JPH08131000A - Large multiplication of alpine plants such as papaver fauriei or aquilegia akiensis - Google Patents

Abstract

PURPOSE: To enable seasonless admiration of alpine plants such as RISIRI HINAGESI (Papaver fauriei) or MIYAMA ODAMAKI (Aquilegia akiensis), very difficult in seed multiplication by effecting a large volume of proliferation using specifically treated tissue sections of these plants. CONSTITUTION: The tissue sections of an alpine plant such as RISIRI HINAGESI or MIYAMA ODAMAKI are inoculated and culture in callus-inducing solid culture medium prepared by adding plant hormones comprising auxins and cytokinins to the basic culture medium A containing a carbon source such as sucrose, preferably of 1.0-3.0wt.% concentration and reduced concentrations of inorganic salts, adjusted in pH to 5.0-6.0. Then, the induced calluses are transplanted to the differentiation solid medium prepared by adding the plant hormones such as auxins and cytokinins to the base culture medium B containing carbon source and reduced concentration of inorganic salts and having an adjusted pH to effect culture in a vessel under aerobic conditions to conduct differentiation and propagation whereby these alpine plants are proliferated in a large volume.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for mass-proliferating alpine plants such as Lycoris sinensis and Rhizobium alba, more specifically, a section of the tissue of an alpine plant such as Rhizophora communis and Rhododendron japonicus is specified. By using a callus induction solid medium, a solid medium for differentiation, by culturing according to a specific culture process, it is conventionally difficult to cultivate on flatland, seeds are hard to form, and a large amount of the same strain can be propagated. It is difficult, and because it grows only locally, it is possible to mass-produce stable and high-yield mass of alpine plants such as Rhododendron japonicus and Miyama columbine, which had been difficult to spread excellent hybrids widely. The present invention relates to a method for mass-proliferating alpine plants such as Rhizoma communis and Rhododendron japonicum by various tissue cultures.

[0002]

2. Description of the Related Art Conventionally, adventitious buds, adventitious roots, flower buds, calli, etc. are induced, formed and grown by aseptically culturing a section of a plant tissue on a medium to propagate the plant. , The growth method by tissue culture of the plant,
Studies have been conducted on various plants, and various techniques have been developed so far.

With the development of such plant tissue culture technology, various plant production methods and growth methods have been developed using the technology, and specific medium composition, culture conditions, etc. suitable for each plant are specific. Some of them have already been put into practical use as production methods for virus-free strains, large-scale breeding methods, etc., and for example, regeneration of disease-free solids by shoot apical tissue culture (growth point culture) of dahlia and potato, orchid (cymbidium). Reproduction of protocomb-like body (original mass-like tissue mass PLB) by shoot apical tissue culture (genus), and various technologies such as mass-propagation technology of genetically uniform seedlings by division growth have been developed and put into practical use. ing.

At present, such tissue culture technology is being applied in various fields from plants such as flowers and vegetables to woody plants such as fruit trees, flowers and trees.
Especially in Japan, orchids, carnations, chrysanthemums, lilies,
The seedling production technology of horticultural crops such as gypsophila, Saintpaulia, gerbera, cyclamen, strawberry, etc. is becoming established.

However, such a tissue culture technique for plants is not yet a completed technique, and there are many unknown fields, and there are many places to wait for future research. With regard to vegetative propagation, except for Cymbidium, which is being researched, the medium composition and culture technology have not yet been completed to the extent that they are generalized. The growth system by tissue culture has not been established.

[0006] Regarding general flowers and ornamental plants, research and development such as formation of multiple buds by liquid shaking culture of proliferating tissue, induction of multiple buds in medium supplemented with hormones, mass multiplication of seedlings, etc. are actively conducted. However, there are still many problems to be solved in the future, such as an extremely high risk of mutation.

[0007] In addition, cyclamen, primula and the like, which are cultivated by seed reproduction in bulbs and perennials, lack genetic homogeneity due to genetic isolation, and the amount collected is small,
Due to problems such as lack of excellent seeds, attempts have been made to grow excellent solids by tissue culture.

Further, storage maintenance and the seed for the mother strains, that the seed propagation and F ₁ solid productive vegetables, F ₁ solid flowers, grown by tissue culture, supplied as seedlings vegetative propagation system Although such attempts have been made, the conventional technique of vegetative growth using such a seed breeding system is only partially put into practical use. In this way, plant tissue culture technology,
As a method for inducing, producing, and propagating various plants, induction and proliferation techniques by tissue culture of various ornamental plants have been proposed.

