JP2010193838A - Method for culturing plant tissue, method for producing useful substance, device for producing culturing solution and device for culturing plant tissue - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for culturing a plant tissue inducing callus and obtaining the effect of production acceleration of a useful substance stably in a high efficiency by using ethylene as a plant hormone, a method for producing the useful substance by using the above method, a device for producing culturing liquid and a device of culturing. <P>SOLUTION: There are provided the method for culturing the plant tissue including culturing the plant tissue by using the culturing liquid containing fine air bubbles consisting of gas containing ethylene, and the method for producing the useful substance by using the method is also provided. Further, the device 1 for producing the culturing liquid is equipped with a culturing liquid-storing part 11, an ethylene-containing gas-storing part 12 and an ethylene-containing air bubble-forming part 13 for forming the fine air bubbles in the culturing liquid. Still further, the device 100 for culturing the plant tissue is equipped with the device 1 for producing the culturing liquid and a culturing vessel 5. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a plant tissue culture method, a useful substance production method, a culture solution production apparatus, and a plant tissue culture apparatus.

  Useful substances produced by plants are widely used in the food, medical and industrial fields. As a method for obtaining the useful substance, a method for extracting the useful substance from a plant grown or grown naturally is known. However, since the method is greatly influenced by the external environment such as weather, it is difficult to secure an industrially stable production amount.

Then, the method shown below is shown as a method of manufacturing the useful substance which a plant produces | generates industrially stably.
(1) A method for producing a useful substance by introducing a gene of an enzyme involved in metabolism of a useful substance in a plant into a host such as Escherichia coli and culturing the host in a large amount (Patent Document 1).
(2) A method for obtaining useful substances from plant cells cultured in large quantities by a plant tissue culture technique (Patent Document 2).

  In the plant tissue culture in the method (2), a plant tissue such as a piece of plant body, cells, organs, etc. is inoculated into a medium (culture solution) containing components necessary for plant growth, and the plant tissue is called a callus. A large amount of plant tissue is cultured by inducing suspension cells after inducing regular cells, or by differentiating the induced callus into another organ. Thereby, the useful substance which a plant metabolizes can be obtained with high efficiency.

In method (2), plant hormones are added to a medium containing inorganic salts, vitamins, saccharides, etc. to induce callus from plant tissues or promote the production of useful substances in plant tissue culture. A method is shown (Patent Documents 3 and 4).
As the types of plant hormones added to the medium, auxins, cytokinins and the like (hereinafter collectively referred to as “auxins and the like”) are mainly used because they are easy to handle. Furthermore, the use of ethylene as a plant hormone has also been shown (Non-patent Document 1).

JP 2005-245242 A Japanese Patent No. 2759656 Japanese Patent Laid-Open No. 60-47678 Japanese Patent No. 3725189

Biochemical Engineering Journal, 27 (2006): 252-263.

  The genetic engineering method of the method (1) has an advantage that the host can be cultured in a large amount in a short period of time compared to the method (2). However, it is necessary to specify the metabolic pathway of the target useful substance, the enzyme involved in the reaction and its gene, etc., and it is difficult to separate and purify the target useful substance from the host as compared with the method (2). .

On the other hand, in the plant tissue culture of method (2), the method using auxins or the like as plant hormones is limited in plant species from which callus induction and the effect of promoting the production of useful substances can be obtained. In addition, when ethylene is used as a plant hormone, the effect of promoting callus induction and the production of useful substances can be obtained even for plant species that are not effective with the auxins and the like. Then, it is difficult to obtain the effect sufficiently and stably. As a solution to the problem, it is conceivable to continuously supply ethylene during the culture, but even if it is continuously supplied, it is difficult to stably obtain the above-described effects by ethylene, and the economy is inferior. .
For the above reasons, there is a demand for a method for sufficiently and stably obtaining the effect of ethylene in plant tissue culture using ethylene as a plant hormone.

  The present invention relates to a method for culturing plant tissue in which the effect of inducing callus and promoting the production of useful substances is obtained with high efficiency and stability using ethylene as a plant hormone, and high productivity using the culturing method. An object of the present invention is to provide a method for producing a stable useful substance, a culture solution production apparatus, and a plant tissue culture apparatus.

The present invention employs the following configuration in order to solve the above problems.
[1] A method for cultivating a plant tissue, comprising culturing the plant tissue in a culture solution containing fine bubbles made of a gas containing ethylene.
[2] The plant tissue culturing method according to [1], wherein the fine bubbles have an average cell diameter of 10 μm or less.
[3] The plant tissue culturing method according to [1] or [2], wherein the number of bubbles of the fine bubbles is 1,000 or more per 1 mL of the culture solution.
[4] A method for producing a useful substance by culturing a plant tissue, the method comprising culturing the plant tissue by the culturing method according to any one of [1] to [3].
[5] A culture solution storage unit for storing a culture solution for culturing plant tissue, an ethylene-containing gas storage unit for storing a gas containing ethylene, and an ethylene-containing bubble for generating fine bubbles made of the gas in the culture solution A culture medium production apparatus comprising: a generation unit.
[6] A plant tissue culture device comprising the culture solution production apparatus according to [5] and a culture tank for culturing plant tissue with the culture solution supplied from the culture solution production apparatus.

According to the culture method of the present invention, by culturing plant tissue using ethylene as a plant hormone, effects of inducing callus and promoting production of useful substances can be obtained with high efficiency and stability.
Moreover, according to the manufacturing method of this invention, the useful substance which a plant tissue produces | generates can be manufactured stably with high productivity.
Moreover, the culture solution manufacturing apparatus of this invention can manufacture the culture solution excellent in the effect of induction | guidance | derivation of callus and the production | generation promotion of the useful substance of a plant tissue.
Moreover, the culture apparatus of the present invention can produce useful substances with high productivity by culturing plant tissues.

It is the conceptual diagram which showed an example of embodiment of the culture solution manufacturing apparatus of this invention. It is the conceptual diagram which showed other embodiment of the culture solution manufacturing apparatus of this invention. It is the conceptual diagram which showed other embodiment of the culture solution manufacturing apparatus of this invention. It is the conceptual diagram which showed an example of embodiment of the culture apparatus of the plant tissue of this invention. It is the graph which showed stability of the bubble which consists of the gas containing ethylene in the culture solution of Example 1 and Comparative Example 1. 4 is a graph showing the results of the production of hydroxynitrile lyase in Example 2 and Comparative Example 2. 4 is a graph showing the results of anthocyanin production in Examples 3 and 4 and Comparative Example 3.

