JP3080784B2 - Mass propagation method of Fuerosou - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for mass-producing a spinach plant. More specifically, the present invention relates to a method for cutting a tissue section of a plant of a spinach plant into a specific callus-inducing solid medium, a split callus growth liquid medium, By culturing by a specific culture process using a differentiation medium, a seedling-induction and growth solid medium, conventionally, seeds are hardly formed,
It was difficult to grow large amounts of the same lineage, and it was difficult to widely spread excellent hybrids. It relates to a mass propagation method.

[0002]

2. Description of the Related Art Conventionally, adventitious buds, adventitious roots, flower buds, calli, and the like are induced, formed, and grown by aseptically cultivating a tissue section of a plant body on a medium, and the plant is proliferated. , The method of propagation of plant tissue culture
Various plants have been studied, and various techniques have been developed so far.

With the progress of such plant tissue culture technology, methods for producing and growing various plants using the technology have been developed, and the medium composition, culture conditions, etc., suitable for each plant are specific. Some have already been put into practical use as a method for producing virus-free strains, mass propagation, and the like. For example, regeneration of disease-free solids by cultivating the shoot tip tissue of dahlia and potato (growth point culture), orchid (cymbidium) A variety of technologies have been developed and put into practical use, such as regeneration of protocomb-like bodies (PLB) by shoot apex tissue culture of the genus) and mass propagation of genetically uniform seedlings by split propagation. ing.

At present, such a tissue culture technique 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 for vegetatively propagated horticultural crops such as Gypsophila paniculata, Saintpaulia, gerbera, cyclamen, strawberries, etc. is becoming established.

[0005] However, such a plant tissue culture technique is not a completed technique, but has many unknown fields, and rather has much to wait for future research. Regarding vegetative propagation, except for Cymbidium, for which research is progressing, the culture medium composition and culture technology have not yet been completed to the point of generalization, and a group of orients such as perilla or perilla has not yet been developed. The growth system by tissue culture has not been established yet.

[0006] With respect to general flowers and houseplants, active research and development are being carried out on the formation of polyblasts by liquid shaking culture of proliferating tissues, induction of polyblasts in a medium supplemented with hormonal agents, and mass propagation of seedlings. However, there are many problems to be solved in the future, such as the extremely high risk of mutation.

[0007] Cyclamen and primula, which are grown in seeds by breeding seeds, such as bulbs and perennials, have a lack of uniformity in their products due to genetic separation and have a small amount of collection.
Due to problems such as lack of seeds in excellent systems, attempts have been made to propagate 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 However, such a technique of vegetatively growing such a conventional seed propagation system is only partially used. Thus, plant tissue culture technology
As a method of inducing, producing, and growing various plants, which has been widely studied, techniques for inducing and growing various ornamental plants by tissue culture have been proposed.

That is, for example, the shoot apex of a useful annual plant is excised, transplanted into an artificial medium containing an inorganic salt composition and a plant growth hormone, and then 0.5 to 10 rpm.
A method for mass-producing useful annual plants, comprising spinning the shoot primordia at a rotation speed of 1 to multiply the shoot primordia and statically culturing the obtained shoot primordia to produce seedlings (Japanese Patent Laid-Open No. 59-
132823). However, this method mainly uses Sho-ot primordia to artificially mass-produce useful substances composed of secondary metabolites such as plastids, vacuoles, oil bodies, and stored substances. Yes, about the growth of ornamental plants, it only refers to petunia, morning glory and the like.

[0010] Further, a method for regenerating a plant that germinates and grows from callus under irradiation of light from a tissue piece of a plant to proliferate, and a growth medium for the same, for example, 0.1-10 ppm of cytokinins as plant hormones, And an MS medium containing 0.01 to 10 ppm of auxins has been proposed (JP-A-3-139224). However,
This study examined a specific medium composition, culture conditions, and the like for asteraceae plants suitable for obtaining a large amount of asteraceae plants in a short period of time.

Further, a method for regenerating a plant, comprising inducing callus from a tissue piece of a plant, culturing the callus to differentiate adventitious buds, and then culturing the adventitious bud to obtain a young plant. And a method of multiplying the seedling have been proposed (JP-A-4-45730). However, this method utilizes differentiated adventitious buds, and is intended for a plant of the genus Pleurotus.

