JPH0223860A - Method of plant cell culture - Google Patents

Abstract

PURPOSE:To provide the title method so designed that plant cells are put to culture in high density and in good efficiency using a light source and an agitator both equipped in a culture tank while irradiating rays and agitating the culture solution, thereby seeking the mass production of the secondary metabolites to be produced by said plant cells. CONSTITUTION:Plant cells are put to culture in a culture tank equipped with a light source and an agitator, thereby growing these cells highly favorably, with the light uniformly irradiated on the entire cells without being shielded by the grown cells, without hampering the agitation even if cell frocculates are sedimented, and with little cell damage due to the stirrer.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention involves culturing plant cells, which form cell aggregates as they grow, in a high-density liquid under light irradiation, and also culturing plants produced from the cultured cells. The present invention relates to a method for obtaining high yields of secondary metabolic compounds.

(Prior Art) For liquid culture of plant cells, aerated agitation culture or air lift culture is generally performed. In the air lift culture method, air etc. are introduced from the bottom of the culture tank, and the rising air bubbles cause
The medium is being stirred.

It is known that irradiation with light promotes the proliferation and growth of plant cells in culture and the production of secondary navy blue products produced by the cultured cells.

Conventionally, when culturing plant cells in liquid by irradiating light for the production of such metabolites, a method of irradiating light from around a glass culture tank has generally been adopted.

On the other hand, a method of irradiating light from inside a culture tank using optical fibers is used for culturing unicellular algae that do not form cell aggregates, such as chlorella and spirulina.

In conventional methods, when culturing plant cells, light is irradiated from outside the culture tank, so as the growth of plant cells is promoted and increases in the culture tank, the penetration of light into the culture tank increases. There was a problem in that the cultured plant cells could not receive sufficient light irradiation because this was hindered by the plant cells themselves. In addition, the production of secondary metabolites produced by plant cells is suppressed due to insufficient light irradiation.
There was a problem in that it was not possible to obtain a large amount of the desired secondary metabolite. Furthermore, even if a sufficient amount of nutrients were present in the culture medium, the cell aggregates that settled at the bottom of the culture tank could not receive a sufficient supply of oxygen, resulting in various problems such as withering and death. Therefore, it is difficult to cultivate large quantities of cultured plant cells under light irradiation using conventional methods, and it is almost impossible to obtain useful secondary metabolites produced by these cells on a commercial scale. There wasn't. In particular, the above-mentioned problems have been severe in cultured land plant cells that form cell aggregates consisting of hundreds to thousands of cells and have a strong tendency to sediment.

(Problems to be Solved by the Invention) The present invention eliminates the above-mentioned obstacles and efficiently cultivates cells of plants that require light for growth and production of secondary metabolic compounds, particularly plants that form cell aggregates, at high density. The purpose is to provide a method for

(Means for Solving the Problems) In order to achieve the above-mentioned object, the present inventors have conducted intensive research and found that when liquid culturing plant cells that form cell aggregates as they grow in a culture tank, When we cultured plant cells by irradiating light from inside the culture tank and forcibly stirring the culture solution using a culture tank that had a light irradiation device installed inside the tank and a stirring device that forcibly stirred the culture solution, we found that the cells did not aggregate. Despite the formation of clumps, plant cells grow extremely well, and
The present invention was completed based on the discovery that the secondary metabolic compounds produced by the plant cells can be produced and obtained in large quantities.

The present invention relates to a method for culturing plant cultured cells, in which plant cells forming cell aggregates are cultured under light irradiation from a light source in a culture tank and stirring of a culture solution by a stirring device, and plant cells cultured by the same culture method. This is a method for producing secondary metabolic compounds that are produced by

There are no particular restrictions on the cultured plant cells that form the cell aggregates used in the present invention, but preferably they are static animals, such as liverwort, greenhorn moss, crested moss,
Examples include livestock such as scaly moss or porcine moss, or yeasts such as sphagnum moss, staghorn moss, silver moss, Japanese moss, Japanese moss, or Japanese moss.