That is, for example, the shoot apex of a useful annual plant is extracted, and this is transplanted to an artificial medium containing an inorganic salt composition and a plant growth hormone, which is 0.5 to 10 rpm.
A method for mass-proliferating useful annual plants has been proposed, which comprises rotating and cultivating shoot primordia at a rotation number of 10 and then statically culturing the resulting shoot primordia to form seedlings (Japanese Patent Laid-Open No. Sho-06-1999). 59-
No. 132823). However, this method mainly uses shoot-primitives (Sho-ot primordia) to artificially mass-produce useful substances consisting of secondary metabolites such as plastids, vacuoles, oil bodies, and stored substances. And, regarding the growth of ornamental plants, it only refers to petunia, morning glory, and the like.

Further, a method of redifferentiating a plant which germinates and roots from callus under light irradiation from a tissue piece of the plant, and a growth medium therefor, as a growth medium, 0.1 to 10 ppm of cytokinins, which are plant hormones, And an MS medium containing 0.01 to 10 ppm of auxins have been proposed (JP-A-3-139224). However,
This is an examination of a specific medium composition, culture conditions, etc. for Asteraceae plants suitable for obtaining a large number of Asteraceae plants in a short period of time.

A method for regenerating a plant, which comprises inducing callus from a tissue piece of a plant, culturing the callus to differentiate adventitious buds, and then culturing the adventitious buds to obtain seedlings , And a method for multiplying seeds and seedlings thereof have been proposed (Japanese Patent Laid-Open No. 4-45730). However, this method utilizes differentiated adventitious buds, and is intended for plants of the genus Astragalus.

Further, a tissue section of a flowering plant or a tissue section of a regenerated plant obtained by culturing the same is cultured in a medium to be redifferentiated to obtain a regenerated plant, and then the regenerated plant is regenerated. A method for culturing flowering plants has been proposed in which a flowering plant is cultured by culturing in a medium having a composition different from that at the time of differentiation (JP-A-4-30733). However, this method is an examination of the medium composition of the medium used for the redifferentiation culture, and the medium used for the flowering culture, and the target plant is a plant belonging to the family Echinacea, Caryophyllaceae, and Solanaceae, especially the genus Torenia. The plants are only illustrated.

Recently, however, there has been proposed a technique related to in-container flowering, in which a plant is cultivated in a hermetically-sealed container to cause it to flower, and the flower can be viewed as it is. As an example of such a proposal, for example, a stem section of a plant is aseptically cultured in a primary basal medium to form adventitious buds, then this is adjusted to a specific inorganic salt concentration, and a dwarfing agent is added. A cultivation method has been proposed, which comprises placing on a secondary basal medium and irradiating with natural daylight color fluorescent lamp for a short day to perform hermetically culturing (Japanese Patent Laid-Open No. 60-221020).
Issue). However, this method utilizes adventitious buds, and the target plant is also limited to Torenia (Japanese name Hanaurikusa).

Further, aseptic whole plants of seedlings are planted in an MS medium containing a specific component as a dwarfing agent in a transparent container, intermittently irradiated with light for a long period of time, and aseptically hermetically sealed. A cultivating method consisting of the following has been proposed (Japanese Patent Laid-Open No. Hei 2)
-200121). However, this method
The whole plant of a young plant is used, and the dwarfing conditions of the larvae belonging to the family Liliaceae are examined.

As described above, in order to establish an in-container cultivation technique in which a plant is cultivated in a closed container and can be viewed as it is, a technique of growing the plant in a closed container in a stable and high yield, Developed dwarfing technology to properly grow in a closed container, flowering technology to differentiate flower buds and bloom
Although it is assumed to be established, in the first place, it is very difficult to cultivate a plant in a closed container and make it bloom, so far, as described above, flowering in a closed container of dwarf torenia, sennichoukou Although there are reports of flowering in a closed container, most of the ornamental plants that grow dwarf plants in a closed container and are used for ornamental use do not have flowers.

[0016] As described above, generally, with the development and widespread use of techniques for propagating various plants by tissue culture, individual culture conditions suitable for various plants have been researched, developed, and are being established. However, there are not many that have reached the level of practical use, let alone cultivate the plant in a closed container or an air permeable container to differentiate flower buds to flower, and to watch as it is in the container container There are few cases of research on this, and it is in a situation where it has not been practically used.

