JPH02268677A - Plant culture device - Google Patents

Abstract

PURPOSE:To prevent adventitious embryos from being damaged in taking out adventitious embryos formed in a culture container by equipping a driving part for a porous plate attached to the culture container and moving the porous plate from a discharge channel of liquid medium during removal of the liquid medium. CONSTITUTION:A plant tissue is cultured in a culture container which has a medium feed opening and an air exhaust vent at the top and an air inlet 26 and a drain hole 27 at the lower part of a porous plate 23. A driving part 25 is driven to take out a product such as out adventitious embryos obtained by the culture and the porous plate 23 is stored in a storage chamber 24. The porous plate is made not to exist in a communicating part from the bottom of a culture chamber 14 to the culture chamber so that the liquid medium is not hindered by the porous plate in flowing in the communicating part.

Description

[Detailed description of the invention] [Purpose of the invention] (Industrial application field) The present invention relates to a plant culturing device.

(Conventional technology) In recent years, plant tissue culture technology has progressed, and plant tissue is dedifferentiated to form a cell mass callus, this callus is multiplied to generate free cells, and the free cells are further grown. A series of tissue cultures to obtain somatic embryos by redifferentiation have been carried out, and generally, such tissue culture consists of a series of steps as shown in FIG. 4.

In other words, the first step shown in Figure (a) is to dedifferentiate the plant tissues to obtain callus, the second step is to proliferate the callus as free cells shown in Figure (B), and the free cells shown in Figure (C). It consists of a third step of forming a somatic embryo by redifferentiation of cells, and a fourth step of removing the obtained somatic embryo.

In the first step, an agar medium containing a dedifferentiation hormone and a substrate is heated to make it liquid, which is injected into a test tube 1 and cooled and solidified to form an agar medium 2. Plants are grown on this agar medium 2. A dot-like tissue 3 is placed on a bed, covered with aluminum foil 4, and waits for callus formation and proliferation of the callus 5.

In the next second step, a liquid medium 7 containing a dedifferentiation hormone is poured into an Erlenmeyer flask 6, the callus 5 obtained in the first step is implanted into this, and the mixture is placed in a shaking incubator (not shown) and agitated. The callus 5 is separated into individual cells, and the free cells 8
and (7) to multiply.

In the next third step, the liquid medium 7 is withdrawn, a liquid medium 7' containing no dedifferentiation hormone is injected, and the culture is performed with shaking and agitation to redifferentiate the cells and form a somatic embryo 9.

Conventionally, each of these steps has been performed manually by an operator.

(Problem to be Solved by the Invention) However, in such a conventional plant culture device, it is necessary to replant the callus on a new agar medium to supply nutrients until the callus has sufficiently proliferated in the first step. In addition, it is necessary to inoculate callus from the first process to the second process, and it is also necessary to change the liquid culture medium when moving from the second process to the third process. Conventionally, these tasks were mostly done manually. Therefore, there was a problem that there was a high possibility of bacterial contamination during culture medium exchange work and replanting work.

At the same time, there is also the problem that there is a risk of damaging the callus during these manual operations.

This invention was made in view of such conventional problems, and it is possible to perform the above-mentioned series of plant tissue culture steps from the first step to the fourth step continuously in one culture container. To provide a plant culturing device capable of reducing bacterial contamination, reducing the risk of damage to callus, and eliminating the risk of damaging somatic embryos when taking out the somatic embryos formed in a culture container.

[Structure of the Invention] (Means for Solving the Problems) The plant culture device of the present invention allows water and air to pass through the bottom of the culture container, which is equipped with a liquid medium supply port and an air exhaust port at the top. A porous plate with countless minute holes is attached, an air supply port and a liquid discharge port are provided at a position below the porous plate in the culture container, and a drive unit for the porous plate is provided to remove the liquid. The porous plate is moved from the liquid medium discharge channel when the medium is discharged.

