JPH01211429A - Method for tissue culture and tissue culture tool used therefor - Google Patents

Abstract

PURPOSE:To supply a large amount of seedlings obtained by tissue culture, by hermetically sealing a tissue culture vessel itself with a plastic bag partially provided with a microcellular film or providing part of the vessel itself with the microcellular film. CONSTITUTION:A vessel is inserted into a bag 6, made of transparent polypropylene and having about 55mu thickness and the inlet is thermally fused. Openings 7 are provided in the bag 6 and microcellular films 8 are then thermally fused to the openings 7.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a tissue culture device with good workability that can efficiently mass-produce tissue culture bacteria using a simple method.

[Conventional technology]

In so-called tissue culture, in which organs, tissues, and cells of a plant are separated from the plant and cultured aseptically under appropriate culture conditions to regenerate a solid with complete plant functions, sterile conditions must be maintained. At the same time, plants perform carbon dioxide assimilation, so a sufficient supply of carbon dioxide gas is necessary, and various methods have been proposed to prevent microorganisms from being mixed in with the passage of these gases.

As culture vessels, Erlenmeyer flasks with small air inlets relative to the culture area are often used, and test tubes, wide-mouth bottles, etc. are also used. Traditionally, the openings of these containers have been covered with cotton plugs made of hardened cotton fibers, or aluminum foil caps that are closed by twisting the protruding part of the container by hand to create wrinkles. This creates a passageway between the outside air and the inside of the container, consisting of a meandering bottleneck around the mouth of the container, and when air passes through this bottleneck, bacteria stays in the bottleneck, preventing contamination by bacteria.

Also, screw lids and fitted lids are used, but in this case no gasket is used, and when outside air flows from bottom to top through the gap between the lid and the container, bacteria can flow from bottom to top. It is kept sterile by taking advantage of the fact that it is difficult to move.

Even in tissue culture, since it is a plant body, it performs photosynthesis and consumes large amounts of carbon dioxide gas. With conventional methods, the supply of carbon dioxide gas is insufficient, and the concentration of carbon dioxide gas drops to 100 ppn in the light season.
At such a low concentration, sufficient photosynthesis does not occur, and 1! of sucrose, glucose, etc. had to be added. Moreover, these seedlings were very weak and had a very low survival rate during acclimation.

The present inventor disclosed in Japanese Patent Publication No. 5B-11717 a breathable cap sheet to which a porous sheet is attached. Furthermore, Japanese Patent Application No. 61-243058 and Japanese Patent Application No. 61-
In No. 243059, microorganisms do not invade even if culture is performed without using a clean room using a wide-mouth bottle with a sealed lid having a microporous film, and in addition, sufficient carbon dioxide gas is supplied to quickly grow strong seedlings. We proposed a technology to grow it.

Tissue culture of plant seedlings is largely a manual process, and labor costs account for most of the production costs, so mechanization, labor savings, and rapid growth were required.

The above-mentioned technology has achieved a method of preventing contamination by microorganisms and supplying sufficient carbon dioxide gas to accelerate the growth of seedlings by using a microporous film, preferably a single-layer microporous film.

[Problem to be solved by the invention]

However, if the flow of air is increased to ensure a sufficient supply of carbon dioxide gas, the flow of water vapor will inevitably improve, and the water vapor in the culture medium will tend to evaporate and dry the culture medium. Drying of the medium has a negative effect on plant growth. Since tissue culture seedlings are usually grown in a sealed container for 2 to 3 months, there is a need for a simple means to prevent the medium from drying out to the extent that it does not adversely affect the growth of the plants during that time.

Therefore, in order to improve productivity and further expand the types of plants that can use Mi-woven cultured seedlings, we have created a system that provides sufficient ventilation for growth, eliminates the risk of medium drying and contamination with microorganisms, and eliminates the use of clean rooms. There was a need for a tissue culture device that could.

[Means of problem solving]

The present invention aims to solve the above-mentioned problem, and has a structure in which a container with a wide entrance into which a medium for growing callus or plantlets is charged and a water source capable of supplying water are housed in the same sealed chamber, Ventilation holes are provided in the sealed chamber by the microporous film to eliminate contamination by microorganisms and supply sufficient carbon dioxide gas, and water vapor that evaporates from the microporous film is replenished from the water source inside the sealed chamber to prevent the culture medium from reaching the extremes. It is characterized by preventing dryness.

The container used in the present invention is preferably a transparent container made of heat-resistant glass, polypropylene, polycarbonate, polystyrene, high-density polyethylene, nylon, or the like that can withstand heat sterilization because it efficiently passes the light necessary for photosynthesis and can withstand repeated use. In particular, it is preferable that the portion above the medium where the stem and leaves grow is transparent.

Furthermore, it is necessary that the medium and water be separated.

