducing somatic embryos of medicinal carrots, characterized by culturing in the following manner, and a treatment time range of 3 to 96 hours in FIG.
Range, a processing temperature range of 30-50 ° C and two straight lines (only
One of the straight lines has a processing time of 3 hours and a processing temperature of 35 ° C.
The point was connected to the point of processing time of 6 hours and processing temperature of 30 ° C.
Straight line, the other straight line is processing time 3 hours and processing temperature
50 ° C point, 96 hours processing time and 35 ° C processing temperature
Between the process temperature and the process
Treating the somatic embryo , and then culturing at 25 ° C. or lower to induce the somatic embryo, and sowing the somatic embryo in a regeneration medium, and regenerating a seedling, which comprises a seedling regeneration step. Fig. 1 shows a method for mass-producing medicinal carrots and the tissue of Panax medicinal carrots in an induction medium .
Time range of 3 to 96 hours, 30 to 50 ° C
Temperature region and two straight lines (however, one straight line
Between 3 hours and processing temperature 35 ° C and processing time 6 hours
This is a straight line connecting the point at the processing temperature of 30 ° C, and the other straight line.
Is the point where the processing time is 3 hours, the processing temperature is 50 ° C., and the processing time is 9
It is a straight line connecting the point of 6 hours and the processing temperature of 35 ° C.)
Process at the processing temperature and processing time in the area between
Somatic embryo induction step of inducing somatic embryos by culturing at 25 ° C. or lower, and subculture of a tissue containing the somatic embryo induced in the somatic embryo induction step in a growth medium containing auxin and / or a natural extract A medicinal carrot characterized by comprising a somatic embryo growth step of growing a somatic embryo, and a seedling regeneration step of regenerating a seedling by germinating the somatic embryo grown in the somatic embryo growth step using a regeneration medium. Mass breeding method. It should be noted that, in the above-mentioned present invention, “FIG.
3 to 96 hours processing time range, 30 to 50 ° C processing temperature
Degree temperature and processing temperature and processing in the area between two straight lines
The “treatment in time” is also referred to as a high-temperature treatment hereinafter. Also this high
The region of the temperature treatment condition includes points on the straight line that defines the region.
No.

The medicinal carrots belonging to the genus Panax in the present invention are carrots used for medicinal purposes among the plants belonging to the phylum Pseudophyta, Umbelliferae, Sarcophagidae, and the genus Panax. Specifically, Panax ginseng CA Meyer
), Panax japonicum CA Meyer
), American carrots (Panax quinquefolium L.),
Three carrots (Panax notoginseng FH Chen) and the like.

Among these Panax medicinal carrots, Panax ginseng is preferred from the viewpoint of high added value.

In the present invention, the tissue of a medicinal carrot treated at a high temperature in an induction medium may be any of roots, stems, leaves, and flower buds of a seedling plant, and callus, adventitious root, and multi-bud, which are culture tissues. It is preferable to once induce a cultured tissue from the viewpoint of preserving the same trait, and it is particularly preferable to induce and use multiple buds from the viewpoint of maintaining the adventitious embryogenic ability and low mutation frequency.

[0015] The somatic embryos referred to in the present invention is a fertilized egg and the same morphological changes of the kind arising from the plant of the body cells to take the process of the embryo, and good embryoid bodies (embr y oid), whole plants It has the ability to grow up to.

The high-temperature treatment referred to in the present invention refers to a treatment in which a plant tissue or an individual itself is subjected to an environment higher than a region of normal suitable temperature for growth and lower than a temperature at which it dies, for a certain period of time. Conditions are defined by temperature and time. Specifically, the normal growth temperature of medicinal carrots, that is, 15 to
25 high have 3 0~50 ℃ in 3-96 hours than ℃ (in particular 3
0 to 40 ° C for 6 to 48 hours ). And in Figure 1
In the region of processing temperature and processing time between the two straight lines.
After that, the conditions for culturing at 15 to 25 ° C. are preferable from the viewpoint of somatic embryo induction efficiency and subsequent growth.

In the mass propagation method of the present invention, the tissue containing the somatic embryo induced in the above-described somatic embryo induction step can be subcultured in a growth medium containing auxin and / or a natural extract . The somatic embryo growth step for growing somatic embryos is not essential, but by providing such a step,
Production management can be facilitated.

