JPH088860B2 - Method and apparatus for culturing plant cells - Google Patents

Description

FIELD OF THE INVENTION The present invention relates to methods and apparatus for producing plant cells in the gas phase.

2. Description of the Related Art Conventional large-scale culture of plant cells or tissues is carried out by liquid culture according to a fermentation method using a tank such as a jar fermenter. In liquid culture, mixing and stirring a liquid medium with a stirring blade or air is indispensable, but stirring consumes a large amount of energy and destroys or damages plant cells. In the case of air agitation, a large amount of air is required, so that the rate of contamination (contamination) increases. Separation of the medium and cells is required when exchanging the medium or taking out the produced substance, but this is not easy.
When the agar medium is used, the prior art has drawbacks such as slow cell growth because the medium is supplied only at the contact point between the agar and plant cells.

Japanese Examined Patent Publication (Kokoku) No. 60-4713 discloses a vapor phase culture method for plant tissue and an apparatus thereof as shown in FIGS. 8 and 9 of the accompanying drawings. This gas-phase culture method comprises a step of inoculating a perforated plate 82 arranged in the gas phase inside a culture tank 80 that has been previously sterilized with a plant tissue 84 as a seed, and a liquid medium 86 that has been sterilized in advance and adjusted to a predetermined culture temperature. Is supplied to the plant tissue on the perforated plate for a predetermined period of time. FIG. 8 shows an example in which the perforated plates are installed in only one stage, and FIG. 9 shows an example in which the perforated plates are installed in multiple stages. However, in this gas phase culture method, the liquid medium 86 falling from the nozzle 88 is retained only as much as it adheres to the plant tissue 84, and the rest falls from the holes of the perforated plate 82 into the liquid medium accommodating portion 90. There is.
Since the liquid medium is not retained in the perforated plate, there are disadvantages that if the liquid medium is not uniformly supplied, the growth of the cells may be uneven, or the cells may be killed at the part where the liquid medium is not supplied.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention An object of the present invention is to provide a method and an apparatus for producing plant cells in the gas phase without physically damaging the plant cells.

Another object of the present invention is to provide a culture method and apparatus that combine the advantages of conventional liquid culture and the advantages of agar culture.

Means for Solving the Problem and Its Action The above-mentioned object of the present invention is, in the first aspect thereof,
In the gas phase of the inside of the pre-sterilized culture tank, a step of installing a water-containing sponge body that horizontally extends integrally from the vicinity of the periphery of the culture tank to the central portion over one or more stages, The method includes placing a plant callus (an aggregate of cells formed by tissue-culturing plant cells) on the sponge body, and dropping a liquid medium from above the sponge body to supply the plant callus. It is achieved by the method for culturing plant cells.

Based on this technique, the present invention provides the following operational effects.

1) A water-containing sponge holds a liquid medium and serves as a conventional agar medium.

2) Small cells released from large cells that become seeds are also retained in the pores of the sponge, the cells are completely separated from the liquid medium, and so-called immobilization (a well-known technique for enzymes and microorganisms) is achieved. This improves the adhesion between the sponge and the cells and improves the absorption rate of the medium from the sponge. Since the cells also grow from the retained free cells, the growth is fast and efficient large-scale culture is possible. because,
Free cells are very important for efficient large-scale cultivation of plant callus, and in the porous plate of the above-mentioned known example, the free cells drop together with the liquid medium into the lower containing portion 90, and then the physical quantity by a pump or a nozzle is used. This is because it is damaged and destroyed.

3) Since the sponge has water content, the cells do not dry even if the medium is not constantly supplied, and the moisture content suitable for the cells can be maintained only by supplying the cells at an appropriate time interval.

4) Since it is static culture, it does not give physical damage to cells.

5) Since the culture is carried out in the gas phase, a large amount of air is not required unlike liquid culture, and the rate of contamination is reduced.

6) When one or more culture methods use two or more types of media such as a cell growth medium and a target substance production medium, the use of the present invention eliminates the step of separating the medium and the cells. Also, replacement of the medium becomes easy.

As a second aspect of the present invention, a water-containing sponge body installed over one or more stages in a gas phase inside a pre-sterilized culture tank, and placed on the sponge body Plant callus, a supply pipe for supplying the plant callus by dropping a liquid medium from above the sponge body, and an air intake part for introducing clean air that has passed through a high-performance filter from the upper part of the culture tank. A bell mouth arranged with its enlarged opening facing upward in the liquid culture medium storage portion at the bottom of the culture tank, and sucking the liquid culture medium and air in the culture tank from the bell mouth and transferring them to the supply pipe There is provided a plant cell culturing device including a pump and an exhaust unit for discharging the sucked air from the middle of the supply pipe to the outside of the system.

