JPH0653034B2 - Raising seedlings of Dicentra spp. - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for raising seedlings of the genus Dicentra, and more particularly to a method for raising seedlings of the genus Dicentra which is suitable for obtaining a large number of seedlings in a short period by organ culture. It is about.

[Prior Art] Dicentra is a perennial of the poppy family, a typical alpine plant, and blooms pink or white flowers in July and August. In other words, Dicentra plants are popular as ornamental plants, and the alkaloid decentrin (C
It is a useful plant containing components having an anesthetic action such as ₂₀ H ₂₁ O ₄ N) and protopin (C ₂₀ H ₁₉ O ₅ N).

There are two kinds of methods for growing the plant of the genus Dicentra: seedling-based growth and stock-based growth. However, it is difficult to germinate from seedlings, and both of these methods of seedling and stock split,
The growth rate was extremely low.

[Problems to be Solved by the Invention] As described above, the growth rate of Dicentra spp. Is extremely low by both seedling and strain dividing methods, and it takes 2 years or more to grow into a flowering strain. For example, mass production of Dicentra plants is extremely difficult.

Furthermore, conventional methods are seasonally sensitive and can only produce seedlings in spring.

At present, a large amount of seedlings are obtained by tissue culture of the plant by western orchid cultivation, etc., but it is not known at all to obtain seedlings from a plant of the genus Dicentra by such a method.

In addition, in tissue culture of normal plants such as cabbage, when the grown callus is transferred to an adventitious bud induction medium, immediately start irradiation of light, but in the culture of the genus Dicentra when light irradiation is started at the same timing, The number of adventitious buds that are induced is small, and the number of days until induction is long, which is problematic in terms of mass production.

The present invention was made in order to solve the above-mentioned problems of the prior art, and a large number of seedlings of Dioscorea plants in a short period of time,
Moreover, it is an object of the present invention to provide a method for raising seedlings of the genus Dicentra which can be produced throughout the year regardless of the season.

[Means for Solving the Problems] In order to achieve the above object, the composition of the method for raising seedlings of the Dioscorea plant according to the present invention has a structure in which a part of the organs of the Dioscorea plant is collected, A step of inducing callus by culturing in a callus induction medium containing at least auxins as plant hormones, a step of repeatedly multiplying the callus using a callus proliferation medium, and inducing adventitious buds of the proliferated callus Transfer to a culture medium for inducing adventitious buds, and the step of transferring the adventitious buds to a rooting medium for rooting to form seedlings, and grow rooted seedlings to regenerate the plant In the method for raising seedlings of the genus Phytolacca, the callus induction step and the callus multiplication step are performed in a dark place as a light condition during culture, and in the adventitious bud induction step, at least 1 week and at most 3 weeks After culturing in a dark place for a while, culturing is performed under light irradiation of 1000 to 15000 lux, and even in the rooting step, 1000 to 150
The culture was performed under the light irradiation of 00 lux.

The drawings are process explanatory diagrams of a seedling raising method for Dicentra plants.

The present invention, as shown in the drawings, is an organ (leaf piece) of Dicentra
Are collected (step), the callus is induced in a callus induction medium (step), and the callus is repeatedly transplanted in large numbers (step). The grown callus is transferred to another adventitious bud induction medium to induce adventitious buds (step), the adventitious buds are transferred to a rooting medium and rooted (step), and the seedlings are grown to produce Dicentra spp. Regeneration (process) is a method consisting of each process.

Here, plant organs include roots, stems, leaves, flowers, fruits, etc. Generally, a group of cells having similar shapes and functions is called a tissue. Become.

In addition, botany callus refers to an amorphous plant cell mass that is experimentally formed by cell division.

In addition, the medium is formed into test tubes or culture vessels.

Next, the method of the present invention will be described more specifically.

First, a part of an organ such as leaves, stems and roots of a Dicentra plant, such as a leaf piece, is cut out and cultured in a callus induction medium to induce callus.

As the callus induction medium, Gamborg's B5 medium, Murashige-Skoog medium, White medium, Rinsmeier-Skoog medium, which is commonly used for plant tissue culture,
And these modified media etc. are used, and plant growth hormones of auxins and cytokinins are added thereto.

In addition, the Gamborg B5 in the above and the following description
Regarding the medium, Gamborg, Miller, Ojima (O,
L., Gamborg, R .; A. Miller, K .; Oj
Exp. Cell Res. 50, pages 151-158, 1968.

Here, at least auxins are always added as plant growth hormones, and the types of auxins include, for example, 2.4-dichlorophenoxyacetic acid (2.4
-D), indole acetic acid (IAA), naphthalene acetic acid (NAA), indole butyric acid (IBA) and the like. The types of cytokinins include, for example, kaitinene, benzyladenine, zeatin and the like.

Auxins are usually always added to the medium at a concentration of 0.01 to 10 ppm. The cytokinins are added to the medium at a concentration of 10 ppm or less, but they are not always necessary. On the other hand, if the added amount (concentration) of these exceeds 10 ppm and is too large, death, inhibition of growth and the like are observed. When the amount of auxin is less than 0.01 ppm, organs such as adventitious roots other than callus are directly differentiated, or killed, and growth inhibition is observed.