[0017]

In such a situation, the present inventors have cultivated a plant in a container to differentiate flower buds, make them bloom, and then to view the plant as it is. With the ultimate goal of establishing training techniques,
The basic technology on which the technology is based, that is, a method for growing a plant in a stable and high yield, dwarfing conditions for stable dwarfing, stable flower bud differentiation, and long-term flowering. We started the basic research to study various cultivation conditions and so on.

According to the research conducted by the present inventors and the findings to date, in order to establish such a plant breeding technique, the above-mentioned various plant breeding techniques and dwarfing techniques, which are the technical bases, are established. Needless to say, the development of basic technologies such as flowering technology is a major prerequisite, but each of these technologies is, in fact, completely different depending on the type of individual plant. Even for Sensiko, even if the cultivation method and conditions are diverted to other plants as it is, it is almost impossible to achieve the intended purpose, and it is unique to each type of plant. It is necessary to develop and establish the cultivation method and cultivation conditions of.

Under such circumstances, the present inventors have found that it is difficult to stably supply seedlings among various plants due to the variation in seeds in the conventional hybrids, and in the hybrids. Since it is difficult to grow a large amount of uniform plants with variations in appearance and color, and it is difficult to popularize excellent varieties, it is strongly requested to develop a new breeding technology that can solve these points. As a result of intensive research conducted with the aim of developing a method for growing stable, high-yielding alpine plants such as Rhinoterium populata and Rhododendron japonicus, a specific culture process and a specific medium It has been found that the intended purpose can be achieved by combining the composition and the culture conditions, and the present invention has been completed.

That is, an object of the present invention is to provide a method for proliferating a large amount of alpine plants such as Lycoris radiculata and Rhododendron japonicus, which were conventionally considered difficult to breed excellent varieties produced by crossing. is there.

[0021] The present invention also provides a method for growing a plant in a stable and high yield by tissue culture using a tissue section of an aseptic plant of an alpine plant such as Rhododendron japonicus and Miyama columbine. The purpose is to do.

Further, the present invention provides an algal plant such as Rhinotorium poplarum, Rhododendron japonicum, which is aseptically cultivated in a container to induce callus, redifferentiate, and can be directly observed in the container. It is intended to provide a method for mass-proliferation.

Furthermore, the present invention utilizes the callus of alpine plants such as Lycoris communis and Rhododendron chinensis formed under a specific culture process, a specific medium composition, and a specific culture condition to utilize the callus of the plant. It is an object of the present invention to provide a method for growing a large amount in a stable and high yield and growing in a container.

[0024]

A first aspect of the present invention for achieving the above object is to provide a carbon source containing a tissue section of an alpine plant such as Lycoris sinensis and Rhizobium alba, A basic medium (A) is a synthetic medium in which pH is adjusted and the concentration of inorganic salts is reduced, and the induced callus is planted and cultured in a callus induction solid medium in which plant hormones composed of auxins and cytokinins are added. ,
A synthetic medium containing a carbon source, pH-adjusted, and having a reduced inorganic salt concentration is used as a basic medium (B), and transplanted to a solid medium for differentiation in which a plant hormone composed of auxins and cytokinins is added, It is a method for mass-proliferating alpine plants such as Rhinotorium communis and Rhododendron chinensis, which is characterized by culturing in a container under air-permeable conditions to allow differentiation and proliferation.

Another embodiment of the present invention is naphthalene acetic acid 0.1-2.0 mg / l, benzyladenine 1.0-
A method for mass-proliferating alpine plants such as Lycoris communis, Rhododendron oryzae, characterized in that a callus-inducing solid medium supplemented with 10.0 mg / l is used.

Another embodiment of the present invention is naphthalene acetic acid 0.0 to 0.1 mg / l, benzyladenine 0.1 to
It is a method for mass-proliferating alpine plants such as Lycoris communis, Rhododendron japonica, which is characterized in that a solid medium for differentiation added with 5.0 mg / l is used.

Further, another embodiment of the present invention contains 1.0 to 3.0% by weight of sucrose as a carbon source and has a pH of 5.0 to
The callus-inducing solid medium, which is a basic medium (A) and is a synthetic medium adjusted to an inorganic salt concentration of 1/5 to 1/2 and adjusted to 6.0.
It is a method for mass-producing alpine plants such as Miyama Dadaki.

Another embodiment of the present invention contains 1.0 to 2.0% by weight of sucrose as a carbon source and has a pH of 5.0 to
A solid medium for differentiation which uses a synthetic medium adjusted to 6.0 and an inorganic salt concentration adjusted to 1/5 to 1/2 as a basal medium (B), and is used for the above-mentioned Rhizopus communis, Rhododendron japonica, etc. Is a method for mass-producing alpine plants.

