JPH03103171A - Culture device - Google Patents

Abstract

PURPOSE:To provide a culture unit high in oxygen feed efficiency, reduced in the shock on plant cells, so designed that an embryoid-inducive culture tank equipped with a draft tube, specific nets located at specified intervals and an air feed device at the end communicates with an adventitious embryoinducive culture tank. CONSTITUTION:An embryoid-inducive culture tank 6 is made up in such a manner that a draft tube 3 with both the ends opened is installed in a culture tank main body 2 with a culture solution 1 reserved therein, and nets 4 differing in mesh number from one another are installed at specified intervals between the outer peripheral surface of the draft tube 3 and the inner peripheral surface of the culture tank main body 2, and the end of the culture tank main body 2 on the side smaller in the number of mesh is equipped with an air feed device 5 consisting of a ring 5a with air feed ports 5c, thereby the culture solution 1 is ensured to circulate from outside of the draft tube 3 toward its inside. And an openable communicating channel 8 is provided between said tank 6 falling in the range of the nets 4 and an adventitious embryo-inducive cnlture tank 7, thus making up the objective culture unit for plant cells.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a culture device.

[Prior art]

When inducing somatic embryos from liquid-cultured cells, culture conditions may need to be changed to increase the induction rate. I had to.

[Problems to be Solved by the Invention] However, filtration, centrifugation, etc. for separating the cells requires a lot of labor and cost.

[Means for solving problems]

The present invention provides a culture device suitable for mass culture of plant somatic embryos, and takes the following technical measures.

That is, (1) a draft tube 3 with both ends open is installed inside the culture tank main body 2 storing the culture solution 1, and a mesh is placed between the outer circumferential surface of the draft tube 3 and the inner circumferential surface of the culture tank main body 2. Different numbers of nets 4 are provided at predetermined intervals, and an air supply device 5 is provided at the end of the culture tank body 2 on the side with fewer meshes to circulate the culture solution 1 from the outside to the inside of the draft tube 3. Constructing an embryoid body induction culture tank 6,
Embryoid body induction culture tank 6 and somatic embryo induction tank 7 between the nets
A culture device (2) comprising a communication path 8 that can be opened and closed between the
) The culture device according to claim 1, wherein the embryoid body induction culture tank 6 and the somatic embryo induction culture tank 7 are provided so as to be able to communicate with each other via a separation tank 9, (3) the separation tank 9 is movable in the upward direction. (3) The culture device according to claim 1 or claim 2 or claim 3, wherein the separation tank 9 is vertically movable.

[Effect]

Plant cells proliferate within the culture tank body and are collected in each net 4 according to the size of cell clusters. Then, the cell mass sent to the separation tank 9 is contracted here. Further, in the somatic embryo induction culture tank 7, culture for somatic embryo induction is performed.

〔Example〕

Examples of the present invention will be described below.

First, to explain its structure, the embryoid body induction culture tank 6 is equipped with a culture tank main body 2, a draft 1-tube 3, etc., and the culture tank main body 2 is formed into a cylindrical shape from a transparent material. The shaft center is located in the vertical direction, and a removable upper Q2a is provided at the upper end, and a bottom Rt2b is provided at the lower end.

Further, the culture tank main body 2 stores a predetermined amount of culture solution 1, and is equipped with a draft tube 3 whose both ends are open and whose axes are arranged in the same direction. A plurality of nets 4 (three in the embodiment) are provided on the outer circumferential surface of the draft tube 3 at predetermined intervals in the axial direction, with the green outer end close to the inner surface of the culture tank body 2. In addition, the net 1.
4 has a larger number of meshes placed on the top side than the bottom side, and the net 1-48 at the top position is placed on the top side.
- Slanted downward.

The air supply device 5 has a ring portion 5a formed by forming a pipe into a ring shape having a diameter larger than the outer diameter of the draft 1 to the tube 3, and one end thereof is connected to this ring portion, and the other end is connected to an air supply means (Fig. An air supply pipe 5b (not shown) is connected to the air pipe 5b. Note that air supply holes 5c are provided at predetermined intervals in the circumferential direction on the upper surface of the ring portion.

Reference numeral 10 indicates an opening/closing valve provided at the middle part of a discharge pipe 11 which is inserted through the bottom u2b of the culture tank main body 2 and communicated with the inside of the tank, and 12 is an opening/closing valve provided at the middle part of the discharge pipe 11 which is inserted through the bottom u2b of the culture tank main body 2, and 12 is a valve 12 which is connected to the culture tank main body 2 in the culture solution-E direction.
This is an air filter that is provided so as to be able to communicate with the space of the air filter.