As the induction medium, growth medium and regeneration medium used in the present invention, any liquid or solid medium used for ordinary plant tissue culture can be used as a basic medium. Particularly preferred basic medium is Murashige and Skoog medium, rinsing Meyer and Skoog medium, Gamborg B5 medium, White medium, Hyponex medium, c Tsu <br/> Dee plant medium, or these modified medium.

In the induction medium, the basal medium may be used as it is, but it is preferable to add auxin, cytokinin and natural extract alone or in combination to the basal medium depending on the tissue properties of the medicinal carrot.

In the growth medium, an auxin and / or a natural extract is added to the above-mentioned basic medium to form a growth medium.

As the auxin to be added to the induction medium or the growth medium as needed, indole-3-acetic acid, indole-3-butyric acid, naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid and the like can be used. Especially indole-3-
It is preferable to use any one of acetic acid, indole-3-butyric acid, and naphthaleneacetic acid from the viewpoint of safety to the human body when finally serving as a medicament.

The natural extracts to be added to the induction medium or the growth medium as required include indole-3-acetic acid (natural auxin), coconut milk, casein hydrolyzate, casein, yeast extract, malt extract, peptone, Tripton or the like can be used. Coconut milk, casein hydrolyzate, casein, which is particularly effective for somatic embryo growth
Alternatively, it is preferable to add a yeast extract.

The regenerating medium may be a basal medium, but it is preferable to add cytokinin to the basal medium, while desirably, it does not contain auxin. As the added cytokinin, kinetin, benzyladenine, isopentenyladenine, zeatin, transzeatin, 4-pyridylphenylurea, etc. can be used, but kinetin or benzyladenine, which is chemically stable and has a high differentiation effect, should be used. Is preferred. The concentration of cytokinin is 0 ppm from the viewpoint of plant regeneration efficiency.
-10 ppm is preferable, especially 0 ppm-2.0 ppm
Is desirable from the viewpoint of improving the later adaptation.

As a support for the regeneration medium used in the seedling regeneration step of the present invention, a gel support such as agar, gellan gum, calcium alginate, glass fiber, ceramic fiber, rock wool or the like may be used. May be used. It is desirable to use inorganic fibers such as glass fiber, ceramic fiber, and rock wool from the viewpoints of improving the later adaptation rate, labor for transplanting to soil, and improving the survival rate in soil.

In the seedling regeneration step, 500 to 1
Irradiation at 000 lux of light for 6 to 24 hours per day is preferable in terms of growth. If the light irradiation amount is less than 500 lux and the irradiation time is less than 6 hours, the growth may be insufficient, and if it exceeds 10,000 lux, the seedlings may die, which are both undesirable.

In order to induce somatic embryos in the present invention, it is preferable to place or suspend a tissue in an induction medium and heat-treat it.

[0027]

According to the method for inducing somatic embryos or the step of inducing somatic embryos of the present invention, it is possible to directly induce somatic embryos without passing through callus from each tissue. Therefore, the efficiency of obtaining normal cloned seedlings is improved. Furthermore, when a multi-blast is once induced and used as a material tissue, the material can be preserved in a genetically stable state, and the ability to form somatic embryos can be maintained for a long period of time. In this way, seedlings with excellent traits can be supplied in large quantities over a long period of time.

Next, since the tissue containing the adventitious embryo after the adventitious embryo induction treatment continues to exhibit the adventitious embryogenic ability for a certain period of time, the embryo is obtained by transplanting it into a growth medium and subculturing it in succession. Can be In this case, the growth medium can be used in any form of solid or liquid, but it is desirable to carry out shaking culture in a liquid medium having a high growth rate.

The primary somatic embryo obtained in the somatic embryo induction step and the secondary somatic embryo obtained in the somatic embryo propagation step as described above are transplanted to a regeneration medium when they become mature embryos. Redifferentiate to seedlings. According to the method of the present invention, since the somatic embryo is directly transformed into a seedling, the rooting process can be omitted, and the above-ground part and the underground part simultaneously differentiate and grow, so that a healthy seedling with well-developed vascular bundles can be obtained.

