JPH02142464A - Culture apparatus - Google Patents

Abstract

PURPOSE:To proliferate plant cells in high efficiency without causing the breakage of the cell by placing a porous member near the bottom wall of a culture vessel containing a culture liquid and introducing air into the culture liquid through the porous member. CONSTITUTION:A gas-permeating porous member 3 made of a ceramics, etc., is placed near the bottom wall of a culture vessel 1 for the culture of plant cell and made of glass, etc. A culture liquid 2 is poured into the culture vessel 1. A plant cell to be cultured is placed at the center of the lid 1c of the culture vessel 1 and air is supplied to the culture liquid through a cylinder 4 from under the porous member 3 with a compression pump 11, etc. The air is exhausted to the outer atmosphere through an air-exhaustion mechanism placed above the vessel while keeping the inside of the vessel in aseptic state with asepticizing filters 7a, 7c, 7c. The proliferation of plant cell can be performed in high efficiency by this process.

Description

DETAILED DESCRIPTION OF THE INVENTION [Object of the Invention] (Industrial Application Field) The present invention relates to a culture device for multiplying plant cells in large quantities using a culture solution.

(Prior art) As a general method for dedifferentiating plant cells and inducing callus, for example, an agar medium containing nutrients and hormones is placed in a Petri dish, and plant cells are placed on the agar medium. There is a method to induce callus by dedifferentiation and proliferation. In this case, in order to efficiently dedifferentiate and proliferate plant cells, it is necessary to frequently replace the agar medium containing nutrients.

There is also a liquid culture method in which the callus is transferred to a culture solution in a culture container and cultured once the cells of the plant are dedifferentiated and become callus on an agar medium.

In this case, for example, the agar medium is replaced every 2 to 3 weeks, and even after being transferred to the culture medium, the culture medium is aerated by shaking or stirring to increase the concentration of molten oxygen in the culture medium. Implement.

However, even in the liquid culture method as described above, there are still problems to be solved as follows. That is, the number of callus-formed plant cells in the liquid culture solution is, for example, 20 to 3.
Grows to 0 μm. In addition, in some cases, 5001
It may even grow to. Plant cells that have grown to such a size become extremely vulnerable to externally applied lateral forces.

Therefore, as described above, when the culture solution is shaken or stirred in order to increase the dissolved oxygen concentration in the culture solution, the problem arises that the grown plant cells are destroyed. As a result, it becomes impossible to dynamically supply oxygen to the culture solution, which in turn causes the problem that plant cells cannot be efficiently grown.

(Problem to be solved by the invention) As described above, in the conventional culture method, the molten oxygen in the culture solution of the plant cells to be propagated can be efficiently supplied without destroying the plant cells. There was a problem that these plant cells could not be sufficiently grown.

The present invention is a method of destroying plant cells by disposing a porous body near the bottom wall of a culture container containing a culture solution and blowing air into the culture solution from the outside through this polyrigid body. It is an object of the present invention to provide a culture device that can maintain the lysin oxygen concentration in the culture solution above a certain value without any problems and can efficiently propagate plant cells.

[Structure of the Invention] (Means for Solving the Problems) In order to solve the above problems, in the culture device of the present invention,
A culture container that stores a culture medium for culturing plant cells, a porous body disposed near the bottom wall of the culture vessel through which gas passes, and a porous body disposed above the porous body within the culture medium. , a reservoir cylinder for convection of the culture solution, an air supply mechanism that opens at the bottom of the culture container and ventilates air into the culture solution from the outside through the porous body, and an air supply mechanism that opens at the top of the culture container. A discharge mechanism, a sterilization filter inserted in each air passage of the air supply mechanism and the air discharger tg, and a humidifier inserted in the air passage between the sterilization filter and the opening in the air supply mechanism. It is prepared.