Leaf pieces of Dicentra were used for callus induction at an ambient temperature of 15-
When cultured in the dark at 30 ° C., callus is formed on the cut surface of the leaf piece after 15 to 30 days.

Next, the cultured callus is transplanted to a callus growth medium to grow the callus.

As a medium for growing callus, a medium usually used for culturing plant tissue such as G5 Borg medium is used, and a solid medium such as agar medium containing agar or a liquid medium containing water is used. Alternatively, the callus induction medium described above may be used.

Auxins and cytokinins as plant hormones that promote proliferation may be added at a concentration of 10 ppm or less, or may be not added. If the amount of these is too large (more than 10 ppm), death, growth inhibition, etc. are observed.

Culturing is usually carried out in the dark at an ambient temperature of 18 to 30 ° C. The proliferated callus is further proliferated by repeating divided transplantation as shown in the process of the drawing.

Then, the grown callus is transferred to a medium for adventitious bud induction to induce adventitious buds.

As the adventitious bud inducing medium, a Muradige-Skoog medium that is commonly used for plant tissue culture is used. Muradige
The composition of the components of the Skoog medium is T. Murashig
e, F. Physiol Plant by Skoog
Arum, 15, pages 473-497, 1962.

The adventitious bud induction medium contains cytokinins such as benzyladenine and kinetin as a plant hormone for promoting adventitious bud induction at a concentration of 5 pp in the above-mentioned Muradige-Skoog medium.
Add at m or less or do not add. The auxins are not added or, if added, are lower than the callus growth medium at 2 ppm or less. here,
The result of adventitious bud induction was better when cytokinins were added, and the better result when auxins were not added. When the concentration of cytokinins exceeds 5ppm and is too much, they become brown and die, and auxins have a concentration of 2ppm.
If the number exceeds the above range, adventitious buds are not differentiated but only callus is propagated.

After callus was placed in such an adventitious bud induction medium and cultured at ambient temperature of 15 to 30 ° C. in the dark for at least 1 week after callus placement and for a maximum of 3 weeks in the dark, 1000-1
When cultured under irradiation of light of 5000 lux, 15-40
Adventitious shoots are induced in the day.

Next, the adventitious buds thus induced are transferred to a rooting medium for rooting.

As the rooting medium, Gamborg's B5 medium usually used for plant tissue culture is used, and indolebutyric acid (IBA) indoleacetic acid (IBA) is used as a root-promoting plant hormone.
Add auxins such as AA) at a concentration of 5 ppm or less, or do not add them. In addition, cytokinins are usually not added or, if added, 0.5 ppm or less. Here, adventitious shoots die when auxins are added in excess of 5 ppm, and rooting is stopped when cytokinins are added in excess of 0.5 ppm.

Adventitious buds are planted in such rooting medium at an ambient temperature of 15 to 3
When the culture is performed by irradiating light of 1000 to 15000 lux under the temperature condition of 0 ° C., rooting takes place in 20 to 50 days, and seedlings of the genus Astragalus are obtained, and the plant body is regenerated by growing the seedlings. It

[Operation] The operation by the above technical means (each step of the seedling raising method) is as follows.

According to the conventional methods of seeding and dividing, the growth rate of Dioscorea plants is extremely low, but in the method of the present invention, callus is effectively induced in a medium containing a plant hormone. Proliferation is rapid in this callus morphology. Therefore,
By inducing adventitious buds from the grown callus and rooting them, a large amount of seedlings of the plant of the genus Dicentra can be obtained in a short period of time.

Further, according to the conventional method, the time when the seedlings are obtained is influenced by the season and can be obtained only in the spring, but according to the method of the present invention, the seedlings can be obtained throughout the year without being influenced by the season.

[Examples] Examples of the present invention will be described below.

Example 1 (1) Collection of leaf pieces of Dicentra japonicum (see the process in the drawing) Seedlings of Dicentra are cut into an appropriate size, and 70% of them are cut.
It was immersed in an aqueous ethanol solution for 60 seconds and further immersed in an aqueous sodium hypochlorite solution (Cl ⁻ concentration of 2.0%) for 12 minutes for sterilization treatment. Wash this with sterile water 3
After repeating this operation three times, the leaves were cut.

(2) Induction of callus (see the process in the drawing) Leaf pieces with three leaves attached were placed on a callus induction medium.

2.4D as auxins for callus induction medium
Was used at a ratio of 0.2 ppm, and as cytokinins, a solid medium (Gellite 0.2%) of Gamborg B5 containing benzyladenine at a ratio of 0.1 ppm was used.

The callus-inducing medium on which the leaf pieces were placed was placed at an ambient temperature of 24
When culture was carried out at 20 ° C in the dark for 20 days, callus was formed at the cuts of the leaf pieces.

(3) Propagation of Callus (Refer to the Steps in the Drawing) The callus formed in (2) was transplanted into a callus growth medium.