When inorganic fibers are used as a support for the regeneration medium, the roots extend well into the support,
Unlike the gel support material, the root development is not inhibited by culture under light irradiation. Further, since the roots are well elongated in the gaps between the fibers, if the support material is transplanted to the soil, the rooting rate can be improved without damaging the roots.

[0031]

Example 1 From the roots of Panax ginseng 5 years, sucrose was 30 g /
1 and Murine Skoog medium (hereinafter abbreviated as K1 medium) supplemented with kinetin at 1 mg / l at 20.degree.
Multiple shoots were induced under light irradiation at 0 lux for 14 hours.
The tissue excluding the leaves and roots of the multibud was placed on a K1 medium and subjected to a high-temperature treatment at 40 ° C. for 12, 24, or 48 hours. Next, the temperature is periodically changed in the range of 12 to 25 ° C. every day, and the light irradiation amount is periodically changed in the range of 0 to 10,000 lux (16 per day).
The irradiation time is 5,000 to 10,000 lux, and the light irradiation is not substantially performed for 8 hours. ) Culture was continued for 6 weeks.

Using six sections of the tissue under each high temperature treatment condition, the number of tissues in which the adventitious embryo was formed was examined.
Four sections were obtained at 12 hours at 40 ° C., one section was obtained at 24 hours at 40 ° C., and one section was obtained at 48 hours at 40 ° C. The number of the sections in which the somatic embryos were formed was 0 in the case where the high-temperature treatment was not performed.

Example 2 The same buds as in Example 1 were placed on the same K1 medium as in Example 1, and the temperature conditions for the high-temperature treatment were 30 ° C., 35 ° C., 50 ° C.
℃, 65 ℃, treatment time 3 hours, 6 hours, 12 hours, 1
Culture was continued under the same conditions as in Example 1 except that the time was changed to 8, 24, and 96 hours, and the results were observed after 4 weeks. Table 1 shows the number of tissues in which the adventitious embryos were formed, using 6 sections of tissue under each high-temperature treatment condition, as in Example 1. In the case of no heat treatment, the number of somatic embryos was 2/9.

FIG. 1 shows the relationship between the treatment temperature and treatment time and the adventitious embryo induction efficiency by combining the results of Example 1 and Example 2. It can be seen that the highest range of somatic embryo induction efficiency is at a treatment temperature of 30 ° C to 40 ° C and a treatment time of about 12 hours to 24 hours. However, it was also suggested that somatic embryos could be induced to some extent by treatment at high temperatures for a short time and at low temperatures for a long time.

In FIG. 1, the horizontal axis indicates the processing time, and the vertical axis indicates the processing temperature. The efficiency of inducing somatic embryos was as follows: ×: less than 30%, Δ: 3
0% or more and less than 50%, □ is 50% or more and less than 65%, and ○ is 65% or more. The region between the two straight lines is a region with a high guiding efficiency, and a diamond-shaped region surrounded by a solid line is a region with a particularly high guiding efficiency. Note that the dotted line is obtained by extending the straight line, and the area between the two dotted lines is expected to have high efficiency.

[0036]

[Table 1]

Example 3 The same buds as in Example 1 were treated at a high temperature of 35 ° C. for 12 hours, and cultured for 6 weeks under the same conditions as in Example 1.
Tissues containing somatic embryos were induced. A somatic embryo alone is separated from the tissue by tweezers, and a medium having the same composition as the K1 medium except that it does not contain kinetin (hereinafter abbreviated as MS medium)
Then, the cells were transplanted to those using ceramic fibers as a support material, and culturing was continued for 8 weeks under the same conditions as in Example 1 to obtain seedlings having a total length of 3 cm to 6 cm.

Example 4 The same buds as in Example 1 were treated at a high temperature under the same conditions as in Example 3 and cultured for 6 weeks to induce somatic embryos. The tissue containing this somatic embryo was treated with 2,4-dichlorophenoxyacetic acid at 1 mg /
l, kinetin 0.1 mg / l, coconut milk 70
ml / ml, M with gellan gum 2 g / l as support material
The cells were transplanted to an S medium (hereinafter abbreviated as a CM medium) and grown in a dark place at 25 ° C. for 3 weeks. The grown tissue was transplanted to a CM liquid medium containing no gellan gum, and 150 rpm in a dark place at 25 ° C.
When the m-rotational shaking culture was continued for 3 weeks, 2.0 g of somatic embryos (about 40 seedlings) were obtained. This somatic embryo was transferred to the same MS medium as in Example 3 and continued to be cultured for 8 weeks. As a result, a seedling similar to that in Example 3 was obtained.