[0012] Furthermore, a tissue section of a flowering plant or a tissue section of a regenerated plant obtained by culturing the flowered plant is cultured and re-differentiated in a medium to obtain a regenerated plant. A method for culturing flowering plants, which is cultured in a medium having a composition different from that at the time of differentiation to cause flowering, has been proposed (JP-A-4-30733). However, this method examines the medium composition of the medium used for the regeneration culture and the medium used for the flowering culture, and the target plant is also a plant belonging to the family Echinacea, Nadesicoceae, and Solanaceae, particularly the genus Torenia. Plants are merely examples.

[0013] Recently, there has also been proposed a technique relating to flowering in a container that allows plants to be cultivated and flowered in a closed container so that they can be viewed as they are. As an example of such a proposal, for example, a stem section of a plant body is aseptically cultured in a primary basal medium to form adventitious buds, which are then brought to a specific inorganic salt concentration, and a stunting agent is added. A cultivation method has been proposed which comprises laying on a secondary basal medium, irradiating a natural daylight fluorescent lamp for a short day, and culturing in a closed state (Japanese Patent Laid-Open No. 60-22210).
No.). However, this method utilizes adventitious buds, and the target plant is also limited to Torenia (Japanese name Hanaurixa).

[0014] In addition, the whole plant of aseptic seedlings is planted in an MS medium to which a specific component is added as a dwarfing agent in a see-through container, irradiated intermittently with light for a long time, and aseptically sealed. A cultivation method comprising
-200121). However, this method
In this study, we used the whole plant of a young plant, and examined the dwarfing conditions of Senichiko, which belongs to the lily family.

As described above, in order to establish an in-container cultivation technique in which plants can be cultivated in a closed container so that they can be enjoyed as they are, a technique for growing plants stably and in a high yield in a closed container can be achieved by: Developed dwarfing technology for properly growing in a closed container, flowering technology for differentiating flower buds and flowering,
It is presupposed that it is established, but in the first place, it is very difficult to grow and flower plants in a closed container, and until now, flowering in a closed container of dwarf torenia, Although there are reports of flowering in closed containers, most of the plants currently growing dwarf plants in closed containers and used for ornamental purposes do not have flowers.

As described above, in general, with the development and widespread use of technology for growing various plants by tissue culture, research and development and establishment of proprietary culture conditions suitable for various plants have been carried out. Few have reached the level of practical use, much more, plant cultivation technology that allows plants to be cultivated in closed containers or air-permeable containers to differentiate flower buds and bloom, and to be viewed as they are. As for, there are few research examples, and it is in a situation where it has hardly been put to practical use.

[0017]

In such a situation, the present inventors cultivated a plant in a container to differentiate flower buds, caused the flower to blossom, and allowed the plant to be viewed as it was. With the goal of establishing breeding technology, the basic technology that is the basis of that technology,
Basic research was undertaken to investigate various methods such as growth methods with high yield, dwarfing conditions for stably dwarfing, stably differentiated flower buds, and cultivation conditions for long-term flowering. .

According to the research of the present inventors and the findings so far, in order to establish such a plant cultivation technique, the above-mentioned various plant growth techniques and dwarfing techniques, which are technical bases, are established. Needless to say, the development of basic technologies such as flowering technology is a major premise. However, each of these technologies is actually completely different depending on the type of individual plant. Even if the cultivation method and cultivation conditions of Senichiko are diverted to other plants as they are, it is almost impossible to achieve the intended purpose, and depending on the type of each plant, It is necessary to develop and establish effective cultivation methods and conditions.

Based on such knowledge, the present inventors have found that among the various plants, the conventional hybrids are difficult to produce seeds from the second generation onward, and it is difficult to multiply the seeds. It is difficult to produce a uniform plant due to variation in color, and it is difficult to spread excellent varieties widely. Therefore, development of a new breeding technique that can solve these points has been strongly demanded. And focused on developing a method for stable and high-yielding growth, the results of intensive research have shown that combining specific culture processes with specific medium compositions and culture conditions has It has been found that the objective of the present invention can be achieved, and the present invention has been completed.

[0020] That is, an object of the present invention is to provide a method for proliferating a large amount of an aneurysm plant that has conventionally been considered difficult to proliferate excellent varieties produced by crossing.

[0021] Another object of the present invention is to provide a method for stably growing a plant at a high yield by tissue culture using a section of a tissue of a plant of Aspergillus aseptic. Things.

Further, the present invention provides a method for mass-producing a plant that can be aseptically cultured in a container to differentiate flower buds, bloom, and be directly viewed in the container. It is intended for.