(Function) In the plant culture device of the present invention, in the first step, the porous plate is positioned at the bottom of the culture container, and the dedifferentiation hormone is applied to the extent that the lower surface of the porous plate is slightly immersed in the liquid. Plant tissue, for example, virus-free growth point cell tissue, is placed on the top surface of the porous plate and cultured for a predetermined period of time. Through this first step of culturing, the plant tissue dedifferentiates, turns into a callus, and proliferates.

Next, in the second step to separate this callus into individual cells and grow them as free cells, a liquid medium is added to the middle of the culture container, and then air is added from the bottom of the porous plate through the air supply port. is supplied, and as it passes through the porous plate, it forms fine bubbles and rises in the liquid medium in the culture container.

As the air bubbles rise, a gentle convection is generated in the liquid medium, and the callus can be agitated in an aerated state, and this agitation allows the callus to become free cells and to proliferate.

When the period for which free cells are sufficiently formed has passed, the process moves to the third step, in which the free cells are redifferentiated using a liquid medium that does not contain dedifferentiation hormones to form somatic embryos. First, it is necessary to replace the liquid medium containing the dedifferentiation hormone in the culture container with a liquid medium that does not contain that hormone. Therefore, while the porous plate is placed in a predetermined position, the liquid medium containing the dedifferentiation hormone in the culture container is discharged from the drain port located below the porous plate. At this time,
Free cells that have grown in the liquid medium up to that point are filtered out by the porous plate and remain inside the container, leaving only the liquid medium to be discharged.

After discharging the liquid medium containing dedifferentiation hormone in this way, without closing the drain port, a liquid medium containing no dedifferentiation hormone is supplied into the culture container from the medium supply port at the top to clean the inside of the container. After washing, close the drain port and inject a liquid medium containing no dedifferentiation hormone to the middle of the culture container.

Thereafter, the free cells are cultured in an aerated state with stirring in the same manner as in the second step, and somatic embryos are formed from each free cell.

Next, after the somatic embryos are sufficiently formed, the drive unit is operated to move the porous plate into the liquid medium in order to remove the somatic embryos as a culture product from the drain port provided at the bottom of the culture container. The somatic embryo is moved or rotated until it is in a state where it does not obstruct the discharge flow path, and in this state, the drain port is opened and the somatic embryo is removed together with the liquid medium.

In this way, a series of plant tissue cultures from the callus formation process by dedifferentiation and proliferation of plant tissues to the formation process of somatic embryos are carried out in one step.
It can be carried out continuously in one culture vessel, and each step can be carried out without human intervention.

(Example) Hereinafter, embodiments of the present invention will be explained in detail based on the drawings.

FIG. 1 shows an embodiment of the present invention, in which a culture container 11 is made of glass or stainless steel with a window for letting in light, and a culture chamber 12 and its lower half are made of glass or stainless steel. It consists of a funnel-shaped part 13 and a bottomed cylindrical culture medium chamber 14 connected to the funnel-shaped part 13 and having a relatively small diameter.

Inside the culture chamber 12, there is a cylindrical draft tube 15 that is approximately half the length and about 40 to 70% of the diameter of the cylindrical culture chamber 12, with its lower end almost aligned with the upper end of the funnel-shaped part 13. and is fixed by a support 16.

A flange 17 is formed at the upper end of the cylindrical culture chamber 12, and a lid 19 is attached to the flange 17 via a packing 18. And M19 has a liquid medium supply port 2.
0, an exhaust port 21, and a seed inoculation port 22 are provided.

The culture medium chamber 14 is formed of a bottomed cylindrical body made of glass or stainless steel with a light intake window.
Near the top of 4, hole diameter 10~1000#m1 thickness 2~2
A porous plate 23 made of stainless steel or ceramics of 0a+i is provided, and can be taken in and out of the storage chamber 24 formed on the side of the culture medium chamber 14.
A drive unit 25 that automatically or manually moves the porous plate 23 into and out of the storage chamber 24 is provided in the storage chamber 24.