[0029] Another aspect of the present invention is that the cultivation of each step is carried out under a temperature condition of 15 to 27 ° C and a light irradiation condition of 8 to 18 hours photoperiod. A method for mass-proliferating alpine plants such as columbine.

Another embodiment of the present invention is a method for mass-proliferating alpine plants such as Lycoris sinensis L., Rhizobium alba, which is characterized in that the plant tissue slices are leaves or petiole tissue slices. is there.

Still another aspect of the present invention is that the synthetic medium is
It is a method for mass-proliferating alpine plants such as Rhizopus communis and Rhododendron chinensis, which is an MS-modified medium.

Next, the present invention will be described in more detail. Papaver fauriei is a perennial herb that is a special product of rocky land in the alpine belt of Rishiri Island, Hokkaido. The leaves are rooted,
It has a stalk, the leaf blade is oval, and it is totally or deeply split into a feather. The flower diameter is 10 to 20 cm, which is taller than the root leaves and has one flower on the top. On the other hand, Aquileg
ia akitensis) is distributed in the boreal zone north of the central region of Honshu,
It is a perennial that grows in the rocky areas of the alpine zone and has thick rhizomes. The stem is 10 to 20 cm in height, the root leaves are bundled and have a long handle. Flowers normally open one flower per stem. The present invention is directed to alpine plants typified by such Lycoris vulgaris and Miyama columbine, and representative examples thereof include Lycoris rhododendron, Miyama columbine, Rhinoceros japonica, Usuyukiso, and Rhododendron.

As a tissue section of an aseptic plant body of an alpine plant such as Lycoris poppyii, Rhododendron japonicus, etc., the stem apex or the sprout portion is preferably used, but other appropriate organs of the plant are suitable. Those obtained by sterilizing the tissue section, or aseptic seedlings obtained by aseptically sowing disinfected seeds and germinated, leaves of the plant in which the seedlings are grown, or petiole sections are appropriately used. In this case, the stem apex and the shoot portion are treated with a sterilizing solution. As the sterilizing liquid, antiformin can be exemplified, but alcohols alone are not preferable. As a treatment condition with the sterilizing solution, a method of immersing in a 2% antiformin solution for 10 to 20 minutes can be exemplified.

A tissue section of an aseptic plant body of an alpine plant such as Lycoris bursaria, Rhododendron japonicus, etc., containing a carbon source based on MS medium (basic inorganic salt medium of Murashige and Skoog) which is a synthetic medium. , A pH-adjusted MS-modified medium in which the inorganic salt concentration was adjusted to 1/5 to 1/2 was used as a basic medium (A), and a phytohormone consisting of auxins and cytokinins was added to and added to the basic medium (A). It is placed on a callus induction solid medium and cultured under specific culture conditions to induce callus.

The MS modified medium has an inorganic salt concentration adjusted to 1/5 to 1/2 based on a normal MS medium,
Folic acid of about 0.5% by weight and biotin of about 0.05% by weight are preferably used, and sucrose as a carbon source, for example, is added at 1.0 to 3.0% by weight, and pH is 5.0.
It is adjusted to ˜6.0, preferably pH 5.8, and used as the basal medium (A). In this case, if the sucrose concentration is out of the above range, it becomes difficult to induce callus and grow. In addition, if the concentration of the medium becomes too thin, it becomes difficult to induce callus and grow. On the other hand, when the concentration of the medium is too high, the browning of the medium becomes prominent and the possibility that the placed slices will die increases.

As described above, as the basic medium (A), the above MS-modified medium and sucrose as a carbon source are preferably used, but the present invention is not limited to this, and a composition equivalent to this,
Alternatively, it goes without saying that other synthetic media having the same effect can be similarly used as long as they are components. As other synergistic synthetic medium, specifically, MS (Murashige, t. And F.
skoog) medium, LS (Linsmaier, EM and F. Skoog
) Medium, NN (Nitsch, C. and JP Nitsch) medium and the like and modified media thereof can be exemplified, but in the case of the present invention, MS modified media is most preferred. In this case, in the case of Rhizobium poppy, a medium with a relatively high concentration,
That is, it is preferable to place the medium in a medium having a concentration of 1/2 of the basic synthetic medium, and, in contrast, in the case of Aedes albicans, a medium having a relatively low concentration, that is, 1/100 of the basic synthetic medium.
It is preferable to place it on a medium having 5 concentrations.