<Culture method of plant tissue>
The plant tissue culturing method of the present invention uses a culture solution (hereinafter referred to as “culture solution (A)”) containing fine bubbles (hereinafter referred to as “ethylene-containing bubbles”) composed of ethylene-containing gas. Is a method of culturing Ethylene contained in the culture solution (A) acts as a plant hormone on the plant tissue to be cultured, and has the effect of inducing callus and / or promoting the production of useful substances.

As the substrate component of the culture solution (A) (medium) in the present invention, those used for normal plant tissue culture can be used, and can be appropriately selected according to the type of plant tissue to be cultured.
Examples of the culture solution (A) include Murashige-Skoog medium (Murashige-Skoog medium), Rinsmeier-Skoog medium (Linsmeier-Skoog medium), White medium (White medium), Gumholg medium (Gamburg medium), Niche medium ( Nitch medium), Niche-Niche medium (Nitch-Nitch medium), Korenbach-Schmidt medium (Kohlenbach-Schmidt medium), or modified media thereof.

  It is preferable that a substance (surfactant) exhibiting a surfactant activity is added to the culture solution (A). The addition of a surfactant to the culture solution (A) makes it easier for the substrate components to be taken into the plant tissue in the culture solution (A), and also makes it easier to stably generate ethylene-containing bubbles. Manufacturing of (A) becomes easy. In addition, the stability of the ethylene-containing bubbles in the culture solution (A) is also improved.

As the surfactant, a nonionic surfactant, a cationic surfactant, an anionic surfactant, an amphoteric surfactant and the like can be used, and a nonionic surfactant is preferable. Examples of nonionic surfactants include polyoxyalkylene polyhydric alcohol fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene sucrose fatty acid esters, alkyl glucosides, polyglycerin fatty acid esters, polyoxyalkylene alkyl ethers. And polyoxypropylene / polyoxyethylene condensates. Examples of polyoxyethylene sorbitan fatty acid esters include polyoxyethylene sorbitan monooleate (commercially available from Atlas Powder under the name Tween 80), polyoxyethylene sorbitan monostearate (Tween 60), polyoxyethylene Examples include sorbitan monopalmitate (Tween 40) and polyoxyethylene sorbitan monolaurate (Tween 20).
Besides these, Triton X-100 (Triton X-100), polyoxyethylene (20) cetyl ether (Brij-58), nonylphenol ethoxylate (Tergitol NP-40), etc. are generally used in the field of biology. Available surfactants are available.
These surfactants may be used alone or in combination of two or more.

  The content of the surfactant in the culture solution (A) varies depending on the type of the surfactant, but is preferably 0.001 to 5% by mass, more preferably 0.01 to 2% by mass. preferable. If the content of the surfactant is 0.001% by mass or more, the intake efficiency of the substrate component by the plant tissue in the culture solution (A) is improved, and the production of the culture solution (A) becomes easy, The stability of the ethylene-containing bubbles in the culture solution (A) is also improved. Moreover, if content of surfactant is 5 mass% or less, it will be easy to prevent the excessive foaming at the time of producing | generating an ethylene containing bubble, and cost can be reduced.

Further, the culture solution (A) may contain plant hormones other than ethylene as plant hormones.
Examples of other plant hormones include auxins, cytokinins, gibberellins, abscisins, brassinosteroids, jasmonic acid, or similar substances thereof, substances having elicitor activity, and substances having growth promoting activity. It is done.
Examples of substances having elicitor activity include polysaccharides, proteins, peptides, lipids, organic acids, chitin, chitosan, pectin, galacturonic acid, alginic acid, glucan, xylan and other oligosaccharides, heavy metals (ions), jasmonic acid and their derivatives. , Mold, yeast, crushed bacteria, and the like.
Examples of the substance having growth promoting activity include coconut milk, casein degradation product, yeast extract, vitamins, inorganic salts, peptidic plant growth factor and the like.
These other plant hormones may be used alone or in combination of two or more.

  The ethylene concentration of the ethylene-containing bubbles in the culture solution (A) is preferably 0.1 to 99.9% by volume, more preferably 2 to 99% by volume, and 5 to 90% by volume. Further preferred. When the ethylene concentration is 0.1% by volume or more, effects such as induction of callus by ethylene and promotion of production of useful substances are easily obtained. Moreover, if the said ethylene concentration is 99.9 volume% or less, cost can be reduced.

The ethylene concentration in the ethylene-containing bubbles can be adjusted by adjusting the concentration of ethylene in the ethylene-containing gas used for generating the ethylene-containing bubbles (hereinafter referred to as “ethylene-containing gas”).
The gas used to adjust the ethylene concentration in the ethylene-containing gas may be any gas that does not adversely affect the cultivation of plant tissue with the culture solution (A). For example, nitrogen gas, inert gas such as rare gas, oxygen Examples include gas, hydrogen gas, and carbon dioxide gas. Of these, an inert gas such as nitrogen gas or rare gas is preferable from the viewpoint of the stability of culture.

In the culture method of the present invention, the ethylene-containing gas is present as fine bubbles in the culture solution (A). Thereby, the ethylene-containing bubbles can be retained in the culture solution (A) for a long period of time, and the effect of ethylene can be obtained with high efficiency and stability.
As a result of studies by the present inventors, in the conventional culture method using ethylene, since ethylene is hardly soluble in water, most of it was diffused into the gas phase, and the effect of ethylene was not sufficiently obtained. I understood. In order to solve this problem, a method of culturing while continuously ventilating ethylene into the culture solution was considered. However, this method imparts stress to the cultured cells and makes the ethylene concentration non-uniform in the culture solution. Therefore, it is difficult to use ethylene effectively. Therefore, the present inventors considered that these problems were due to the use of bubbles having a large bubble diameter (several mm to several cm), and attempted to use ethylene-containing gas as fine bubbles. As a result, it was found that ethylene can be stably maintained in the culture medium, and that the effect of ethylene can be obtained with high efficiency and stability without continuous supply.