According to such a device, in addition to the above-described action and effect, a large amount of air can be supplied to the callus as compared with a device that is simply opened to the atmosphere, and a pump for circulating the liquid medium is also used for air suction. Advantages such as not requiring special power are obtained.

Other features and advantages of the present invention will become apparent from the following description with reference to the embodiments of FIGS. 1 to 7 of the accompanying drawings.

Example FIG. 1 shows a culture vessel 10 for applying the method according to the present invention.
The heat sterilization and temperature control means 11 and the circulation pump 12 are arranged on the outside thereof, and the plant mouth 13 for introducing plant callus and the vent 14 are provided in the upper part of the tank 10. A liquid culture medium storage portion 15 is provided in the lower portion.
According to the present invention, at least one stage of sponge body 16 is arranged in the upper gas phase of the containing portion 15, and is held by the stopper 17 on the inner surface of the tank. The stopper may be omitted when the sponge body 16 does not slip due to its elasticity. The sponge body is formed of, for example, a polymer such as urethane or butylene in the shape of a sponge, and a cotton-like material can be preferably used as long as it has water content.

The plant callus 18 is placed on the sponge body 16, and the liquid medium 20 drops from the nozzle of the supply pipe 19 arranged above and is supplied to the plant callus 18. The size of voids in the sponge body is appropriately selected according to the type of plant callus to be treated. The supply of the medium from the nozzle is continuous only for the first time, and intermittent supply is sufficient thereafter. As the supply form, various methods such as dripping, spraying, and spraying can be selected. The liquid medium is absorbed and retained by the plant callus 18 or the water-containing sponge body 16, but the excess medium passes through the voids of the sponge body and flows into the liquid medium storage portion 15 below, and by the pump 12. It is circulated again.

FIG. 2 shows an embodiment in which the sponge body 16 is installed in the culture tank 10 over four stages.
Each time the plant is attached, the plant callus 18 is placed on it to form a multi-stage structure. The liquid medium 20 is dropped from the nozzle of the supply pipe 19 arranged above the uppermost sponge body. By flowing through the voids of the sponge body to the sponge body in the lower stage, the medium 20 can be evenly distributed from the upper stage to the lower stage. Furthermore, by providing a plurality of protrusions 25 on the lower surface of the sponge body 16 corresponding to the positions of the callus placed on the lower step, the position of dropping the liquid medium can be determined, and a better result can be obtained.
The protrusion may be a sponge part of which is protruded, or a thorn-like member may be separately attached.

In the above-mentioned publicly known example, since a perforated plate is used, it is necessary to arrange a nozzle for each stage when it is configured in multiple stages, but in the present invention, since the water-containing sponge body is used, the nozzle is the uppermost portion. It only needs to be installed in.

3 and 4 show the sponge body 21 of the first stage (bottom stage).
The plant callus 18 is placed on the first-stage sponge body 21, and then the second-stage sponge body 22 is attached to the first stage with its outer periphery free. In the following, an example is shown in which the sponge body is loaded on the plant callus, and the placement of the plant callus and the free loading of the sponge body are repeated in multiple stages in the same manner. Since the liquid medium 20 is supplied from above the uppermost sponge body and falls along the plant callus, the medium is absorbed from the entire plant callus.
FIG. 3 shows a state in which culture has started, and each plant callus 18 is vertically sandwiched by a water-containing sponge body.
It can be seen in FIG. 4 that the plant callus has grown and the sponge bodies after the second stage have moved upward.

By using the method shown in FIGS. 3 and 4, it is possible to realize a state closer to the liquid culture in which the liquid medium is constantly in contact with the plant cells, and further, since it is static culture, the cells may be damaged. Since it is also in contact with air, there is no need to consider the problem of dissolved oxygen (DO).

FIG. 5 shows an example in which the plant callus 18 on the sponge body 16 is surrounded by fine sponge pieces 30 so that the same effects as those of the embodiments of FIGS. 3 and 4 can be obtained. ing. That is, the sponge piece 30 is a liquid medium
By absorbing and retaining 20, a state close to liquid culture is realized. However, it is necessary to keep a certain amount of air around the plant callus 18.