In the medium, auxin of 0.1 to 2.0 mg / l and cytokinin of 1.0 were added as plant hormones.
~ 10.0 mg / l is added. That is, as the plant hormone added to the basic medium (A), naphthalene acetic acid (NAA) 0.1 to 5.0 mg / l, preferably 1.0 mg / l, benzyladenine (BA) 1.0 to 1
0.0 mg / l, preferably 1.0-5.0 mg / l
Is preferably used, but in this case, it is added under the condition of NAA ≦ BA. Further, not limited to these, other auxins and cytokinins having the same physiological activity as these can be similarly used in combination. Other auxins include indole acetic acid (IAA), indole butyric acid,
Examples of cytokinins include 2,4-dichlorophenoxyacetic acid and the like, and examples of cytokinins include kinetin, zeatin, and 2-isopentenylaminopurine.

If the amount of auxin added is too large,
The problem of dying will occur. On the other hand, if the amount of addition is too small, it will be difficult to induce callus and the induced callus will die. Similarly, in the case of cytokinin, if the amount added is too large, the problem of dying occurs, and conversely, if the amount added is too small, callus is difficult to be induced and induced. The problem of callus dying will occur. As a supporting material, for example, about 0.7 to 1.0% by weight of agar is added to the medium containing such components, the pH is adjusted to the above range, and sterilization is performed by a conventional method, and then an appropriate amount is added. Aseptically dispense into a container and solidify to prepare a callus induction solid medium. The support material is not limited to agar,
Appropriate substances such as gellan gum can be used as long as they have the same effect.

Callus is induced by placing a section of the tissue of an aseptic plant body of an alpine plant such as Lycoris bursaria, Rhizobium alba, etc. in the callus-inducing solid medium and inducing callus. By culturing under a temperature condition of 27 ° C., preferably 20 ° C. ± 1 ° C. and a light irradiation condition of 8 to 18 hours day, preferably 16 hours day, for about 1 month, stable and high yield can be obtained. Callus can be induced with a rate.

Next, the callus induced by the above-mentioned step was used as a basic medium (B) in a synthetic medium containing a carbon source, pH-adjusted, and reduced in the concentration of inorganic salts, to which auxins and cytokinins were added. The buds and roots are differentiated from the induced callus by placing and culturing in a solid medium for differentiation containing a plant hormone. That is, after the callus is induced, the callus is separately cultured under a sterile condition in a medium having a sucrose concentration of 2.0% by weight and a concentration of 1/5 to 1/2 of the basic synthetic medium. . The medium is redifferentiated in a medium containing auxin at 0.0 to 0.1 mg / liter, cytokinin at 0.1 to 5.0 mg / liter, and auxin <cytokinin. In this case, if the amount of auxin added is too large, the callus will not be differentiated, and in some cases, it will die. In addition, if the amount of cytokinin added is too large, callus proliferation will increase and there is a high possibility of death. On the contrary, if this addition amount becomes too small,
The problem arises that it is difficult to obtain a plant body because roots are not differentiated only by rooting. Furthermore, regarding the relationship between the amount of auxin added and the amount of cytokinin added, it is necessary to set auxin <cytokinin, otherwise rooting will be prosperous, or only callus growth will occur, making it impossible to obtain a plant body. Problem will occur.

As the solid medium for differentiation, the basic medium (B) is prepared by adding 0.0-0.1 mg / l of naphthalene acetic acid.
Preferably 0.1 mg / l, benzyladenine 0.1-
A solid medium supplemented with 5.0 mg / l, preferably 0.1-1.0 mg / l is preferably used, but the plant hormone to be added is not limited to this, and the same effects as those mentioned above can be obtained. Other auxins and cytokinins having the physiological activity of can be similarly used in combination. The culture temperature condition and the light irradiation condition are set in the same manner as in the above step.

In this case, a solid medium for differentiation containing a carbon source, pH-adjusted, inorganic salt concentration adjusted to ⅕ to ½ and a reduced MS modified medium as a basal medium (B). Culture using.

As the solid medium for differentiation, for example, 2.0 wt% of sucrose as a carbon source is used, pH is adjusted to 5.8, and MS modified medium in which the concentration of inorganic salt is adjusted to ½ is a basic medium. The material (B) is preferably used. Needless to say, the basal medium (B) is not limited to this, and can be similarly used as long as it is a synthetic medium having the same effect as described above.