Furthermore, an air supply port 26 and a liquid drain port 27 are provided at the bottom of the culture medium chamber 14 .

One end of a liquid supply pipe 28 is connected to the liquid supply port 20,
The other end of this liquid supply pipe 28 is connected to the outlet of a pump 29.

The inlet side of the pump 29 includes piping 30a, 30b and a valve 31. a, 31b, and piping 32a, 32b, respectively, to a first medium tank 33a for a first liquid medium containing dedifferentiation hormone, and a second medium tank 33a for a second liquid medium not containing dedifferentiation hormone. 33b, respectively.

A filter 34 is attached to the exhaust port 21 to prevent germs from entering when outside air directly enters the culture chamber]2. A stopper 35 is attached to the seed opening 22.

Further, one end of an air supply pipe 36 is connected to the air supply port 26 on the culture medium chamber 14 side, and the other end of this air supply pipe 36 is connected to a sterile water tank 37, piping 38, an air filter 39, a flow meter 40, a valve 41, etc. is connected to a source of pressurized air 42 via.

A drain pipe 43 is connected to the drain port 27.
It is connected to the valve 44 at the other end.

Next, the operation of the plant culturing apparatus having the above configuration will be explained.

This plant culturing device is capable of performing each step from the first step to the fourth step, which are explained based on FIG. 4 as a conventional example, with one device.Hereinafter, the first step,
The operation will be explained separately in the second step, third step, and fourth step.

First Step In the first step, the plant tissue is dedifferentiated to form a callus, and the valve 31. a, start the pump 29, and supply the first liquid medium containing the dedifferentiation hormone from the first medium tank 33a to the medium supply port via the pipe 32a, the valve 31a, the pipe 303% pump 29, and the pipe 28. 2
The liquid is supplied to the culture container 11 from 0 to the extent that the lower surface of the porous plate 23 is slightly immersed in the liquid in the culture medium chamber 14.

By supplying this liquid medium, the porous plate 23 sucks up the liquid medium to its upper surface by capillary action, and forms a thin liquid curtain of the liquid medium here.

Next, a set m43 of plants to be cultured from the seed opening 22, for example, a growing point, is placed on the porous plate 23, and then a stopper 35 is attached to the seed opening 22.

If culture is carried out in this state, callus formation will begin in about one week, and then the callus will proliferate to a predetermined size in about one month.

Second Step This third step is a step in which callus is grown as free cells.

First, the valve 31a is opened and the pump 29 is driven 1. to supply the first liquid medium from the first medium tank 33a to the liquid supply port 20.
The liquid level is supplied to the culture chamber 12 through the draft pipe 1-5.
feed until it reaches above the second side.

Next, open the valve 41 and supply air from the pressurized air source 42 to the culture medium chamber]
4 through the air supply port 27.

At this time, the amount of air supplied from the pressurized air source 42 is
Adjusted by 0. In addition, the water is sterilized by the filter 39 and humidified when passing through the sterile water tank 137.
4 from the air supply port 26.

The air sent into the culture medium chamber 14 becomes fine bubbles when passing through the porous plate 23 from bottom to top, enters the culture chamber 12, and ascends inside the draft pipe 15. When ascending through the draft tube 15, an upward flow occurs inside the draft tube 15, and a downward flow occurs outside the draft tube 15, causing convection of the liquid medium in the culture chamber 1-2 along with air bubbles, which causes the callus to grow. After aeration and agitation, the callus separates into individual cells and then divides and proliferates as free cells.

Normally, this second step also lasts about a month, and the cells are continued to be cultured to grow and homogenize them.

Third step The third step is a step of redifferentiating the free cells to form a somatic embryo.

First, the valve 44 is opened to drain the first liquid culture medium from the drain port 27 at the bottom of the culture medium chamber 14. At this time, the porous plate 23 acts as a filter to filter out the free cells dispersed in the liquid, and they are not discharged together with the liquid medium.