FIG. 6 shows a preferred embodiment of the culturing apparatus according to the present invention, and the vent hole in the multi-stage type apparatus shown in FIG.
An air intake section 34 for introducing clean air that has passed through a high-performance filter 32 such as a HEPA filter from the upper part of the culture tank 10.
Have been replaced with. Further, the bell mouth 36 is arranged in the liquid culture medium containing section 15 with its enlarged opening facing upward and connected to the circulation pump 12, and an adjustment tank 38 is provided in the middle of the pipe from the circulation pump 12 to the nozzle of the supply pipe 19. Provide the exhaust part 40 above it
The air is exhausted from the system. The bell mouth 36 sucks the air in the tank together with the liquid medium, and the sucked air is discharged from the exhaust unit 40. Therefore, since the pressure in the tank becomes negative, clean air is sucked from the air intake portion 34. The circulation pump 12 controls the rotation speed so as not to cause cavitation. Thus, according to the apparatus of FIG. 6, a large amount of air can be supplied to the plant callus without requiring special power for inhaling air.

FIG. 7 shows that a small compartment 50 is provided at the inlet portion of the circulation pump 12 to the culture tank 10, and a liquid medium is made into a mist by an ultrasonic transducer 52 arranged at the bottom of the compartment, thereby producing a fine medium. It shows an embodiment in which the water is evenly distributed inside the tank. A ventilation pipe between each step of the sponge body
54 is provided for communication, and the misted liquid medium reaches the plant callus in the lower stage. Further, a fan 56 for stirring the air in the tank is arranged to introduce air from the lower part of the tank to the upper part of the compartment 50, and at the same time, the high performance filter 32
The clean air that has passed through is additionally introduced from the supply unit 58. Since only the ventilation pipe 54 depends on the gravity settling of the mist,
By forcibly stirring with Juan 56, uniform culture conditions from the upper stage to the lower stage can be obtained.

EFFECTS OF THE INVENTION As described in detail above, according to the present invention, the above-described effects can be obtained, and in particular, a culture method and apparatus that combine the advantages of liquid culture and the advantages of agar culture can be realized. , Its technical effect is extremely remarkable.

[Brief description of drawings]

FIG. 1 is a vertical sectional view showing a single-stage type culture tank to which the method according to the present invention is applied, FIG. 2 is a vertical sectional view showing a multi-stage type embodiment, and FIG. 3 is a free-loading type embodiment. Longitudinal section,
FIG. 4 is a longitudinal sectional view showing the state after growth of FIG. 3, and FIG.
FIG. 6 is a vertical sectional view showing another embodiment of the sponge body, FIG. 6 is a vertical sectional view showing a multistage type embodiment of the device according to the present invention, and FIG. 7 is a vertical sectional view showing an ultrasonic type embodiment. , 8th
FIG. 1 is a vertical sectional view showing a conventional single-stage culture device, and FIG. 9 is a vertical sectional view showing a conventional multi-stage culture device. 10 …… Culture tank, 11 …… Sterilizing means 12 …… Pump, 15 …… Liquid medium storage unit 16 …… Sponge body, 18 …… Plant callus 19 …… Supply pipe, 20 …… Liquid medium 32 …… Filter, 34 …… Air intake section 36 …… Bell mouth, 40 …… Exhaust section

Claims (4)

[Claims] 1. A water-containing sponge body which is integrally stretched in the gas phase inside a pre-sterilized culture tank in the horizontal direction from the periphery of the culture tank to the central portion is installed in one or more stages. A method of culturing plant cells, comprising: a step of placing a plant callus on the sponge body; and a step of dropping a liquid medium from above the sponge body and supplying the liquid callus to the plant callus. 2. The method according to claim 1, wherein the sponge bodies are installed in multiple stages, and the sponge bodies in each stage are installed so that their outer peripheral edges are held on the inner surface of the culture tank. 3. A sponge body of the second stage is installed by holding the first stage of the sponge body on the inner surface of the culture tank by the outer peripheral edge thereof, and placing the plant callus on the sponge body of the first stage. A body is loaded on the plant callus on the first-stage sponge body with its outer peripheral edge free, and the placement of the plant callus and the free loading of the sponge body are repeated in multiple stages in the same manner. The method according to item 1. 4. A water-containing sponge body installed in a gas phase inside a pre-sterilized culture tank in one or more stages, a plant callus placed on the sponge body, and a sponge body of the sponge body. A supply pipe for dropping the liquid medium from above to supply it to the plant callus, an air intake part for introducing clean air that has passed through a high-performance filter from the upper part of the culture tank, and a liquid medium at the lower part of the culture tank. A bell mouth arranged with its enlarged opening facing upwards in the accommodation section, a pump for sucking the liquid medium and air in the culture tank from the bell mouth and transferring the air to the supply pipe, and the sucked air An apparatus for culturing plant cells, comprising: an exhaust unit that discharges the system from the middle of the supply pipe.