For seed culture, a photoheterotrophic culture method in which plant cells are cultured in a modified Murashige-Skoog medium containing 2% glucose, or a modified Murashige-Skoog medium without glucose enriched with 1 to 5% carbon dioxide gas. Cultured cells cultured by a photoautotrophic culture method that involves aeration of air and irradiation with light can be used.

As the culture tank, a culture tank used for general plant culture can be modified as necessary, and one of an appropriate size is used depending on the amount of plant cells to be cultured.

The culture medium that can be used in the culture method of the present invention is a nutrient medium that is generally used for culturing plant cells, and contains inorganic components and carbon sources as essential components, to which plant hormones and vitamins are added, and further added as necessary. This is a medium to which amino acids have been added. Inorganic components include inorganic salts containing elements such as nitrogen, phosphorus, potassium, sodium, calcium, magnesium, sulfur, manganese, zinc, boron, molybdenum, chlorine, iodine, and cobalt. Specifically, potassium cum nitrate, sodium nitrate, ammonium nitrate, ammonium chloride, potassium chloride, calcium chloride, phosphoric acid.
Examples include potassium hydrogen.

Examples of plant hormones that may be added to this medium as needed include naphthaleneacetic acid, indoleacetic acid, p-chlorophenoxyacetic acid, and the like. Examples of vitamins include biotin, thiamine, pyridoquine, and ascorbic acid.

Suitable media for use in culturing the cultured plant cells of the present invention include Murashige-Skoog medium or modified Murashige-Skoog medium.

The light irradiation device installed in the culture tank used in the present invention includes optical fibers inserted into a glass protection tube, fluorescent tubes, or heat-resistant optical fibers that do not require a protection tube, depending on the size of the culture tank. Ten to several hundred pieces are used. The optical fiber may have a diameter of, for example, about 3 mm, and may have light emitting portions at multiple locations in the longitudinal direction, with the tip thereof being positioned slightly above the stirring device at the bottom of the culture tank. You can also insert it like this.

In the present invention, when culturing plant culture cells, an artificial light source such as a xenon lamp or a solar concentrator is used as a light source to collect and transmit light. is applied to the cultured plant cells, and the stirring device is operated at 100 to 300 revolutions for 1 minute under ventilation. This allows the cells to float uniformly and receive effective light irradiation.

When plant cells are cultured using such a culture method, they can be cultured to a high density.

As for the stirring device, a floating magnetic stirrer is used in a small-scale culture device, and a stirring device having a stirring blade connected to an external motor by a mechanical coupler is used in a large-scale culture device. It is convenient to use a porous material such as sintered glass or Teflon with many pores processed as an aeration pipe (sparger) for the aeration device used in the culture tank.

Note that one of the reasons why a method of irradiating light from inside a tank under forced stirring has not been used in the past is that suitable culture tanks were not available. In other words, strong stirring is necessary to sufficiently suspend the cell aggregates, but when optical fibers are introduced at various locations in the tank for single-light irradiation, stirring is impossible in a culture tank equipped with a normal stirrer. Met.

Therefore, the inventors have also created a stirring device that is particularly suitable for the method of the invention. This stirring device consists of a framework that can be fixed inside the culture tank main body and a stirring blade that rotates within the framework. Preferably, a magnetic rotor is used as the stirring blade. By placing the rotor within the framework in this manner, stirring is not hindered even if cell aggregates settle, and damage to the cells due to the movement of the rotor (stirring blades) can be prevented.

In the preferred stirring device shown in FIG. 1, a magnetic rotor 1 is provided coaxially with the shaft 3 of the framework 2. This is placed on the bottom of the culture tank, and the rotor inside the tank is rotated using a magnet installed outside the tank. The upper arm 4 and lower arm 5 of the framework are extendable and retractable, and their outer tips 6 have a shape that contacts the inner surface of the culture tank and prevents rotation of the framework 2.

Furthermore, it is desirable to maximize the stirring effect while minimizing cell damage by selecting the shape of the blades of the stirring device. Furthermore, cells can be cultured at an extremely high concentration by appropriately replenishing consumed nutrients i.