The above-mentioned callus was transplanted to the above-mentioned solid medium aseptically dispensed in an appropriate container, sealed with a sterile filter, and aerated under aeration conditions, for example, at about 20 ° C.
Incubate under light irradiation conditions for a time period of day.

As the container in this case, a test tube, an Erlenmeyer flask, a glass bottle, a plastic container, or the like is preferably used, but any appropriate container can be used as long as it has sufficient light transmittance. Culturing is performed under the condition of intermittently irradiating light for a long period of time, that is, under the light irradiation condition of 8 to 18 hours day, preferably 16 hours day, and the temperature condition is 1
5 to 27 ° C., particularly 20 ° C. ± 1 ° C. are preferable.

By accurately controlling the culture environment such as temperature and sunshine duration, it is possible to grow the plant with high reproducibility. Especially, the temperature condition of culture is an important requirement. The temperature condition of ℃ ± 1 ℃ is particularly preferable because the plant can be uniformly and stably regenerated and propagated. Then, it was confirmed that the growth slowed down when the temperature condition of the culture was lowered to, for example, 15 ° C. or lower. Further, in this case, the air permeability condition is an essential requirement, and if a tightly closed container is used, the growth will be slow and the plant body will be lengthened.

As described above, the present invention requires the culture process of the above-mentioned first to the third step, and the medium composition and culture conditions in each step as essential requirements. Alpine plants such as columbine cannot be normally grown in the container, and the intended purpose of the present invention cannot be achieved. Then, such culture process, and the medium composition in each step, the culture conditions are peculiar to alpine plants such as Lycoris sinensis, Rhododendron japonicus, these are tissue culture techniques of various plants, or the Torenia, Even with the conventionally known matters such as the cultivation technique in a closed container such as S. persicae, it cannot be expected at all. The method for differentiating shoots and roots from such callus of alpine plants such as Rhinotari poppy and Rhododendron has been developed by the present inventors.

Next, test examples will be shown to verify the unique effects of the present invention. Test Example 1 (Callus Induction Test) As test plants,
Callus induction test was performed using (Papaver fauriei). A material of about 5 mm to 10 mm was collected from a petiole tissue of an aseptic plant obtained by culturing aseptically by a conventional method to prepare a section.

As the medium, an MS-modified medium was used as the basic medium (A), to which naphthalene acetic acid and benzyl adenine were added at predetermined concentrations. As the basal medium (A), an MS-modified medium obtained by adding 0.05% by weight of biotin and 0.5% by weight of folic acid to MS medium and adjusting the inorganic salt concentration to 1/2 to be used, To this, 2% by weight of sucrose and 0.7% by weight of agar were added to adjust the pH to 5.8, which was used as a medium. 20m of basal medium sterilized by pressure steam by a conventional method in a test tube with a diameter of 25mm
l was dispensed.

Then, the petiole sections of Lycoris poppyum were placed on the basal medium in the test tube and cultured for 25 days under irradiation conditions of natural daylight fluorescent lamps at room temperature of 20 ° C. and illuminance of 3,000 Lux for 16 hours. Induced callus. Table 1 shows the results. As is clear from Table 1,
It was confirmed that when 0.1-1.0 mg / l of naphthalene acetic acid and 0.1-1.0 mg / l of benzyladenine were added, callus was formed stably and in high yield.

[0051]

[Table 1]

Test Example 2 (Callus Induction Test) As a test plant, Japanese linden
Callus induction test was performed using (Aquilegia flabella var. Pumilla). A material of about 5 mm to 10 mm was collected from a petiole tissue of an aseptic plant obtained by culturing aseptically by a conventional method to prepare a section.

As the medium, an MS-modified medium was used as the basic medium (A), and naphthalene acetic acid and benzyl adenine were added to the medium at predetermined concentrations. As the basal medium (A), an MS-modified medium obtained by adding 0.05% by weight of biotin and 0.5% by weight of folic acid to MS medium and adjusting the inorganic salt concentration to 1/2 to be used, To this, 2% by weight of sucrose and 0.7% by weight of agar were added to adjust the pH to 5.8, which was used as a medium. 20m of basal medium sterilized by pressure steam by a conventional method in a test tube with a diameter of 25mm
l was dispensed.