As a culture medium for callus growth, 2.4 as auxins
The solid medium (gellite 0.2%) of Gamborg B5 containing 0.2 ppm of D and benzyladenine of 0.2 ppm as cytokinins was used.

The callus growth medium in which the callus was transplanted was set at an ambient temperature of 2
Incubate for 30 days in the dark at 4 ℃,
The callus was repeatedly transplanted as shown in FIG.

(4) Induction of adventitious buds (see the process in the drawing) The proliferative callus obtained in (3) was transplanted to an adventitious bud induction medium.

As a medium for adventitious bud induction, 0.1% casamino acid was added to Murashige-Skoog solid medium (gellite 0.2%) containing benzyladenine as a cytokinin at a ratio of 0.2 ppm and containing no auxin. , Which contained 25 ppm of adenine sulfate was used.

The adventitious bud-inducing medium to which the growth callus was transplanted was cultivated at an ambient temperature of 24 ° C. under a dark condition for 12 days and then under irradiation of 3000 lux. After placing the floor, about 20
Induction of adventitious buds started in a day, and 20 or more adventitious buds were induced in a test tube having a diameter of 25 mm in about 30 days.

(5) Rooting (Refer to the process in the drawing) The adventitious bud induced in (4) was cut from the root and transplanted onto a rooting medium.

As the rooting medium, a solid medium of Gamborg B5 containing indolebutyric acid as an auxin at a ratio of 0.2 ppm and containing no cytokinins (gellite 0.2
%) Was used.

The rooting medium to which the adventitious bud was transplanted was placed at an ambient temperature of 20 ° C.
The culture was performed under a light condition of 000 lux. About 2 after bed
Rooting started in 0 days, several roots of 20 to 40 mm were generated in 40 days, and seedlings of Dicentra communis that could be transplanted into pots were obtained.

As described above, according to this example, seedlings can be obtained in a shorter period of time as compared with the conventional method of germinating seedlings or the method of dividing the seedlings.

Example 2 (2) Collection of leaf pieces of Dicentra esculenta This is the same as in Example 1 (1).

(2) Callus induction The same as in Example 1 (2).

(3) Propagation of callus The callus formed in (2) is used as an auxin.2.
4D was contained at a ratio of 0.2 ppm and transferred to a liquid medium of Gamborg B5 containing no cytokinins, and cultured in a reciprocating orbital shaking incubator.

By replanting callus in the same medium every two weeks,
Callus is suspended in a liquid medium and its growth rate is
The value was larger than that on the solid medium. Other culture conditions are the same as in Example 1 (3).

(4) Induction of Adventitious Bud The callus in the suspended state obtained in (3) above was used in Example 1.
The suspension was transplanted onto the adventitious bud medium used in (4) in a suspended state and cultured under the same temperature and light conditions as in Example 1 (4).

Induction of adventitious buds started about 20 days after transplantation, and after about 30 days, a large number of adventitious buds of almost uniform size were induced.

(5) Rooting When the adventitious buds obtained in (4) above were cultured in the same manner as in Example 1 (5), seedlings of Dicentra communis that could be transplanted into pots were obtained in about 40 days.

[Effects of the Invention] As described in detail above, according to the present invention, there is provided a method for raising seedlings of the genus Dicentra which can produce a large number of seedlings of the genus Dicentra in a short period of time and are not affected by the season, and can be produced throughout the year. Could be provided.

[Brief description of drawings]

FIG. 1 is a process explanatory diagram of a seedling raising method for Dicentra plants.

Claims (5)

[Claims] 1. A part of an organ of a plant of the genus Dicentra is collected,
Inducing callus by culturing the collected one in a callus-inducing medium containing at least auxins as plant hormones, a step of repeatedly dividing and dividing the callus by using a callus-proliferating medium, and multiplying The step of inducing adventitious shoots by transferring the callus to the adventitious shoot induction medium, and the step of transferring the adventitious shoots to a rooting medium and rooting them as seedlings, and growing the rooted seedlings In the method for raising seedlings of the genus Dicentra which regenerates the plant body, the callus induction step and the callus multiplication step are performed in a dark place as a light condition during the culture.
After culturing in the dark for a week, culturing under light irradiation of 1000 to 15000 lux, and also in the rooting step, culturing under light irradiation of 1000 to 15000 lux. Method. 2. The callus induction medium according to claim 1, wherein the concentration of auxins is 0.01 to 10 ppm and the concentration of cytokinins is 10 ppm or less. . 3. The method for raising seedlings of the genus Dicentra according to claim 1, wherein each of the auxin and cytokinin concentrations in the callus growth medium is 10 ppm or less. 4. The adventitious bud induction medium has an auxin concentration of 2 ppm or less and a cytokinin concentration of 5 ppm.
The method for raising a Dicentra plant according to claim 1, characterized in that: 5. The rooting medium has an auxin concentration of 5 pp.
2. The method for raising seedlings of the genus Dicentra according to claim 1, wherein the concentration of cytokinins is 0.5 ppm or less and 0.5 ppm or less.