The average bubble diameter of the ethylene-containing bubbles in the culture solution (A) is preferably 10 μm or less, more preferably 5 μm or less, and even more preferably 1 μm or less. However, an average bubble diameter means the number average of the bubble diameter of the ethylene containing bubble in a culture solution (A).
If the bubble diameter is 10 μm or less, the rate at which the ethylene-containing bubbles rise in the culture solution (A) decreases, so the residence time until the bubbles are released from the culture solution (A) into the gas phase The effect of inducing callus by ethylene and promoting the production of useful substances is easily obtained.
The average bubble diameter of the ethylene-containing bubbles is preferably 0.05 μm or more, and more preferably 0.1 μm or more, from the viewpoint of ease of bubble formation.
Examples of the method for measuring the average bubble diameter of ethylene-containing bubbles include LS 13 320 (manufactured by Beckman Coulter, Inc.) measured by a laser diffraction scattering method, Multisizer 3 (manufactured by Beckman Coulter, Inc.) measured by a pore electrical resistance method, and the like. It is done.

The number of ethylene-containing bubbles in the culture solution (A) is preferably 1,000 or more, more preferably 10,000 or more, and 50,000 per mL of the culture solution (A). More preferably, it is the above. If the number of bubbles is 1,000 or more per 1 mL of the culture solution (A), it is easy to obtain callus induction by ethylene and the effect of promoting the production of useful substances. The effect obtained by improving the contact efficiency of the plant tissue is further increased.
The number of ethylene-containing bubbles is preferably 10 million or less, more preferably 1,000,000 or less per 1 mL of the culture solution (A), from the viewpoint of easy bubble formation.
Examples of the method for measuring the number of bubbles of ethylene-containing bubbles include Multisizer 3 (manufactured by Beckman Coulter, Inc.).

The culture solution (A) generates ethylene-containing bubbles by aerating ethylene-containing gas to the culture solution containing the substrate component of the culture solution (A) and, if necessary, a surfactant and other plant hormones. Can be manufactured. Hereinafter, the culture solution before aeration with the ethylene-containing gas is referred to as a culture solution (a).
The method for venting the ethylene-containing gas to the culture solution (a) is not limited as long as it is a method capable of generating fine bubbles, and is a known swirling method, pressure dissolution method, mechanical stirring method, ultrasonic method, membrane. A transmission method or a combination of these methods can be used.

Hereinafter, an example of the culture solution production apparatus for producing the culture solution (A) will be shown and described in detail.
[Culture medium production equipment]
(First embodiment)
As shown in FIG. 1, the culture solution production apparatus 1 of the present embodiment includes a culture solution storage unit 11 that stores a culture solution (a), and an ethylene-containing gas storage unit 12 that stores an ethylene-containing gas (hereinafter, simply “ A gas storage unit 12 "), an ethylene-containing bubble generation unit 13 (hereinafter simply referred to as" bubble generation unit 13 ") for generating ethylene-containing bubbles by aerating ethylene-containing gas into the culture solution (a). And a culture solution supply unit 14 for supplying the culture solution (A).
Further, the culture solution storage unit 11 and the bubble generation unit 13 are connected by a pipe 21, and the gas storage unit 12 and the bubble generation unit 13 are connected by a pipe 22, and the bubble generation unit 13 and the culture solution supply unit are connected. 14 is connected by a pipe 23.

The culture solution storage unit 11 stores the culture solution (a) that does not contain the ethylene-containing bubbles having the necessary components of the culture solution (A).
The magnitude | size of the culture solution storage part 11 is not specifically limited, It can set arbitrarily considering the quantity of the culture solution (A) manufactured according to a culture scale. Moreover, the shape of the culture solution storage part 11 is not specifically limited.
The material of the culture solution storage unit 11 is not particularly limited, and examples thereof include a storage tank made of ceramic, glass, metal, or fluororesin.
The culture medium storage unit 11 may be a single tank or a combination of a plurality of tanks.

The gas storage unit 12 stores an ethylene-containing gas.
As the gas storage part 12, it is preferable to store ethylene as a gas, and a pressure vessel such as a gas cylinder is preferable. The gas storage unit 12 may be a container that contains a liquid or solid substance and generates and stores ethylene by reacting the substance chemically or biologically.

The bubble generating unit 13 generates an ethylene-containing bubble by ventilating the ethylene-containing gas supplied from the gas storage unit 24 into the culture solution (a) supplied from the culture solution storage unit 11 to form fine bubbles. is there.
The bubble generation unit 13 may be any unit that can generate fine bubbles, and in this example, a pump 15 that feeds the culture solution, and a bubble generation nozzle 16 that generates a fine bubble by generating a swirling flow. It is a bubble generation part by the swirl flow system which has. Examples of the bubble generating nozzle 16 include those described in JP-A-2006-142300.

The culture solution supply unit 14 supplies the culture solution (A) containing the ethylene-containing bubbles obtained by the bubble generation unit 13.
The structure of the culture solution supply unit 14 is not particularly limited, but a structure that can supply and stop the culture solution using a valve or the like is preferable.
Further, the culture solution supply unit 14 is preferably provided with a liquid feeding mechanism such as a pump for supplying the culture solution (A). However, in the case where the bubble generating unit 13 is provided with a culture solution feeding mechanism (pump 15 in this example) such as a pump, the culture solution supply unit 14 may not be provided with a solution feeding function. .