Then, the petiole sections of Amaranthus edulis were placed on the basal medium in the test tube, and cultured for 25 days under irradiation conditions of a natural daylight fluorescent lamp at room temperature of 20 ° C. and an illuminance of 3,000 Lux for 16 hours. Then, callus was induced. The results are shown in Table 2. As is clear from Table 2,
It was confirmed that callus was stably formed when 0.1 to 1.0 mg / l of naphthalene acetic acid and 0.1 to 5.0 mg / l of benzyladenine were added.

[0055]

[Table 2]

Test Example 3 (Container Proliferation Test) The callus induced in Test Examples 1 and 2 was sampled with tweezers. On the other hand, to the MS medium, 0.05 wt% of biotin and 0.5 wt% of folic acid were added, and the MS modified medium modified by adjusting the inorganic salt concentration to ⅕ was used as the basic medium (B), and as a control, Inorganic salt concentration as it is 1/1
MS modified medium was used, to which naphthalene acetic acid 0.
0-1.0 mg / l, benzyladenine 0.0-1.0
mg / l was added, 0.7% by weight of agar was added, and pH was 5.8.
Then, 50 ml was dispensed into a glass container having a volume of 200 ml to form a solid medium. Then, the above-mentioned induced callus was placed on the medium in the glass container and sealed at room temperature under an air-permeable condition of 20 with a sterile filter MilliSeal (Teflon filter manufactured by Millipore) at room temperature.
℃, natural daylight fluorescent lamp illuminance 3,000 Lux, 1
After statically culturing under irradiation conditions of 6-day photoperiod, the growth of the induced callus and the state in which shoots and roots were differentiated from the callus were observed. The results are shown in Tables 3-6.

[0057]

[Table 3]

[0058]

[Table 4]

[0059]

[Table 5]

[0060]

[Table 6]

As is apparent from Tables 3 to 6, naphthalene acetic acid of 0.
0-1.0 mg / l, benzyladenine 0.0-1.0
When mg / l was added, good leaves, petiole formation, and rooting were observed within 30 days after the start of culture, and a plant having good morphology of leaves and petiole was obtained.

[0062]

EXAMPLES Next, the present invention will be specifically described based on Examples. Example 1 Cultivation of Papaver fauriei Papaver fauriei The shoot apex of the plant of the above Papaver faunai was cut off,
After sterilizing with a 2% antiformin solution for 15 minutes, it was washed several times with sterile water. Then cut the stem apex into small pieces,
An MS modified medium containing 2% by weight of sucrose, 0.05% by weight of biotin and 0.5% by weight of folic acid and adjusted to pH 5.8 was used as a basic medium (A), and the section was treated with naphthalene acetic acid. 1.0 mg / l, benzyladenine 1.0 mg
/ L was added, and the mixture was further placed on a callus induction solid medium containing 0.7% by weight of agar, and 3,000 Lux at 20 ° C.
The cells were cultured for 1 month under a 16-day photoirradiation condition with a natural daylight fluorescent lamp.

MS-modified medium having a sucrose content of 2% by weight and a pH of 5.8 and adjusted to ½ was used as the basic medium (B), and naphthalene acetic acid 0.1 mg / l and benzyladenine 1.0 mg / 1%, agar 0.7% by weight, adjusted to pH 5.8, placed on a solid medium for differentiation formed, and exposed to 3,000 Lux natural daylight fluorescent lamp at 20 ° C. for 16 hours photoperiod. The shoots and roots were differentiated from the divided callus by culturing under irradiation conditions.

Then, the shoots differentiated from the callus induced as described above were placed together with the roots in a seedling-inducing and growing solid medium which had been aseptically dispensed into a container and placed at 20 ° C. for 3,0.
The seedlings were induced and grown by culturing them under the light irradiation condition of 00 Lux natural daylight fluorescent lamp with 16-day photoperiod.

As the seedling induction and growth solid medium, 1% by weight of sucrose and 0.7% by weight of agar were added to adjust the pH.
The MS modified medium adjusted to 5.8 and the inorganic salt concentration adjusted to 1/2 is used as the basic medium, sterilized by a conventional method, and dispensed into a glass flask container with a volume of 200 ml to be used. did. The flask container was sealed with a sterile filter MilliSeal (Teflon filter manufactured by Millipore), and the culture was performed under an aeration condition. 2 after the start of culture
In 0 to 30 days, the roots were elongated, the leaves were differentiated, the rooting state was good, and the morphology of leaves and petiole was also good.