Furthermore, the present invention provides a stable and high-yielding plant by utilizing the split callus of a Fusarium plant formed under a specific culture process, a specific medium composition, and a specific culture condition. It is an object of the present invention to provide a method of growing a large amount at a high rate and growing in a container.

[0024]

The structure for achieving the above object of the present invention comprises the following technical means (1) to (8). (1) Fusarium plant (Geranium
Genus, plant of the genus Erodium) in a container to grow flower buds
Differentiation, flowering, and appreciation
A method for mass-producing a plant in a container of the plant, wherein 1) a section of a tissue of a plant of a plant of the anthracnose plant is a synthetic medium containing a carbon source and having a pH adjusted as a basic medium (A), and an auxin and Planting and culturing in a callus-derived solid medium to which a plant hormone consisting of cytokinins is added; 2) a liquid medium obtained by adding the obtained callus to the basic medium (A) to which a plant hormone consisting of auxins and cytokinins is added; In the medium, the cultured callus was grown under the condition of a shaking rotation speed of 100 rpm or more to grow the divided callus. 3) The grown callus was differentiated by adding a plant hormone consisting of auxins and cytokinins to the basic medium (A). and cultured were transferred to solid medium, 4) together buds differentiated from callus and root, contains a carbon source, pH adjusted, basal medium synthetic medium with reduced mineral salt concentration ( ) And were transplanted to the induction and growth solid media of plantlets were cultured in a vessel under conditions breathable, mass propagation method Fuurosou plant characterized by growing.

(2) 0.1 to 2.0 mg of naphthaleneacetic acid
/ 1 and a callus-derived solid medium to which 0.1 to 6.0 mg / l of benzyladenine has been added, wherein the method for mass-proliferation of the Anthracnose plant according to the above (1), is used.

(3) 0.1-5.0 mg of naphthaleneacetic acid
/ 1 and a method for mass-producing a spinach plant according to the above (1), wherein a liquid medium containing 0.5 to 6.0 mg / l of benzyladenine is used.

(4) Naphthalene acetic acid 0.1 to 5.0 mg
/ 1 and a solid medium for differentiation supplemented with 0 to 1.0 mg / l of benzyladenine, wherein the method for mass-proliferation of the Antelope plant according to (1) above is used.

(5) It contains sucrose as a carbon source and has a pH
It is adjusted to 5.0 to 6.0, and the inorganic salt concentration is 1/2 to 1 /
5. The method according to (1) above, wherein a solid medium for inducing and growing young plants is used as a basic medium (B) using the synthetic medium adjusted to 5.

(6) The method according to (1) above, wherein the cultivation in each step is carried out under a temperature condition of 22 to 27 ° C. and a light irradiation condition of 12 to 18 hours of light. .

(7) The method according to (1) above, wherein the plant tissue section is a leaf or petiole tissue section.

(8) The method according to (1) above, wherein the synthetic medium is a modified MS medium.

Next, the configuration of the present invention will be described in detail. In general, Fuerosou (wind dew grass) means a general term for plants of the genus Geranium and Erodium in the family Anthracidae, and their scientific names are Geranium (G-er
anium), called Erodium. In addition, a kind of the Anthracaceae native to Europe is sometimes simply referred to by this name, as in the case of Dutch furo and Ibuki furo. It grows naturally in the grasslands of the mountains, and its leaves have palmar fissures, autumn leaves, and five-petaled flowers. The present invention is intended to target such Fuurosou plants, Ha Kusanfuuro, geranium genus such Asamafuuro, Erodiumu genus and Holland Fuuro, the
For plants.

As a section of a tissue of a plant body of these angiosperm aseptic plants, a tissue section of an appropriate organ of a normal plant body is sterilized, or a sterilized seed is aseptically sowed and germinated. Aseptic seedlings, leaves of the plant body in which the seedlings have been grown, or sections of petiole are preferably used.

A tissue section of such an aseptic plant body of the anthracnose plant is prepared by synthesizing an MS medium (a basic inorganic salt medium of Murashige and Skoog) which is a synthetic medium and containing a carbon source and adjusting the pH of the MS. The modified medium is a basic medium (A), to which a plant hormone consisting of auxins and cytokinins is added, placed on a callus-inducing solid medium containing the medium, and cultured under specific culture conditions to induce callus. .