Hereinafter, the operation of the culture broth production apparatus 1 will be described.
The composition and amount of the culture solution (a) can be appropriately determined according to the type of plant tissue to be cultured by the culture solution (A), the culture scale, and the like.
First, the culture solution (a) stored in the culture solution storage unit 11 is sent by the pump 15 to the bubble generation unit 13 through the pipe 21 and is passed through the bubble generation nozzle 16. At the same time, the ethylene-containing gas stored in the gas storage unit 12 is supplied to the bubble generation nozzle 16 of the bubble generation unit 13 through the pipe 22. The supply mechanism of the ethylene-containing gas may be a mechanism that is sent to the bubble generation unit 16 by pressurization, or may be a mechanism that is sucked into the bubble generation unit 16 by decompression.
In the bubble generation nozzle 16, a swirling flow is formed by the culture solution (a) to be passed, and a gas cavity containing ethylene is formed at the center of the vortex generated by the swirling flow. When the tip of the gas cavity containing ethylene is torn off by the shearing force generated by the swirling flow of the culture solution (a), fine bubbles containing ethylene are generated.
The average bubble diameter and the number of bubbles of the ethylene-containing bubbles in the obtained culture solution (A) are the shape of the bubble generation nozzle 16, the degree of swirling flow to be generated, and the culture solution (a) flowing through the bubble generation nozzle 16. It can be controlled by adjusting the liquid flow rate, the flow rate of the ethylene-containing gas, the type and addition amount of the surfactant.
The culture solution (A) containing ethylene-containing bubbles generated by the bubble generation nozzle 16 is fed to the culture solution supply unit 14 through the pipe 23 and supplied to a predetermined container or the like by operating a valve or the like.

The bubble generating unit 13 is not limited to the above-described swirling flow method, and may employ a pressure dissolution method, a mechanical stirring method, an ultrasonic method, a membrane permeation method, or a combination thereof. May be.
For example, the membrane permeation method includes a porous glass module (bubble generating part) that has a porous glass membrane and can pass a culture solution inside. In the porous glass module, ethylene-containing bubbles are introduced into the culture solution by ventilating the ethylene-containing gas through the porous glass membrane having many fine pores with respect to the culture solution passed through the module. Can be generated.

  The average bubble diameter of the ethylene-containing bubbles in the culture solution (A) in the membrane permeation method is the average pore diameter of the pores through which the ethylene-containing gas is vented, the pressure of the ethylene-containing gas, the flow rate of the culture solution to be passed, It can be controlled by adjusting the type and amount added. The number of bubbles can be controlled by adjusting the surface area (number of pores) of the porous glass membrane, the aeration time of the ethylene-containing gas, the flow rate of the culture solution to be passed, the type and addition amount of the surfactant.

In order to sufficiently generate ethylene-containing bubbles having an average bubble diameter of 10 μm or less in the module, the average hole diameter of the holes through which the ethylene-containing gas is vented is preferably 0.05 to 1 μm.
The pressure of the ethylene-containing gas at the time of aeration usually depends on the average pore size of the membrane, the amount of surfactant added, etc. For example, when the average pore size of the membrane is 0.08 μm, the gas pressure is 2.5 to It is preferably 3.5 MPa.
The flow rate of the culture solution varies depending on the amount of the culture solution to be prepared, but in the case of 1 L of the culture solution, it is preferably 0.5 to 10 L / min.

(Second Embodiment)
Next, a culture solution production apparatus 2 which is another embodiment of the culture solution production apparatus of the present invention will be described with reference to FIG. In the culture broth production apparatus 2, the same parts as those in the culture broth production apparatus 1 are denoted by the same reference numerals and description thereof is omitted.
As shown in FIG. 2, the culture medium production apparatus 2 includes a culture medium storage unit 11, a gas storage unit 12, a bubble generation unit 13, a culture solution supply unit 14, and a switching valve 17, and the culture solution storage unit 11. And the bubble generation unit 13 are connected by a pipe 21, the gas storage unit 12 and the bubble generation unit 13 are connected by a pipe 22, and the bubble generation unit 13 and the switching valve 17 are connected by a pipe 24. The culture solution storage unit 11 and the switching valve 17 are connected by a pipe 25, and the switching valve 17 and the culture solution supply unit 14 are connected by a pipe 26.

In the culture solution production apparatus 2, when the ethylene-containing gas is generated by aeration of the ethylene-containing gas into the culture solution (a), the switching valve 17 causes the pipe 24 and the pipe 25 to communicate with each other and the liquid is sent from the culture solution storage unit 11. The culture medium (a) to be circulated is returned to the culture medium storage section 11 through the pipe 21, the bubble generating section 13, the pipe 24, and the pipe 25 in order. Accordingly, the number of ethylene-containing bubbles can be easily improved by continuously venting the ethylene-containing gas.
Then, after the culture solution (A) containing sufficient ethylene-containing bubbles is obtained, the switching valve 17 is switched so that the pipe 24 and the pipe 26 communicate with each other, and the culture solution (A) is supplied from the culture solution supply unit 14. .

(Third embodiment)
Next, a culture solution production apparatus 3 which is still another embodiment of the culture solution production apparatus of the present invention will be described with reference to FIG. In the culture broth production apparatus 3, the same parts as those in the culture broth production apparatus 1 are denoted by the same reference numerals and description thereof is omitted.
As shown in FIG. 3, the culture medium production apparatus 3 includes a culture medium storage unit 11, a gas storage unit 12, a bubble generation unit 13, and a culture solution supply unit 14, and the culture solution storage unit 11 and the bubble generation unit. 13 is connected by a pipe 21, the gas storage part 12 and the bubble generation part 13 are connected by a pipe 22, and the downstream side of the bubble generation part 13 and the culture solution storage part 11 are connected by a pipe 27. The culture solution storage unit 11 and the culture solution supply unit 14 are connected by a pipe 28.

In the culture solution production apparatus 3, the culture solution (a) fed from the culture solution storage unit 11 is passed through the pipe 21, the bubble generation unit 13, and the pipe 27 in order and returned to the culture solution storage unit 11 for circulation. . Accordingly, the number of ethylene-containing bubbles can be easily improved by continuously venting the ethylene-containing gas. Then, the culture solution (A) containing ethylene-containing bubbles is supplied to the culture solution supply unit 14 through the pipe 28 and supplied from the culture solution supply unit 14.
It is preferable that the culture solution supply unit 14 in the culture solution production apparatus 3 is connected to a portion near the bottom of the culture solution storage unit 11.