[0066]

INDUSTRIAL APPLICABILITY As described in detail above, according to the present invention, a tissue section of a plant body of an alpine plant such as Rhododendron japonicus and Rhododendron chinensis is cultured in a specific culture process under a specific medium composition and culture conditions. Utilizing the callus induced by that, alpine plants such as Lycoris sinensis, Miyama columbine, is to grow stably, and in high yield, thereby, the present invention, the seed breeding difficult alpine rhododendron, It is possible to breed Miyama Dadaki etc. regardless of the season.

In the conventional hybrids, alpine plants such as Rhizoma communis and Rhododendron chinensis that were difficult to mass-produce uniform plants due to variations in appearance and color,
Only the excellent strain can be selectively grown in stable and high yield, and the parent strain can be maintained.

Further, according to the present invention, it is possible to grow a uniform plant body from one strain in a stable and large amount in a large amount even if the seeds were difficult to grow since the second generation and the growth was difficult. In addition to enabling, it induces alpine plants such as Lycoris poppy, Miyama daimaki in a breathable container,
It has been made possible to grow, and it has the possibility that it can be flowered in a container and used as it is for ornamental use.

Further, the method for mass-proliferating alpine plants such as Rhododendron japonicus and Rhododendron japonicus of the present invention is aseptic culture by tissue culture from the collection of the material to the growth of the plant body, so that the parent plant Since it is possible to stably grow a plant genetically identical to the body in a virus-free state, it is possible to grow easily and in large quantities the excellent mating species that have been difficult to grow in the past. , Its industrial utility is extremely high.

Furthermore, according to the present invention, since the water, nutrients and the like necessary for the growth of the plant are added to the solid medium, the plant can be grown without special fertilizer. Therefore, maintenance such as watering is not required. Further, since it is inside the container, it is possible to perform complete environmental control.

Claims (8)

[Claims] 1. A section of a tissue of an alpine plant such as Rhinotorium communis, Rhododendron japonicus, etc., containing a carbon source, p
A synthetic medium in which H was adjusted and the concentration of inorganic salts was reduced was used as a basic medium (A), which was planted and cultured in a callus-inducing solid medium in which plant hormones composed of auxins and cytokinins were added to induce induction of callus. Contains a carbon source,
A basic medium (B), which was pH-adjusted and had a reduced inorganic salt concentration, was used as a basic medium (B), which was transplanted to a solid medium for differentiation in which plant hormones composed of auxins and cytokinins were added, and the medium was aerated. A method for mass-proliferating alpine plants such as Rhododendron japonicus and Rhododendron chinensis, which is characterized by culturing in a container, differentiation and proliferation. 2. Naphthalene acetic acid 0.1-2.0 mg /
2. A method for mass-proliferating alpine plants such as Rhododendron japonicus and Miyama columbine according to claim 1, characterized in that a callus-inducing solid medium supplemented with 1.0 and benzyladenine 1.0-10.0 mg / l is used. 3. Naphthalene acetic acid 0.0 to 0.1 mg /
2. A method for mass-proliferating alpine plants such as Lycoris sinensis and Rhizobium alba, according to claim 1, characterized in that a solid medium for differentiation added with 0.1 to 5.0 mg / l of benzyladenine is used. 4. A synthesis containing 1.0 to 3.0% by weight of sucrose as a carbon source, adjusted to pH 5.0 to 6.0, and an inorganic salt concentration adjusted to 1/5 to 1/2. 2. A method for mass-proliferating alpine plants such as Lycoris sinensis L. and Rhododendron japonica according to claim 1, wherein a callus-inducing solid medium having the medium as a basic medium (A) is used. 5. A synthesis containing 1.0 to 2.0% by weight of sucrose as a carbon source, adjusted to pH 5.0 to 6.0, and an inorganic salt concentration adjusted to 1/5 to 1/2. 2. A method for mass-proliferating alpine plants such as Rhododendron japonicus and Rhododendron chinensis according to claim 1, characterized in that a solid medium for differentiation using the medium as a basic medium (B) is used. 6. Cultivation in each step is carried out under a temperature condition of 15 to 27 ° C. and a light irradiation condition of 8 to 18 hours photoperiod, and the alpine plants such as Lycoris sinensis and Rhizoma perforatum according to claim 1. Mass proliferation method. 7. The method for mass-proliferating alpine plants such as Rhododendron japonicus and Rhododendron japonicus according to claim 1, wherein the plant tissue slice is a leaf or petiole tissue slice. 8. The method for mass-proliferating alpine plants such as Rhododendron japonicus and Rhododendron chinensis according to claim 1, wherein the synthetic medium is an MS-modified medium.