The modified MS medium is based on a normal MS medium and contains about 0.5% by weight of folic acid and about 0.05% of biotin.
% Is preferably used, and as a carbon source, for example, 2% by weight of sucrose is added, and pH 5.0 to 5.0 is added.
The pH is adjusted to 6.0, preferably pH 5.8, and this is used as a basal medium (A).

As described above, in the basic medium (A), the above-mentioned MS modified medium and sucrose as a carbon source are preferably used. However, the present invention is not limited thereto.
Alternatively, it is needless to say that another synthetic medium of the same effect can be similarly used as long as it is a component. Specific examples of other synthetic media having the same effect include MS medium, B5 medium, and Nit-sc medium.
h medium (1965).

The plant hormone to be added to the basic medium (A) is naphthalene acetic acid 0.1 to 2.0 mg /
1, preferably 1.0 mg / l, benzyl adenine 0.1.
1-6.0 mg / l, preferably 1.0-5.0 mg / l
1 is preferably used, but not limited thereto, and other auxins and cytokinins having the same physiological activity can be used in combination in the same manner. Other auxins include indoleacetic acid, indolebutyric acid, 2,4-
Examples include dichlorophenoxyacetic acid, and examples of cytokinins include kinetin, zeatin, and 2-isopentenylaminopurine.

As a supporting material, for example, about 0.7 to 0.9% by weight of agar is added to a medium containing such components, the pH is adjusted to the above range, and the medium is sterilized by a conventional method. Is aseptically dispensed into an appropriate container and solidified to prepare a callus-derived solid medium. The support material is not limited to agar, and any suitable component such as gellan gum can be used as long as it has the same effect.

The callus is induced by placing a section of the tissue of the aseptic plant of the anthracnose plant on the callus-inducing solid medium and culturing it.
By culturing for about one month under a temperature condition of 22 to 27 ° C., preferably 25 ° C. ± 1 ° C., and a light irradiation condition of 12 to 18 hours, and preferably 16 hours, photoperiod, it is stable and Callus can be induced with high yield.

The callus obtained in the above step is added to a liquid medium based on the basic medium (A), and the number of shaking rotations is 100 rpm or more, preferably 130 to 150 rpm.
The cultured calli are grown under the conditions of m.
Such divided calli are collected, for example, with a pipette every week and subcultured for about one month.

As the liquid medium, naphthalene acetic acid 0.1 to 5.0 mg / l, preferably 1.0 mg / l, benzyl adenine 0.5 to
A liquid medium supplemented with 6.0 mg / l, preferably 1.0 to 5.0 mg / l, is suitably used, but the plant hormone to be added is not limited thereto, and may be the same as described above. Other auxins and cytokinins having effective physiological activity can also be used in combination in the same manner. Culture temperature conditions and light irradiation conditions are set in the same manner as in the above steps.

In this step, the divided callus is not propagated by a normal culture method, but it is possible to grow the divided callus only by applying such a high-speed rotation condition. It is. Therefore, providing such a high-speed rotation condition is an essential requirement as a means for growing the divided callus in this step.

Next, the callus that has undergone division and proliferation obtained in the above step is placed on a solid medium for differentiation based on the basic medium (A) and cultured to differentiate shoots and roots from the divided callus. Let it. As the solid medium for differentiation, naphthaleneacetic acid 0.1 to 0.1 is added to the basic medium (A).
5.0 mg / l, preferably 1.0 mg / l, benzyladenine 0-1.0 mg / l, preferably 0-0.1 m
The solid medium to which g / l is added is preferably used, but the plant hormone to be added is not limited thereto, and other auxins and cytokinins having the same physiological activity as those described above are also used. Can be used in combination. Culture temperature conditions and light irradiation conditions are set in the same manner as in the above steps. The method of differentiating shoots and roots from such split and proliferated callus of a Flourium plant has been developed by the present inventors.

Next, the sprouts differentiated from the split callus obtained by the above-mentioned step are combined with a root, and contain a carbon source,
pH is adjusted and the inorganic salt concentration is 1/1 or less, preferably,
Culture is performed using a seedling-induction and growth solid medium using the MS modified medium adjusted to 1/2 to 1/5 and reduced as the basic medium (B).

As the solid medium for inducing and growing seedlings, for example, 1% by weight of sucrose is used as a carbon source.
MS adjusted to H5.8 and the inorganic salt concentration adjusted to 1/2
The modified medium used as the basic medium (B) is suitably used. The basic medium (B) is not limited to this, and it goes without saying that the same basic medium as described above can be used as well.