(Other embodiments)
In addition, the culture solution manufacturing apparatus of this invention is not limited to the above-mentioned culture solution manufacturing apparatuses 1-3. For example, the culture solution storage unit stores the solvent component (a component obtained by adding a surfactant to water or the like as necessary) and the solute component (nutrient component) of the culture solution (a). The solvent storage unit may be divided into a solute storage unit, and the solvent storage unit may be disposed on the upstream side of the bubble generation unit 13 and the solute storage unit may be disposed on the downstream side of the bubble generation unit 13. With such an arrangement, the bubble generation unit 13 is less likely to be contaminated or clogged with the solute component of the culture solution (a), so that the maintenance frequency of the apparatus can be reduced.
Moreover, you may provide the sterilization part which suppresses the biological contamination (contamination) of a culture solution (A) in the downstream of the bubble generation part 13. FIG.

[Culture method]
The culture method of the present invention is a method for culturing plant tissue using the culture solution (A) described above.
The plant tissue used in the culturing method of the present invention may be a tissue of a plant species that can induce callus and / or promote the production of useful substances with ethylene. For example, useful substances such as organic compounds such as pigments and enzymes And moss, lichens, lichens, fungi, algae, microalgae, seaweeds, seaweeds and the like that are similar to these plants.
The plant tissue can be used in various forms such as a piece of plant tissue, a cell, an organ, a cultured cell, a protoplast, and the like. However, the culture method of the present invention is not limited to the cultivation of the plant tissue of the above-described form.

Specific examples of plant tissues in which callus induction is promoted by an appropriate concentration of ethylene include, for example, almonds (Prunes amygdulicis) that produce the enzyme hydroxynitrile lyase, and Nichinichi grass (Cathanthanthus roseus) that produces Indole alkaloids. Etc.
Specific examples of plant tissues whose production of useful substances is promoted by ethylene include, for example, taxoid compounds such as strawberry (Fragaria ananassa), rose (Rosa hybrids), grape (Vitis vinifera); Genus yew plants (for example, Taxus cuspidata) that produce Indoalkaloids; Catharanthus roseus that produces Indole alkaloids; almonds (Prunes amygdulcis) that produce hydroxynitrile lyase, an enzyme, and the like.

In the culture method of the present invention, a normal plant tissue culture method can be used except that the culture solution (A) is used.
Although the culture temperature varies depending on the type of plant tissue to be cultured, the culture temperature normally used in plant tissue culture is 10 to 40 ° C, preferably 20 to 37 ° C.
In the culture method of the present invention, the culture may be performed while supplying oxygen gas, hydrogen gas, carbon dioxide gas or the like to the culture solution (A) as necessary.

  Conditions for the pH and dissolved oxygen concentration (DO) of the culture solution (A) in plant tissue culture can be appropriately selected according to the plant tissue to be cultured. As for the pH of the culture solution (A), the pH of the culture solution (a) before generating the ethylene-containing bubbles may be adjusted in advance, and after the generation of the ethylene-containing bubbles, the pH of the culture solution (A) is adjusted. You may adjust. The DO of the culture solution (A) may contain oxygen at a predetermined concentration in an ethylene-containing gas that generates ethylene-containing bubbles, or oxygen gas may be separately supplied during the culture.

In addition, conditions such as the amount of light irradiation, agitation, and shaking during cultivation can be appropriately selected according to the plant tissue to be cultured, and it is not necessary to set special conditions as compared to normal plant tissue culture. . In the culture solution (A) of the present invention, even if stirring or shaking is performed under conditions normally employed for plant tissue culture, the stability of the ethylene-containing bubbles is hardly lowered, so that the effect of ethylene is stable and sufficient. It is possible to get to.
Moreover, culture | cultivation of this invention can use methods, such as a batch type, a semibatch type, a semi-continuous type, a continuous type, suitably according to the kind and planting scale of a plant tissue.

  Although the culture method of the present invention described above is a method of supplying ethylene, which is a gas hardly soluble in water, to the culture solution, the microbubbles are cultured by forming microbubbles made of a gas containing ethylene. It can remain in the liquid (A) for a long time. Therefore, the ethylene concentration can be maintained for a long time by suppressing the release of ethylene from the culture solution (A) into the gas phase, and the effects of inducing callus and promoting the production of useful substances are highly efficient and Obtained stably. Further, the culture method of the present invention is excellent in economic efficiency because it is not necessary to continuously supply ethylene during the culture.

[Plant tissue culture equipment]
The plant tissue culture of the present invention can be performed, for example, with the plant tissue culture apparatus shown below.
As shown in FIG. 4, the plant tissue culture apparatus 100 of the present embodiment (hereinafter simply referred to as “culture apparatus 100”) includes the above-described culture liquid production apparatus 1 for producing a culture liquid (A) and a culture liquid ( A sterilizer 4 for sterilizing A) and a culture tank 5 for culturing plant tissue with the culture solution (A) are provided. In the culture apparatus 100, the culture solution supply unit 14 of the culture solution production apparatus 1 and the sterilization apparatus 4 are connected by a pipe 29, and the sterilization apparatus 4 and the culture tank 5 are connected by a pipe 30.
The connection position of the piping 30 in the culture tank 5 is not particularly limited, but is preferably near the bottom of the culture tank 5. By connecting the pipe 30 to such a position, the culture solution (A) can be supplied to the culture vessel 5 more gently, and the residence time of the ethylene-containing bubbles in the culture solution (A) is improved. This makes it easier to obtain the effect.

  The sterilizer 4 is a device that sterilizes the culture solution (A) in order to suppress biological contamination (contamination) of the culture solution (A). The sterilizer 4 may be any device that can sterilize the culture solution (A) and suppress biological contamination, and is a known sterilizer that uses ultraviolet rays, radiation, photocatalysts, metal catalysts, discharge, plasma, ozone, filters, and the like. The device can be used.

In the culture tank 5, a stirrer 51 that stirs the culture solution (A), an aeration member 52 that supplies oxygen to the culture solution (A), and various items of the culture solution (A) as necessary. A sensor 53 is provided.
The shape and size of the culture tank 5 are not particularly limited, and can be appropriately determined according to the culture scale. For example, the culture tank 5 can be applied to the case where a plant is cultivated (cultured) in a form such as hydroponics by making the shape of the culture tank 5 into a water channel shape or a flat plate shape.
Moreover, the material of the culture tank 5 is not specifically limited, The product made from a ceramic, glass, metal, and a fluororesin is preferable.