Next, the somatic embryo induction culture tank 7 and the somatic embryo induction culture main body 13
, draft tube l4, etc. The somatic embryo induction culture main body 13 is made of a material that can be seen through and has a cylindrical shape, and has an axis located in the downward direction, and has an upper wall L3 at the upper end. a and a bottom wall 13b is provided at the lower end. The somatic embryo induction culture main body 13 stores a predetermined amount of a somatic embryo induction medium 15, and also houses the draft tube 14 with both ends open and whose axes are positioned in the same direction. . On the lower side of the draft tube 14, there is a ring part 16a formed in a ring shape that is smaller than the outer diameter of the tube 14, one end part of which is connected to this ring part lea, and the other end part of which is connected to an air supply means ( The somatic embryo inducing air supply device 16 is provided with an air supply pipe 16b communicating with the somatic embryo inducing air supply device 16 (not shown). Note that air supply holes 3-Ga are provided at predetermined intervals in the circumferential direction on the upper surface of the ring portion 16a.

The soil 7 is a discharge pipe installed in the bottom wall work 3b of the somatic embryo induction culture main body 13 and communicates with the somatic embryo induction culture main body, 1
8 is this exclusion:. L Q? 1. This is an opening/closing valve placed in the middle of 7.

In addition, the upper part L 3 a of the somatic embryo induction culture main body 13 has an additional supply pipe 1 that can additionally supply the somatic embryo induction medium 15 into this main body.
9, and an air filter 20 provided so as to be able to communicate with the space of the somatic embryo induction culture main body above the somatic embryo induction medium.
has been established.

The separation tank 9 has two walls 9a at the upper end and a bottom a at the lower end.
'l! 9b, and is formed into a cylindrical shape from a see-through material. The single-nucleus separation tank 9 contains a predetermined somatic embryo induction medium 21 at 11? ffi'! In addition, a somatic embryo induction medium supply tube 22 and an air filter 23 are provided on the wall 9a. Further, a guide cylinder 25 equipped with a valve 24 is provided on the lower wall 9b of the separation tank 9, and this guide cylinder 25
is connected to the space between the net 4a at the uppermost position and the net immediately below, and is provided so as to be able to communicate with a guide tube 27 having a valve 26 provided in the outer chamber of the culture tank main body 2 via a flexible hose 28. There is. A guide tube 30 having a valve 29 is provided on the lower outer wall of the separation tank 9, and the guide tube 30 is connected to a guide tube 32 having a valve 31 provided on the outer wall of the intermediate portion of the somatic embryo induction culture main body l3. They are provided so that they can communicate via a flexible hose 33. In addition,
The flexible hoses 28, 33 are detachably attached to the guide tubes 25, 27, 30, 32 via couplers 34.

35 is composed of a discharge tube 36 provided at the bottom W9b of the separation tank 9 and a recirculation tube 37 provided in the culture tank body 2 above the net 4a at the top, which are detachably installed. Reflux hose 38
It is provided so that it can communicate via the. Note that valves 39, 40, and 41 are provided in the protrusion tube 36 and the reflux tube 37, respectively. In addition, the bottom 1 &
9b is provided with a net 42, and the separation tank 9 is provided so as to be swingable in the vertical direction via a vertical swinging means (for example, a forward/reverse motor).

Next, its effect will be explained.

First, a predetermined amount of culture solution 1 containing seedling-cultured plant cells is supplied to the culture tank main body 2 in a flask or the like, and then air is fed into the air supply pipe 5b of the air supply device 5 through an appropriate means to start the culture. Then, the air flows through each air supply hole 5C of the ring portion 5a.
The cell mass is ejected upwards and creates a water flow, so the cell mass increases and the cell mass riding the water flow moves upward. Among these moving cell clusters, the cell clusters that have not yet passed through the bottom net stay there, while the other cell clusters pass through and move to the upper net side. Cell clusters that cannot pass through the intermediate net stay here, while other cell clusters pass through and move toward the uppermost net 4a. In this case, for example, in the case of carrots, the cell size is 40 to 60
μm is said to be optimal, so the net at the lowest level is made into a 149 μm mesh, and the net in the middle is made into a 149 μm mesh.
3μm mesh, and the top net is 37μm
If it is shaped like a mesh, the desired cell mass will be received by the uppermost nets 1 to 4a6.In addition, the air that has passed through the culture solution l and reached the upper space of the culture tank body 2 will be filtered through the air filter l2. You will be guided to a designated location. A part of the culture solution 1 enters the inside at the upper end of the draft tube 3 and moves downward, and then comes out from the lower end and moves upward again. Hereafter, the same action is repeated.

In this action, each net 4 increases the efficiency of dispersing air bubbles, so that the supply of oxygen can be improved. In addition, by providing the draft tube 3, the enzyme supply efficiency is improved, so the amount of ventilation is reduced, and the shearing force when air is blown out from the ring portion 5a can be reduced, reducing the impact on the plant cells. be able to. Since the net 4 is provided on the outside of the draft tube 3, it is possible to easily remove a cell mass suitable for embryoid body induction.