Next, the valve 30b is opened and the pump 29 is driven to supply the second liquid medium containing no dedifferentiation hormone to the second medium tank 3.
3b to the culture chamber 12 to wash the inner metal body of the culture container 1-1 and the cells inside the main device, and discharge the washed liquid medium from the drain port 27.

Subsequently, the valve 31b is opened again to drive the pump 29, and the second liquid medium is supplied to the top of the draft pipe 15 of the culture chamber]-2.

At the same time, air is sent from the pressurized air source 42 to the culture medium chamber 14 via the flow meter 40, filter 39, sterile water tank 37, and air supply port 26, and the porous plate 23 is fed into the medium chamber 14 as in the second step.
The inside of the draft tube 15 is raised by passing minute air bubbles (-), and the liquid medium is cultured while being aerated and stirred by the flow of the air bubbles.

Usually, each cell dedifferentiates and a somatic embryo is formed in about one month.

Fourth Step In the fourth step, as shown in FIG. 2, in order to take out the product obtained as a result of culturing, such as the somatic embryo obtained in the third step, the drive unit 25 is driven to form a porous membrane. The plate 23 is housed in the storage chamber 24, and the porous plate 23 is present in the communication part leading from the funnel-shaped part 3 at the bottom of the culture chamber 12 to the culture medium chamber 14, and the flow when the liquid culture medium flows down this part is controlled. Avoid becoming a hindrance.

After this, the valve 44 is opened and the second liquid medium in the culture container 11 is discharged from the drain port 27, and the somatic embryo is also removed at the same time.

In this way, a series of steps from the first step to the fourth step for culturing plants can be carried out in one culture container 11-, and somatic embryos can be taken out from the culture container 1 as the final product.

Note that the present invention is not limited to the above-described embodiment, and in particular, the drive unit 25 of the porous plate 23 is configured to be able to rotate the porous plate 23 within the culture medium chamber 14 as shown in FIG. Normally, the porous plate 23 is held horizontally, but
When removing a somatic embryo, the porous plate 2 is moved by the drive unit 25.
It is also possible to rotate the porous plate 3 so that the porous plate 23 becomes straight so that it does not interfere with the flow of the liquid medium when it is discharged.

Furthermore, in the above embodiment, the porous plate 23 is manually accommodated in the storage chamber 24 from the drive unit 25, but the drive unit 25 automatically slides and rotates the porous plate using electromagnetic force or a motor. [Let them do it] 7 is fine.

[Effects of the Invention] As described in 2 below, according to the present invention, a porous plate is provided at the bottom of the culture container, a culture medium chamber is formed in the lower part, and an air supply port and a culture medium drainage are provided in the culture medium chamber. A drive unit is provided to rotate or move the porous plate in the culture medium chamber so that the porous plate does not obstruct the flow of liquid during culture medium discharge. The somatic embryos formed in the process are not strained out by the porous plate when the somatic embryos are not removed from the drainage port of the culture medium chamber, and the plant culture can be carried out effectively. In addition to being able to take out the cultured plants (7) smoothly.

[Brief explanation of drawings]

FIG. 1 is a partially cutaway front view of one embodiment of the present invention, FIG. 2 is a partially cutaway sectional view showing the state of movement of the porous plate at the time of somatic embryo retrieval 17 in the above embodiment, and FIG. The figure is a plan sectional view of a culture medium chamber showing a porous plate drive unit according to another embodiment of the present invention, and FIG. 4 is an explanatory diagram showing a conventional plant culture process.

Claims (1)

[Claims] A porous plate with countless fine holes through which water and air can pass is attached to the bottom of a culture container equipped with a liquid medium supply port and an air exhaust port at the top, and the porous plate in the culture container An air supply port and a liquid discharge port are provided at a lower position than the above, and a drive unit for the porous plate is provided to move the porous plate from the liquid medium discharge channel when the liquid medium is discharged. Device.