  The stirrer 51 plays a role of improving the contact efficiency between the plant tissue (cells), ethylene, and the substrate component by stirring the culture solution (A). The blade 51a of the stirrer 51 may be any blade that can improve the contact efficiency between the plant tissue and the culture solution (A) by stirring. In this example, the blade 51a is a two-blade type.

The diffuser member 52 plays a role of supplying a desired gas such as oxygen gas, hydrogen gas, carbon dioxide gas or the like to the culture solution (A).
The diffuser member 52 is connected to, for example, a blower (not shown) for sending a gas (oxygen gas or a gas with a high oxygen concentration) supplied to the culture tank 5, and the gas is used as a filter or the like. The form supplied through the sterilizing member is included.
The shape of the diffuser member 52 is preferably a porous plate or the like from the viewpoint that the bubble diameter is reduced and the oxygen dissolution efficiency is easily improved.
The material of the diffuser member 52 is not particularly limited, and it is preferable to use a material made of ceramic, glass, metal, fluororesin or the like.

Examples of the sensor 53 include sensors that can detect items such as pH, DO, oxidation-reduction potential (ORP), liquid temperature, cell amount, ethylene concentration, useful substance amount (metabolite amount), etc. according to each culture object and purpose. It is done.
In addition, when the sensor 53 is provided in the culture tank 5, it is preferable to provide a control device that can feedback control the optimum conditions for the culture according to the measured value.

  In the culture apparatus 100 described above, the culture liquid (A) manufactured by the culture liquid manufacturing apparatus 1 is sent to the sterilization apparatus 4 through the pipe 29 to be sterilized and supplied to the culture tank 5. Next, the plant tissue is inoculated into the culture solution (A), stirred by the stirring device 51, supplied with oxygen by the aeration device 52, and cultured while detecting various items by the sensor 53.

In addition, the plant tissue culture apparatus of the present invention is not limited to the culture apparatus 100 described above. For example, instead of the culture medium production apparatus 1, an apparatus including the culture medium production apparatuses 2 and 3 may be used, or an apparatus including other culture liquid production apparatuses may be used.
Further, although the aeration and stirring type culture tank 5 is shown in the culture apparatus 100, the aeration type, the air lift type, the bubble column type, the rotary drum type, etc. are generally used depending on the type of plant tissue to be cultured and the culture scale. The culture tank currently used can be used suitably. Moreover, you may have a mechanism which raises the dissolved density | concentration of ethylene and oxygen in a culture solution (A) by raising the pressure in the culture tank 5. FIG.

Moreover, the apparatus which does not have the aeration member 52 may be sufficient depending on the plant tissue to culture, for example, using the apparatus similar to the culture solution manufacturing apparatus 1, it is the same composition as culture solution (A), and oxygen is used. The culture apparatus which manufactures the culture solution containing the microbubble which consists of a gas to contain, and supplies this culture solution to the culture tank 5 may be sufficient.
In addition, the apparatus which does not have the stirring apparatus 51 may be sufficient, for example, arrange | position a vibration member etc. inside the culture tank 5, or an outer surface, and culture medium (A) by vibrating the culture tank 5 itself. It may be a device that performs the stirring. Moreover, the culture apparatus which does not use a sensor may be sufficient.
Furthermore, the culture apparatus of the present invention may employ any system such as a batch system, a semi-batch system, a semi-continuous system, and a continuous system.

<Method for producing useful substances>
The method for producing a useful substance of the present invention is a method for producing a useful substance produced by a plant tissue, and includes a step of culturing the plant tissue by the above-described culture method.
Examples of useful substances produced by the production method of the present invention include Taxoid compounds such as strawberry (Fragaria ananassa), rose (Rosa hybrids), grape (Vitis vinifera), and Paclitaxel that produce anthocyanin as a pigment. Examples include yew plants (for example, Taxus cuspidata); Nichinichi grass that produces Indole alkaloids; Almonds that produce hydroxynitrile lyase, an enzyme, and the like.

The plant tissue culturing step is as described in the above-described culturing method.
The method for recovering the useful substance from the plant tissue grown by the culture is not particularly limited, and a suitable method can be used according to the target plant tissue and the kind of the useful substance from the conventional recovery methods.

  Since the production method of the useful substance of the present invention described above can maintain the ethylene-containing bubbles in the culture solution (A) for a long time during the culture, the ethylene concentration in the culture solution (A) can be maintained high, The production of useful substances is promoted by ethylene, and the desired useful substances can be produced stably with high productivity.

Hereinafter, the present invention will be described in detail with reference to Examples and Comparative Examples. However, the present invention is not limited by the following description. In this example, “%” means “mass%” unless otherwise specified.
<Stability of ethylene-containing bubbles>
A culture solution containing ethylene-containing bubbles was produced, and the stability of the ethylene-containing bubbles in the culture solution was evaluated.
[Example 1]
The culture solution production apparatus 3 shown in FIG. 3 has a bubble generation unit employing a membrane permeation method using a porous glass membrane (average pore size 0.08 μm, manufactured by SPG Techno) as the bubble generation unit 13. (Hereinafter referred to as “manufacturing apparatus 3A”).
The culture solution was a culture solution (a1) in which 0.2% of Tween 80 (manufactured by Wako Pure Chemical Industries, Ltd.) was added as a surfactant to the Gamborg B5 medium, and the solution temperature was set to 25 ° C.
In the production apparatus 3A, the culture solution (a1) is circulated (liquid flow rate 1.3 L / min) inside the bubble generation unit, and the ethylene concentration diluted with nitrogen in the culture solution (a1) in the bubble generation unit is 5.0. A culture solution (A1) containing ethylene-containing bubbles was produced by venting 10% by volume of ethylene-containing gas through the porous glass membrane at a pressure of 3.5 MPa.
The average bubble diameter and number of bubbles of ethylene-containing bubbles remaining in the culture solution (A1) immediately after preparation of the culture solution (A1) were measured with Multisizer 3 (detection limit 0.4 μm, manufactured by Beckman Coulter, Inc.). Was 1.0 μm, and the number of bubbles (measured bubble diameter: 0.4 to 2 μm) was about 57,000 per 1 mL of the culture solution (A1).