Next, when air is sent into the air supply pipe 16b through an appropriate means, this air is ejected upward from each air supply hole 16c of the ring portion 16a to generate a water flow. After the air reaches the upper space of the somatic embryo induction and culture main body 13, it passes through the air filter 20 and is guided to a predetermined location. Then, the somatic embryo induction medium 15 moving upward inside the draft tube 14 exits from the upper end into the somatic embryo induction culture main body, moves downward, and enters the inside again from the lower end of the draft tube 14. enter. Hereafter, the same cycle is repeated. Therefore, when the valve 26 is opened, the small cell mass received by the net 4a passes through the guide tube 27 and enters the somatic embryo induction culture main body and is cultured there.
When opened, the somatic embryo induction medium 21 and the like can be discharged from the discharge tube 17 to the outside of the somatic embryo induction culture main body.

Next, when the valve 26 and the valve 24 are opened in the embodiment shown in FIG. It is sent to tank 9. In addition, since the net 4a has a raised guide tube side, it becomes easier to guide the collected small cell clusters toward the guide tube side. In addition, by providing a separation tank 9, it is possible to measure the cells to be separated (for example, the number of cells, etc.)
Since it becomes easy to attach sensors, equipment, etc. for this purpose to this separation tank 9, it becomes easy to measure the number of cells and biological activity.

Then, when the bottom surface of the separation tank 9 is pulled up close to the liquid level of the culture tank main body 2, the liquid depth of the separation tank 9 becomes shallower and cells can be concentrated and separated. In addition, the return of the liquid when pulling up the separation tank 9 and the flow of the liquid from the culture tank Honkyu 2 to the separation tank 9 are balanced so that the cells accumulated in the separation tank 9 do not return to the culture tank main body 2. Manipulate. Therefore, it is possible to separate and concentrate cells without once removing them from the culture tank main body 2, thereby preventing bacterial contamination.

Then, by repeating this operation, when the cells are concentrated and the flow rate of the separation tank 9 decreases, the valve 39 is opened and the other valves 24, 26, 29, and 3 are closed to drain the liquid in the separation tank.

Next, close valves 24, 26, and 39, and close valves 29 and 3.
1, then pour the medium for inducing somatic embryos from the supply tube 19 and rinse it out.
Send the cells to 3.

Thereafter, when the somatic embryo induction culture tank main body 19 is filled with cells and medium suitable for somatic embryo culture, culture for somatic embryo induction is carried out. In addition, when the reflux cylinder 37 is provided as shown in FIG. 4, the valves 24, 26, 39, 40, and 41 are closed, and the valves 29 and 3l are opened. This allows for efficient collection of cells with almost no loss of culture fluid during concentration in the separation tank 9. In addition, since the flexible hoses 28, 33, and 38 can be easily removed, each tank body can be easily attached and detached even during cultivation, making cleaning and sterilization easy, and there is no need to worry about bacterial contamination even during continuous operation. few. [Effect] Oxygen can be efficiently supplied and the impact on Tln cells can be reduced. In addition, it becomes easy to extract a cell mass suitable for inducing embryoid production. By providing the separation tank 9, the number of cells and biological activity can be easily measured.

[Brief explanation of drawings]

The figures show an embodiment of the present invention, in which Fig. 1 is a conventional process diagram, Fig. 2 is a front view, and Fig. 3 is a separation tank between the culture tank main body and the somatic embryo induction culture main body. FIG. 4 is a diagram in which a reflux cylinder is provided in the configuration of FIG. 3. 1 is the culture solution, 2 is the culture tank body. 3 is the draft tube,
4 is a net 1-, 5 is an air supply device, 6 is an embryoid body induction culture tank,
7 is a somatic embryo induction culture tank, 8 is a communication path, and 9 is a separation tank.

Claims (4)

[Claims] (1) A draft tube 3 with both ends open is installed in the culture tank main body 2 storing the culture solution 1, and a mesh number is set between the outer peripheral surface of the draft tube 3 and the inner peripheral surface of the culture tank main body 2. Different nets 4 are provided at predetermined intervals, and an air supply device 5 is installed at the end of the culture tank body 2 on the side with fewer meshes.
is provided to constitute an embryoid body induction culture tank 6 in which the culture solution 1 is circulated from the outside to the inside of the draft tube 3, and between the embryoid body induction culture tank 6 and the somatic embryo induction culture tank 7 between the nets. A culture device provided with a communication path 8 that can be opened and closed. (2) The culture apparatus according to claim 1, wherein the embryoid body induction culture tank 6 and the somatic embryo induction culture tank 7 are provided so as to be able to communicate with each other via a separation tank 9. (3) The culture apparatus according to claim 2, wherein the separation tank 9 is provided so as to be movable in the vertical direction. (4) Claim 1 in which the net in the communication path 8 is installed obliquely.
Or the culture device according to claim 2 or claim 3.