The shoots differentiated from the divided callus are transplanted to the solid medium aseptically dispensed into an appropriate container,
Sealed with a sterile filter, under breathable conditions, for example,
Incubate at about 25 ° C. for 16 hours under light irradiation conditions.

As the container in this case, a test tube, an Erlenmeyer flask, a glass bottle, a plastic container and the like are suitably used, but any suitable container having sufficient light transmittance can be used. The culture is performed under conditions of intermittently irradiating light for a long period of time, that is, under light irradiation conditions of a day length of 12 to 18 hours, and preferably a day length of 16 hours.
A temperature of 22 to 27 ° C., particularly 25 ° C. ± 1 ° C., is preferred.

By precisely controlling the culture environment such as temperature and sunshine duration, the plant can be grown with high reproducibility. In particular, the temperature condition of culture is an important requirement. The temperature condition of ° C. ± 1 ° C. is particularly preferable since the plant can be uniformly and stably regenerated and propagated. Then, it was confirmed that when the temperature condition of the culture was reduced to, for example, 20 ° C. or less, the growth was slowed down. Furthermore, in this case, the ventilation condition is an essential requirement, and if a hermetically sealed container is used, the growth will be slowed down and the plant will become prolonged.

As described above, the present invention requires the culturing process of the first to third steps, the medium composition in each step and the culturing conditions as essential requirements. It cannot grow normally in the container and cannot achieve the intended purpose of the present invention. And such a culture process, the medium composition in each step, the culture conditions are specific to the Anthraciana plant, these are tissue culture techniques of various plants,
Alternatively, it cannot be expected at all even with conventionally known matters such as the above-mentioned cultivation technique in a closed container such as Torenia or Sennichiko.

Next, test examples will be shown to verify the specific effects of the present invention. Test Example 1 (Callus Induction Test) A callus induction test was carried out using a dung beetle (Erodium chamaedryoides) as a flies plant. A material of about 5 mm to 10 mm was collected from a petiole tissue of a germ-free plant obtained by aseptically culturing by a conventional method, and a section was prepared.

As a medium, a modified MS medium was used as a basic medium (A), and a medium to which naphthaleneacetic acid and benzyladenine were added at predetermined concentrations was used. As the basic medium (A), an MS modified medium modified by adding 0.05% by weight of biotin and 0.5% by weight of folic acid to an MS medium was used, and 2% by weight of sucrose and 0% of agar were added. 0.7% by weight
Was added, and the pH of the mixture adjusted to 5.8 was used as a medium. A basic medium sterilized by pressurized steam using a conventional method is used to remove
20 ml was dispensed into a 5 mm test tube.

Next, the petiole slices of the above-mentioned Hollandia americana were placed on a basal medium in the above-mentioned test tube, and were placed at room temperature for 2 hours.
The callus was induced by culturing for 30 days under irradiation conditions of a natural daylight fluorescent lamp having an illuminance of 4,000 Lux at 5 ° C. for 16 hours. Table 1 shows the results. As is clear from Table 1, when naphthalene acetic acid 0.1-2.0 mg / l and benzyladenine 0.1-6.0 mg / l are added,
It was confirmed that calli were formed in a stable and high yield.

[0053]

[Table 1]

Test Example 2 (Split Callus Proliferation Test) 7.0 g of the callus induced in Test Example 1 was added to the above-mentioned basic medium (A) by adding 1.0 mg / l of naphthaleneacetic acid and 1.0 mg / l of benzyladenine. The culture was carried out in a 300-ml Erlenmeyer flask containing 50 ml of the added liquid medium and irradiating with a 4,000 Lux natural daylight fluorescent lamp at 25 ° C. for 16 hours under daylight irradiation conditions, while changing the shaking rotation speed. Then, the proliferation test of the divided callus was performed. Table 2 shows the results.

[0055]

[Table 2]

As is clear from Table 2, the shaking rotation speed was 10
It was found that the divided callus proliferated under the high-speed rotation condition of 0 rpm or more.

Table 3 shows the results of tests conducted by adding predetermined concentrations of naphthaleneacetic acid and benzyladenine, and setting the shaking speed to 130 rpm. As is clear from Table 3, it was found that the split callus proliferated at a high yield when the concentration of naphthalene acetic acid was 1.0 mg / l and benzyladenine was 1.0 to 5.0 mg / l.