[Comparative Example 1]
The culture broth (B1) was produced by ventilating the culture broth (a1) of Example 1 with an ethylene-containing gas diluted with nitrogen and having an ethylene concentration of 5.0% by volume using a tube tube having an inner diameter of 5 mm for 10 minutes. .
Immediately after preparation of the culture solution (B1), the average bubble diameter and the number of bubbles of ethylene-containing bubbles remaining in the culture solution (B1) were measured with Multisizer 3 (manufactured by Beckman Coulter). B1) The number of bubbles per mL (measured bubble diameter: 2 to 80 μm) was about 300. Bubbles with a diameter of several millimeters to several centimeters generated at the time of aeration immediately floated and were diffused into the gas phase, and could not be measured.

[Evaluation methods]
The culture solution (A1) when the culture solutions (A1) and (B1) containing ethylene-containing bubbles obtained in Example 1 and Comparative Example 1 were allowed to stand for 1 hour, 3 hours, 24 hours, and 72 hours. , (B1) each contained ethylene concentration was measured by gas chromatography (GC-18A, manufactured by Shimadzu Corporation, column: Porapak Q (Mesh 60/80), measurement temperature 75 ° C.). The result is shown in FIG.

As shown in FIG. 5, in the culture solution (A1) of Example 1, the ethylene concentration was maintained at 5 mg / L even after 72 hours, and the culture solution (A1) was obtained by using fine bubbles containing ethylene. ) Ethylene was retained for a long time.
On the other hand, the culture solution (B1) of Comparative Example 1 already had an ethylene concentration of approximately 0 mg / L after 24 hours, and after 72 hours, all the ethylene-containing bubbles in the culture solution (B1) were diffused into the air. Was.

<Production of hydroxynitrile lyase (HNL)>
A production test of hydroxynitrile lyase (HNL) using cultured cells of almond (Prunus amygdulcis) was performed.
[Example 2]
Almond cells to be inoculated are sterilized using 70% ethanol solution and 5% NaClO solution (0.2% Tween 80 added), washed with distilled water 3 times, and then finely cut tissue pieces are removed. , Gamburg B5 medium (2,4-dichlorophenoxyacetic acid (1 mg / L), benzylaminopurine (1 mg / L)) was used for shaking culture (25 ° C., 120 rpm, dark condition), and induced callus cells were used. It was.
The culture broth was added to 2,4 dichlorophenoxyacetic acid (1 mg / L), benzylaminopurine (1 mg / L), and Tween 80 (0.2%) in Gamburg B5 medium, and the filter had a pore size of 0.2 μm. The culture solution (a2) filtered with (25CS020AS, manufactured by Advantech Toyo Co., Ltd.) was used.
In the same production apparatus 3A as in Example 1, the culture solution (a2) was circulated (liquid flow rate 1.3 L / min) inside the bubble generation unit, and the ethylene concentration diluted with nitrogen was added to the culture solution (a2) in the bubble generation unit. 5.0% by volume of ethylene-containing gas was aerated through the porous glass membrane for 10 minutes at a pressure of 3.5 MPa to produce a culture solution (A2) containing ethylene-containing bubbles.
When the average bubble diameter and the number of bubbles of the ethylene-containing bubbles in the culture solution (A2) were measured by the Multisizer 3, the average bubble size was 0.9 μm, and the number of bubbles (measured bubble size: 0.4 to 2 μm) was the culture solution ( A2) About 55,000 per mL.
Subsequently, the callus cells were inoculated into 200 mL of the culture solution (A2), and cultured under shaking at 120 ° C. for 7 days under dark conditions at 25 ° C.

[Comparative Example 2]
The callus cells were cultured with shaking in the same manner as in Example 2 except that the ethylene-containing gas was not used in the culture solution (a2) of Example 2 as it was.

[Evaluation methods]
The amount of HNL produced was evaluated by measuring the HNL enzyme activity by the following method.
Before each culture (immediately after the callus cell inoculation) and after each culture, callus cells are ground in a mortar, 50 mL of distilled water is added, and the upper layer water is centrifuged twice (3000 rpm, 10 minutes). Clear water was used as sample water. Next, a solution is prepared by adding DL-mandelotril (0.931 μL) as a substrate to 0.2 M sodium citrate buffer (7 mL, pH 5.5), and the sample solution (10 μL) is added to 3 mL of the solution. The change in absorbance (20 ° C.) due to benzaldehyde produced by the decomposition of the substrate was measured at a wavelength of 249.6 nm for 30 seconds. The HNL activity was calculated from the initial reaction rate in the measurement. The results for callus cells before culture are shown in FIG. 6 (A), and the results for callus cells after culture (after 7 days) are shown in FIG. 6 (B).

As shown in FIGS. 6 (A) and (B), in the callus cells of the culture solution (A2) of Example 2, the HNL activity after the culture was 0.3 (U / g-fresh cell). This is because the production | generation of HNL was accelerated | stimulated because the culture solution (A2) contains the fine ethylene containing bubble.
On the other hand, in the callus cells of Comparative Example 2, the HNL activity of the callus cells could not be confirmed even after 7 days of culture, and the production of HNL was not promoted.

<Manufacture of anthocyanin (red pigment)>
An anthocyanin (red pigment) production test using cultured cells of rose (Rosa hybrids) was performed.
[Example 3]
The rose cells to be inoculated were obtained by carrying out callus culture of tissue pieces collected from rose petals by shaking culture (25 ° C., 120 rpm, dark conditions) using MS medium (Murashige-Skoog medium). Mutant strains were used.
As the culture solution, a culture solution (a3) obtained by adding 0.2% by mass of Tween 80 to the MS medium and filtering with a filter having a pore size of 0.08 μm (Track Etch New Clipore, manufactured by Whatman) was used.
The culture medium production apparatus 3 (FIG. 3) has a bubble generation unit employing a swirling flow method using a microbubble generation apparatus (BT-50, manufactured by Bubble Tank Co., Ltd.) as the bubble generation unit 13. Hereinafter, “manufacturing apparatus 3B”) was used.
Using the production apparatus 3B, the culture solution (a3) is circulated (liquid flow rate: 1.3 L / min) in the microbubble generator which is the bubble generating unit 16, and the ethylene concentration is 5.0% by volume diluted with nitrogen. Gas was aerated for 10 minutes to produce a culture solution (A3) containing ethylene-containing bubbles.
When the average bubble diameter and the number of bubbles of ethylene-containing bubbles remaining in the culture solution (A3) immediately after preparation of the culture solution (A3) were measured by the Multisizer 3, the average bubble size was 10 μm, and the number of bubbles (measured bubble size: 2). ˜80 μm) was about 3,200 per 1 mL of the culture solution (A3).
Next, 200 mL of the culture solution (A3) was inoculated with the mutant strain, and cultured with shaking (120 rpm) at 25 ° C. under dark conditions for 3 days.