The present invention is applicable not only to cultured cell systems that require light for both cell growth and production of secondary metabolic compounds, such as cultured livestock cells, but also to light for growth, such as the production of anthocyanins by cultured grape cells. It can also be applied to higher plant cell systems where production of secondary metabolic compounds is promoted by light, although the production of secondary metabolic compounds is not required.

Therefore, the cultivation method of the present invention has the advantage of being applicable to both photoheterotrophic and photoautotrophic plants.

Examples of secondary metabolic compounds produced by the method of the present invention include terpene compounds such as 1°4-dimethylazulene, or vitamin E and β
-There are many vitamins such as carotene.

Since the method of the present invention irradiates light from a light source inside the culture tank, even if the plant cells forming a cell cluster gradually grow during culture, the irradiated light continues to grow and is blocked by two cells. Since the entire cultured cells are uniformly irradiated without any irradiation, high-density cell growth and high-yield production of secondary metabolic compounds can be achieved. In addition, since the culture solution is stirred by a stirring device, even if cell clusters form, they will float in the tank without settling, so that these clusters will be uniformly irradiated with light and oxygen will be supplied. Can receive.

Example 1 Liverwort cells were grown in modified Murashige-Skoog medium at 22.5
Preculture was carried out by photoheterotrophic cultivation method at ℃.

Next, 200 ml+ of the precultured cell suspension is inoculated into a 7a volume culture tank and culture is started.

The culture conditions were as shown in Table 1 using MSK-2 medium.
A 300W xenon lamp was used as an artificial light source, and the light from the light source was divided into 35 plastic optical fibers with a diameter of 3 mm with light leakage treatment on the sides installed in the culture tank and irradiated into the culture tank, as shown in Figure 1. Culture was carried out by stirring at 200 revolutions per minute using the indicated blades and aerating at an aeration rate of 1 to 3.511/min.

Measure the glucose concentration in the culture solution and replenish glucose, ammonium nitrate, and potassium nitrate if glucose is consumed.

After culturing for 228 days, the dry weight at the start of culture was 0°5g/
lit' increased to 40 g/Il (approximately 80 times).

Example 2 When culturing was carried out in the same manner as in Example 1 except that Acropora moss cells were used instead of Liverwort cells and MSK-4 in Table 1 was used as the medium, 0.5 g/
After 24 days of culture, the cell concentration at Q was approximately 10 g/
1.4-dimethylazulene was produced at approximately 1% of the cell dry ItIk.

(Effects of the Invention) The method for culturing plant cells of the present invention can extremely efficiently culture plant cells that form cell aggregates, which were conventionally difficult to culture in large quantities at high density, and can produce the cells. This is a useful invention that allows a large amount of useful secondary metabolic compounds to be obtained.

[Brief explanation of the drawing]

FIG. 1 is a plan view of a preferred stirring device used in culture; FIG. 2 is a side view of a stirring device used in culture. 1...Rotor, 2...Framework, 3...Axis, 4...
・Upper arm, 5... Lower arm, 6... Frame tip/tail/A Diagram 2

Claims (1)

[Claims] 1. In liquid culturing plant cells that form cell aggregates as they grow, the plant cells are irradiated with light from a light source placed in a culture tank and cultured while stirring the culture solution using a stirring device. A method for culturing plant cells characterized by the following. 2. The culture method according to claim 1, wherein the stirring is performed under conditions that do not damage the cells and that most of the cell aggregates are suspended. 3. The culturing method according to claim 1 or 2, wherein the plant cells are bryophyte cells. 4. Cultivating plant cells under light irradiation from a light source in a liquid culture tank and stirring the culture solution using a stirring device, causing the cultured cells to produce secondary metabolic products, and A method for obtaining plant secondary metabolic products, the method comprising recovering them from a liquid medium. 5. The culture method according to claim 4, wherein the stirring is performed under conditions that do not damage the cells and that most of the cell aggregates are suspended. 6. A plant cell culturing device that forms cell aggregates, characterized by having a framework that can be fixed to the culture tank body and a stirrer that rotates within the framework. 7. The culture device according to claim 6, wherein the framework can expand and contract according to the internal diameter of the culture tank main body.