[0058]

[Table 3]

Test Example 3 (Incubation Test in Containers) The split callus formed in Test Example 2 was collected with a pipette. On the other hand, 0.1 to 5.0 mg / l of naphthalene acetic acid and 0 to 1.0 mg / l of benzyladenine were added to the basic medium (A), and the agar was added at 0.
After adding 7% by weight and adjusting the pH to 5.8, 50 ml was dispensed into a 200 ml glass container to form a solid medium. Next, the divided callus is placed on a culture medium in this glass container, and left standing for 30 to 40 days at room temperature of 25 ± 1 ° C., irradiance of 4,000 Lux by natural daylight fluorescent lamp, and irradiation condition of 16 hours day length. After culturing, shoots and roots were differentiated from the divided calli.

Next, 1% by weight of sucrose and 0.7% by weight of agar were added, the pH was adjusted to 5.8, and the MS modified medium in which the inorganic salt concentration was adjusted to 1/2 was subjected to pressurized steam sterilization by a conventional method. The medium was dispensed in a volume of 50 ml into a heat-resistant glass flask container having a volume of 200 ml.

The sprouts differentiated from the divided callus are placed on a solid medium in a flask, sealed under a sterile filter Milli-seal (Teflon filter manufactured by Millipore) under air-permeable conditions, and sealed with a polypropylene stopper. At 25 ± 1 ° C. at room temperature.
By static culture under irradiation conditions of 000 Lux natural daylight fluorescent light for 16 hours in a day length, the state of plant growth and rooting was observed. Table 4 shows the results.

[0062]

[Table 4]

As is clear from Table 4, in the group using the air-permeable container sealed with a sterile filter, good leaf and petiole formation and rooting were observed 20 to 40 days after the start of culture. In addition, a plant having good leaf and petiole morphology was obtained. In the group using the container sealed with a stopper made of polypropylene, the petiole was prolonged and could not be grown into a normal plant.

[0064]

Next, the present invention will be specifically described based on examples. Example 1 Dutch Elephant spinach (white) (Erodium chamaedryoid
es) Cultivation The petiole of the plant of the Anthracnose plant was cut out, and 7
The solution was sterilized for 15 minutes with the filtrate of the solution containing the mashed powder, and washed several times with sterile water. Next, cut the petiole part small,
The petiole slice was used as a basic medium (A) containing 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. Acetic acid 1.0 mg / l, benzyl adenine 1.0 m
g / l, further placed on a callus-derived solid medium containing 0.7% by weight of agar, and 4,000 Lu at 25 ° C.
x was cultured for one month under the condition of light irradiation for 16 hours in a natural daylight fluorescent lamp.

Next, the callus 5.5 induced as described above was obtained.
g of naphthalene acetic acid 1.0 m in the basic medium (A).
g / l and 1.0 mg / l of benzyladenine in a liquid medium (50 ml), and a shaking rotation speed of 130 rpm at 25 ° C. under a light irradiation condition of 4,000 lux natural daylight fluorescent light for 16 hours. Was performed. Every week after the culture, the divided callus formed in the culture solution was collected with a pipette and subcultured.

Next, the split callus collected as described above was added to the basic medium (A) in an amount of 1.0 mg / ml naphthaleneacetic acid.
l, benzyladenine (1.0 mg / l), 0.7% by weight of agar, pH 5.8, placed on a solid medium for differentiation formed, and 4,000 Lux natural daylight fluorescence at 25 ° C. The shoots and roots were differentiated from the divided calli by culturing under light irradiation for 16 hours in the light.

Next, the shoots differentiated from the calli that had been divided as described above are placed together with the roots on a seedling-derived and growing solid medium that has been aseptically dispensed into a container.
Seedlings were grown by culturing under a light irradiation condition of a natural daylight fluorescent lamp of 00Lux for 16 hours.

The seedling-inducing and growing solid medium was supplemented with 1% by weight of sucrose and 0.7% by weight of agar.
The base medium is an MS modified medium adjusted to 5.8 and the inorganic salt concentration adjusted to １ ／, which is sterilized by an ordinary method, and is formed by dispensing into a 200 ml glass flask container. did. The flask container was sealed with a sterile filter Milli-seal (Teflon filter manufactured by Millipore), and the culture was performed under aeration conditions. 2 after start of culture
In 0 to 30 days, the root elongated, the leaves and petiole grew, the rooting condition was good, and a plant with good leaf and petiole morphology was obtained.