[0039]

Industrial Applicability According to the present invention, somatic embryos of Panax medicinal carrots are directly induced from various tissues by high-temperature treatment, and seedlings are directly obtained from the somatic embryos to obtain seedlings. Can be. In addition, the growth of somatic embryos in a growth medium has the excellent effect of efficiently mass-producing seedlings, and the use of multiple buds as a material makes it possible to maintain excellent traits genetically and stably. The effect is that it is possible to supply excellent seedlings over a long period of time.

[Brief description of the drawings]

FIG. 1 is a diagram showing the relationship between the treatment temperature and treatment time of high-temperature treatment and the efficiency of somatic embryo induction.

Continuation of the front page (58) Fields investigated (Int. Cl. ⁷ , DB name) A01H 4/00 BIOSIS (DIALOG) JICST file (JOIS)

Claims (8)

(57) [Claims] 1. A treatment time region of 3 to 96 hours in FIG.
A processing temperature range of 30 to 50 ° C and two straight lines (however,
One straight line indicates the point where the processing time is 3 hours and the processing temperature is 35 ° C.
A straight line connecting the points of processing time 6 hours and processing temperature 30 ° C
And the other straight line represents a processing time of 3 hours and a processing temperature of 50 hours.
℃ point and processing time 96 hours and processing temperature 35 ℃
Between the processing temperature and the processing time in the area between
A method for inducing somatic embryos of medicinal carrots , wherein the treatment is performed at 25 ° C. or lower . 2. A treatment time region of 3 to 96 hours in FIG.
A processing temperature range of 30 to 50 ° C and two straight lines (however,
One straight line indicates the point where the processing time is 3 hours and the processing temperature is 35 ° C.
A straight line connecting the points of processing time 6 hours and processing temperature 30 ° C
And the other straight line represents a processing time of 3 hours and a processing temperature of 50 hours.
℃ point and processing time 96 hours and processing temperature 35 ℃
Between the processing temperature and the processing time in the area between
And then culturing at 25 ° C. or lower to induce somatic embryos, and a seedling regeneration step of regenerating seedlings by causing the somatic embryos to germinate in a regeneration medium. Mass breeding method of medicinal carrot. 3. A treatment time region of 3 to 96 hours in FIG.
A processing temperature range of 30 to 50 ° C and two straight lines (however,
One straight line indicates the point where the processing time is 3 hours and the processing temperature is 35 ° C.
A straight line connecting the points of processing time 6 hours and processing temperature 30 ° C
And the other straight line represents a processing time of 3 hours and a processing temperature of 50 hours.
℃ point and processing time 96 hours and processing temperature 35 ℃
Between the processing temperature and the processing time in the area between
Embryo induction step of inducing somatic embryos by culturing at 25 ° C. or lower, and a growth medium containing auxin and / or a natural extract containing a tissue containing the somatic embryos induced in the somatic embryo induction step. And a seedling regeneration step of regenerating seedlings by regenerating the somatic embryo grown in the adventitious embryo growth step using a regeneration medium by subculturing the somatic embryo by subculture. A mass breeding method for medicinal carrots, characterized in that: 4. The method according to claim 3 , wherein the growth medium is a medium containing coconut milk. 5. The method according to claim 5, wherein the seedling regeneration step is 500 to 10,000.
Irradiate with a light of 00 looks for 6 to 24 hours per day,
The method according to claim 2 , 3 or 4 . 6. The method according to claim 2 , wherein the regeneration medium is an auxin-free medium. 7. The method according to claim 2 , wherein an inorganic fiber is used as a support for the regeneration medium. 8. The tissue of ginseng to be processed by the induction medium is a multiple shoots, claim 1, 3, 4, 5, 6 or 7
The method described in.