(Function) In the culture apparatus configured as described above, a porous body through which gas is ventilated is disposed near the bottom wall of the culture container containing the culture solution. A cylinder is disposed above this porous body. Then, when air is supplied from the bottom of the porous body using an air supply device (1) using a compression pump, for example, the air becomes minute bubbles in the porous body and flows into the culture medium in the cylinder. Then, it is exhausted to the outside air through an air exhaust mechanism that opens above the culture container.

At this time, the culture solution in the cylinder moves in the - direction as the bubbles rise, and when it reaches near the liquid level, it descends on the outside of the cylinder. That is, the culture solution convects around the side wall of the cylinder. In addition, sterilization filters are inserted in each air passage of the air supply mechanism and air discharge mechanism, so the inside of the culture container remains sterile (1)! do.

(Example) An embodiment of the present invention will be described below with reference to the drawings.

FIG. 1 is a schematic cross-sectional view showing a culture apparatus of an example. 1
is a culture container made of glass or the like and formed into an A-bottom cylindrical shape.

A culture solution 2 for growing plant cells is stored in this culture container 1. Then, the bottom wall 1aJ of the culture container 1 &:
The side portion is formed to have a small diameter, and a disc-shaped porous body 3 is fixed to the upper end of this small diameter portion 1b. This porous body 3 is made of, for example, ceramics, sintered metal, a fluororesin filter, etc., and when pressurized air is applied from one side, the air fills the minute gaps existing in the porous body 3. The property of passing through and exiting to the opposite side is nailed.

At a position above the porous body 3, a cylinder 4 whose axis is oriented in the vertical direction is supported 14j on the culture container 1 by a support member (not shown). A planting port 5 for introducing plant cells to be cultured is formed in the center of the lid C of the culture container 1. Further, an exhaust pipe 6 as an air exhaust mechanism is attached to the lid IC. A sterilization filter 7a is inserted in the middle of the exhaust pipe 6 to prevent bacteria in the atmosphere from entering the culture container 1.

Further, one end of an air supply pipe 8a is opened in the bottom wall 1a of the culture container 1, and the other end of this air supply pipe 8a is opened into a humidifier 10 in which sterile water 9 is sealed.

Furthermore, one end of another air supply pipe 8b is connected to the sterile water 9 in the humidifier 10.
The other end of this air supply pipe 8b is opened to the outside air via a sterilization filter 7b and a pressurizing pump 11. Therefore, the air supply pipe 8a.

8b and the pressurizing pump 11 constitute an air supply mechanism.

In addition, a liquid supply pipe 12 is located near the lid IC of the culture container 1.
One end is open, and the other end of the liquid supply pipe 12 is connected to the pump 13.
A culture solution supply tank 1 that stores a spare culture solution 2 via the
It opens into the culture medium 2 in 4. An air vent pipe 15 equipped with a sterilization filter 7C is attached to this culture solution supply tank 14.

Further, a drain pipe 16 is attached to the side wall of the culture container 1 near the porous body 3 for draining the culture solution 2 in the culture container 1. Note that a valve 17 is attached to the drain pipe 16.

When growing plant cells using the culture apparatus configured as described above, first, the pump 13 is driven to supply the culture solution 2 in the culture solution supply tank 14 via the solution supply pipe 12 to the liquid level. is fed into the culture container 1 until it rises above the upper end position of the cylinder 4.

Next, plant cells to be propagated are introduced into the culture solution 2 through the planting port 5.

Next, the pressure pump 11 is activated to supply outside air to the lower surface of the porous body 3 in the culture container 1 via the sterilizing filter 7b and the humidifier 10. Note that the air that has reached the lower surface of the porous body 3 is sterilized by removing bacteria contained in the air by the sterilization filter 7b. Furthermore, dehumidifier 1
The humidity of the air pressurized to the lower surface of the porous body 3 is increased to almost the limit humidity.

Air pressurized to the lower surface of the porous body 3 by the pressurizing pump 11 passes through minute gaps within the porous body 3 and exits to the upper surface of the porous body 3. Then, they become minute bubbles 18 and rise two widths of the culture solution in the cylinder 4 toward the water surface. The air in the bubbles 18 flying out from the water surface is released into the atmosphere through the exhaust pipe 6 and the sterilization filter 7a that open in the lid 1c.