[0069]

Industrial Applicability As described above in detail, the present invention provides a method for culturing a tissue section of a plant of a Fusarium plant by a specific culture process.
Using a specific medium composition, split callus formed by tissue culture under culture conditions, to grow a plant of the genus Furoso in a stable and high yield,
Thus, the present invention provides a second hybrid in the conventional hybrid.
It has the effect of easily, stably, and growing a high-yield plant of Furonium, which has been difficult to produce seeds since the generation and is propagated only by strain division and difficult to mass-produce.

Further, the present invention provides a method for producing a hybrid plant which has been difficult to mass-produce a uniform plant body because of its variation in appearance and color in the conventional hybrids. It has the effect of being able to grow at high yield.

The present invention also provides a method for stabilizing a uniform plant from one strain, even if it is difficult to produce seeds from the second generation and it is difficult to grow the seeds, as in the case of Hime-Furo, a hybrid of Fusarium plants. In addition to being able to grow in large quantities at high yields, it is also possible to induce and grow Fusarium plants in a ventilated container, and to flower in the container and use it for ornamental purposes There is a possibility that can be.

Further, the method of the present invention for mass-producing a spinach plant is aseptically cultivated by tissue culture from the collection of the material to the growth of the plant, so that it is genetically identical to the parent plant. Can be stably grown in a virus-free state, and excellent breeding species, which had been particularly difficult to grow in the past, can be easily and mass-proliferated, so that they are industrially useful. Sex is extremely high.

Further, according to the present invention, since 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, according to the culture technique of the present invention,
The plant having a good form can be obtained easily and in large quantities without requiring special management.

──────────────────────────────────────────────────続 き Continuation of front page (56) References Amer. Soc. Hort. S ci. , Vol. 106 [1] (1981), p. 114-116 (58) Field surveyed (Int. Cl. ⁷ , DB name) A01H 4/00 BIOSIS (DIALOG) JICST file (JOIS)

Claims (8)

(57) [Claims] We claim: 1. foster zero Raniumu genus or Erodiumu genus plant <br/> vessel to differentiate flower buds, is flowering, a container mass propagation method of the plant as you can appreciate, ( 1) A section of a tissue of a plant of the Geranium or Elodium spp. Plant was used as a basic medium (A) containing a carbon source and a pH-adjusted synthetic medium. Planted and cultured in a callus-derived solid medium to which a plant hormone consisting of cytokinins has been added,
(2) The obtained callus is cultured in a liquid medium obtained by adding a plant hormone consisting of auxins and cytokinins to the basic medium (A) at a shaking rotation speed of 100 rpm or more, and the divided callus is propagated. (3) The grown calli are transferred to a solid medium for differentiation obtained by adding a plant hormone consisting of auxins and cytokinins to the basic medium (A), and cultured. (4) The shoots differentiated from the callus are rooted. And a synthetic medium containing a carbon source, pH-adjusted, and having a reduced inorganic salt concentration, transplanted to a solid medium for the induction and growth of seedlings as a basic medium (B) under aeration conditions. A method for mass-producing the Anthracnose plant, comprising culturing and growing in a container. 2. Naphthalene acetic acid 0.1 to 2.0 mg /
1. A callus-inducing solid culture medium containing 0.1 to 6.0 mg / l of benzyladenine is used.
A method for mass-producing the Anthracnose plant according to the above. 3. Naphthalene acetic acid 0.1-5.0 mg /
2. The method according to claim 1, wherein a liquid medium containing 0.5 to 6.0 mg / l of benzyladenine is used. 4. Naphthalene acetic acid 0.1-5.0 mg /
2. The method according to claim 1, wherein a solid medium for differentiation containing 0 to 1.0 mg / l of benzyladenine is used. 5. It contains sucrose as a carbon source and has a pH of 5.
It is characterized by using a solid medium for the induction and growth of seedlings, wherein the synthetic medium adjusted to 0 to 6.0 and the inorganic salt concentration adjusted to 1/2 to 1/5 is used as the basic medium (B). A method for mass-producing a spinach plant according to claim 1. 6. The method according to claim 1, wherein the cultivation in each step is carried out under a temperature condition of 22 to 27 ° C. and a light irradiation condition of 12 to 18 hours in photoperiod. 7. The method according to claim 1, wherein the plant tissue section is a leaf or petiole tissue section. 8. The method according to claim 1, wherein the synthetic medium is a modified MS medium.