[Example 4]
Using the same production apparatus 3A as in Example 1, in the production apparatus 3A, the culture solution (a3) was circulated (liquid flow rate 1.3 L / min) inside the bubble generation unit, and the culture solution (a3) was produced in the bubble generation unit. Then, an ethylene-containing gas diluted with nitrogen and having an ethylene concentration of 5.0% by volume was aerated through the porous glass membrane at a pressure of 3.5 MPa for 10 minutes to produce a culture solution (A4) containing ethylene-containing bubbles.
When the average bubble diameter and the number of bubbles of ethylene-containing bubbles remaining in the culture solution (A4) immediately after preparation of the culture solution (A4) were measured by the Multisizer 3, the average bubble size was 1.0 μm, and the number of bubbles (measured bubble size : 0.4-2 μm) was about 52,000 per 1 mL of the culture solution (A4).
Next, 200 mL of the culture solution (A4) was inoculated with the mutant strain, and cultured with shaking (120 rpm) at 25 ° C. under dark conditions for 3 days.

[Comparative Example 3]
An ethylene-containing gas having an ethylene concentration of 5.0% by volume diluted with nitrogen was bubbled through the glass aeration filter (glass ball filter G4, Kinoshita Rika Kogyo Co., Ltd.) with respect to the culture solution (a3) of Example 3. A culture solution (B2) was obtained.
When the average bubble diameter and the number of bubbles of ethylene-containing bubbles remaining in the culture solution (B2) immediately after preparation of the culture solution (B2) were measured by the Multisizer 3, the average bubble size was 48 μm and the number of bubbles (measured bubble size: 2). ˜80 μm) was about 700 per 1 mL of the culture solution (B2). Bubbles with a diameter of several millimeters to several centimeters generated at the time of aeration immediately floated and were diffused into the gas phase, and could not be measured.
Next, 200 mL of the culture solution (B2) was inoculated with the mutant strain, and cultured with shaking (120 rpm) for 3 days at 25 ° C. in the dark.

[Evaluation methods]
The anthocyanin concentration is determined by extracting the pigment from each culture solution before culture (immediately after the inoculation of the mutant strain) and after culture by adding an HCl-methanol solution to the culture solution and shaking the suspension. After filtering with a 0.45 μm discic filter (25CS045AS, manufactured by Advantech Toyo Co., Ltd.), the absorbance at a wavelength of 530 nm was measured.
FIG. 7A shows the anthocyanin concentration of each culture solution before culturing, and FIG. 7B shows the anthocyanin concentration of each culture solution after culturing (after 3 days).

As shown in FIG. 7, the anthocyanin concentration of Example 3 using the culture solution (A3) containing ethylene-containing bubbles having an average bubble diameter of 10 μm was 0.2 g / L before the cultivation, whereas after the cultivation. Increased to 0.7 g / L, and the amount of anthocyanin produced increased. Similarly, the anthocyanin concentration of Example 4 using the culture solution (A4) containing ethylene-containing bubbles having an average bubble diameter of 1.0 μm was 0.2 g / L before the cultivation, whereas it was 1 after the cultivation. It increased to 0.1 g / L, and the amount of anthocyanin produced increased. This is because the production of anthocyanins was promoted by the culture solutions (A3) and (A4) containing fine ethylene-containing bubbles.
Further, from comparison between Example 3 and Example 4, it was found that the smaller the average bubble diameter of ethylene-containing bubbles, the greater the effect of promoting the production of anthocyanins. This is because ethylene-containing bubbles are more easily maintained in the culture solution as the average bubble size is smaller, and the effect of ethylene is more easily obtained.

On the other hand, the anthocyanin concentration of Comparative Example 3 using the culture solution (B2) in which ethylene-containing bubbles having an average bubble diameter of 48 μm were generated was 0.2 g / L before the culture, but was 0. It did not increase as much as 4 g / L, and the effect of promoting the production of anthocyanins was low.
From the above results, according to the production method of the present invention, it was shown that the production of useful substances was promoted in the cultivation of plant tissues, and the intended useful substances could be produced with high productivity.

  1, 2, 3 Culture fluid production device 4 Sterilization device 5 Culture tank 11 Culture fluid reservoir 12 Ethylene-containing gas reservoir 13 Ethylene-containing bubble generator 14 Culture fluid supply unit 15 Pump 16 Bubble generator nozzle 17 Switching valve 21, 22, 23, 24, 27, 28, 29, 30 Piping 51 Stirring device 51a Blade 52 Air diffuser member 53 Sensor 100 Plant tissue culture device

Claims (6)

  A plant tissue culturing method comprising culturing a plant tissue in a culture solution containing fine bubbles made of a gas containing ethylene.   The plant tissue culturing method according to claim 1, wherein an average bubble diameter of the fine bubbles is 10 μm or less.   The plant tissue culturing method according to claim 1 or 2, wherein the number of fine bubbles is 1,000 or more per 1 mL of the culture solution. A method for producing a useful substance by culturing plant tissue,
The manufacturing method of a useful substance which has the process of culture | cultivating a plant tissue with the culture | cultivation method in any one of Claims 1-3.   A culture solution storage unit for storing a culture solution for culturing plant tissue, an ethylene-containing gas storage unit for storing a gas containing ethylene, and an ethylene-containing bubble generation unit for generating fine bubbles made of the gas in the culture solution; A culture medium production apparatus comprising:   A plant tissue culture apparatus comprising: the culture solution production apparatus according to claim 5; and a culture tank for culturing a plant tissue with a culture solution supplied from the culture solution production apparatus.

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