Then, the culture solution 2 existing in the cylinder 4 rises as the bubbles 18 rise, as shown by the arrow in the figure, moves to the outside of the cylinder 4 near the liquid level, and moves downward on the outside of the cylinder 4. Move to. Then, it passes under the cylinder 4, enters the cylinder 4, and rises again at the bubble 18. That is, the culture solution 2 convects around the side wall of the cylinder 4.

In this way, the microscopic air bubbles 18 containing oxygen are vented into the culture solution 2 stored in the culture container 1, so that the culture solution 2
Oxygen is sufficiently supplied inside the culture solution 2, and the dissolved oxygen concentration in the culture solution 2 is maintained at a substantially constant level.

Furthermore, since the culture solution 2 convects around the cylinder 4, oxygen is uniformly supplied. In addition, due to the convection, the plant cells to be proliferated that have been introduced into the culture solution 2 do not remain in the lower part of the culture container 1, but are evenly dispersed in the culture solution 2.

Therefore, the plant cells introduced into the culture solution 2 are efficiently grown under the best conditions.

Further, since only minute air bubbles 18 pass through the culture solution 2, excessive shear stress is not applied to the grown plant cells, so that they are not destroyed during the growth process.

In addition, since the humidifier 10 is used to increase the humidity of the air supplied into the culture medium 2 via the porous body 3 to the limit humidity, when dry air is used, it evaporates as water vapor and is exhausted. It is possible to suppress the culture solution being released into the outside air through the tube 6. That is, a decrease in the volume of the culture solution 2 and a change in the concentration of the culture solution 2 can be prevented. Therefore, plant cells can be grown under even better conditions.

In addition, a container tube 6°8b that communicates the inside of the culture container 1 with the outside air,
Since the sterilization filters 7a, 7b, and 7c are inserted in the filter 15, germs in the atmosphere are prevented from entering the culture container l.

In addition, since the humidifier 10 is inserted into the air passage between the sterilizing filter 7b and the porous body 3, it is possible to prevent the sterilizing filter 7b from getting wet, and to suppress clogging of the filter and proliferation of various bacteria. It becomes possible to maintain the sterilization function of the sterilization filter 7b for a long period of time. In other words, the life of the sterilizing filter 7b can be extended.

[Effects of the Invention] As explained above, according to the culture device of the present invention, a porous body is disposed near the bottom wall of a culture container containing a culture solution,
Air is introduced from the outside into the culture solution through this multi-hard body. Therefore, the lysin oxygen concentration in the culture solution can be maintained above a certain value without destroying the plant cells, and the plant cells can be efficiently grown. Further, by disposing the humidifier downstream of the air flow path of the sterilizing filter, the life of the sterilizing filter can be extended.

[Brief explanation of the drawing]

FIG. 1 is a schematic cross-sectional view showing a culture apparatus according to an embodiment of the present invention. DESCRIPTION OF SYMBOLS 1...Culture container, 2...Culture solution, 3...Porous body, 4...Cylinder, 6...Exhaust pipe, 7 a r 7
b r 7 c...Bacterial filter, 8a, 8b...
・Air supply pipe, 10... Humidifier, 11... Pressure pump,
14...Culture solution supply tank.

Claims (1)

[Claims] A culture container that stores a culture medium for culturing plant cells, a porous body disposed near the bottom wall of the culture vessel through which gas passes, and a porous body disposed above the porous body within the culture medium. a cylinder provided therein for causing convection in the culture solution; an air supply mechanism that opens at a bottom position of the culture container and aerates air into the culture solution from the outside through the porous body; and the culture container. an air discharge mechanism opened at an upper position; a sterilization filter inserted into each air passage of the air supply mechanism and the air discharge mechanism; and air between the sterilization filter and the opening in the air supply mechanism. A culture device equipped with a humidifier inserted in a passage.