As a means of separation, a two-tiered structure is used in which an upper box serving as a culture medium is placed above a lower box serving as an aquarium, and a notch is provided at the top end of the lower box as shown in Figure 2, or a lower box as shown in Figure 1 is used. There are methods such as drilling a through hole above the water surface of the box, or providing a pipe-shaped through hole projecting above the culture medium from the bottom of the upper box as shown in FIG. Furthermore, there is a method in which moisture in the lower box is directly sucked into the culture medium in the upper box using a collection of capillary tubes such as threads or strings, or a method in which water vapor in the lower box is directly absorbed into the culture medium without the culture medium or moisture falling onto the bottom of the upper box. It is also possible to provide a porous support plate for absorption. It is also possible to arrange the culture medium and aquarium side by side. In short, in addition to the culture medium surface, it is sufficient to have a water surface separated from this.

A container with as wide a mouth as possible is preferable for implanting plant tissue into a culture medium, both manually and mechanically. Furthermore, it is preferable that the side surface of the container above the medium be shallow enough to prevent the medium from overflowing. However, if the container is shallow, the plant will come into contact with the bag after planting, making it difficult to grow straight. Therefore, when the upper box containing the culture medium is made shallow, it is preferable to use various protrusions that have the function of lifting the bag so that the plants do not come into contact with the bag. As the protrusion, a frame that can fit into the upper box, a partition plate, a rod, or a tube can be used. For example, a partition plate may be made to protrude from the center of the bottom of the upper box, screen-like partition plates may be made to protrude from the bottom of both ends of the upper box, or a single rod may be made to protrude from the center of the upper box.
One method is to have four rods protrude from the four corners of the upper box.

The amount of water should be sufficient to replenish the water evaporated from the medium during the culture period and the evaporation water surface.

The culture medium container and water tank are simultaneously stored and sealed airtight in a bag.

A portion of this bag must be made of a microporous film, which will be described later. Furthermore, it is preferable that the bag is transparent, and even if the bag is not entirely transparent, it is sufficient if the part above the medium where the plantlets grow is transparent when the above-mentioned container is housed.

In this way, when a container is stored in a bag, the container is less likely to be soiled, and the durability of the container is improved so that it can withstand repeated use.

Any method may be used to seal the plastic bag as long as it remains completely airtight throughout the culture period, but heat-sealing is reliable and simple. In addition, as shown in FIG. 4, a clip can be used for sealing. If a clip is used, the bag can be reused.

The microporous film used in the present invention has pores of a size of 0.02μ or more, preferably 0.01μ or more that cannot allow microorganisms to pass through, and when the difference between the major axis and the minor axis is small, the minor axis of the pores is is 0.1μ or less, preferably 0.05μ or less. In addition, in the case of a drainboard-like elongated hole, the maximum short axis is 0.
．． If the diameter is 1 μ or less, preferably 0.05 μ or less, and more preferably 0.02 μ or less, passage of microorganisms can be prevented even if the major axis is large. The porosity is 50% or less, preferably 4
It is 0% or less. In short, any film may be used as long as it has pores as small as possible within a range that allows oxygen and carbon dioxide gas to flow without resistance.

A single-layer film is preferred in order to reduce gas flow resistance and supply carbon dioxide gas more smoothly.

In general, microporous films often have multiple layers with large pores, forming bottlenecks.

Such a microporous film with a multilayer structure inevitably has a large ventilation resistance, which tends to impede smooth supply of carbon dioxide gas. Moreover, although microorganisms that do not have self-mobility cannot pass through the bottleneck and are blocked, it is not possible to completely prevent the invasion of self-mobile microorganisms such as microscopic mites.

The microporous film material is preferably a water-repellent film made of polypropylene, polyethylene, nylon, fluorocarbon resin, or the like. Since microorganisms mainly move with water, it is difficult for microorganisms to pass through pores of the same size if the microporous film material is water-based. In addition, drying of the culture medium is due to the evaporation of water contained in the culture medium, but in the case of a water-repellent microporous film, hydrophobic carbon dioxide and oxygen can easily pass through it, but water vapor cannot pass through it easily. (That is, it has sufficient air permeability and has some effect of preventing medium drying.

In order to use a microporous film for a part of a plastic bag, for example, a part of the bag, preferably the upper part when a container is stored, is cut at one or more places to provide through holes, and these through holes are reinforced with nonwoven fabric or the like. The resulting microporous film may be heat-sealed. Alternatively, as shown in FIG. 5, holes may be made intermittently in the bag, and a reinforced microporous film may be heat-sealed or adhered to cover the holes.

When the culture medium container and the water tank are not sealed in a bag, the culture medium container and the water tank are hermetically sealed using a lid, as shown in FIG. 3, and the microporous film is attached to the lid. Furthermore, the upper space of the culture medium container and the upper space of the water tank are required to be in communication. When the culture medium container and the water tank are stacked in two layers, upper and lower, it is sufficient that the upper space of the water tank in the lower box and the culture medium are in communication with each other.

In this case, if the container is separated into an upper box and a lower box, it is necessary to reliably seal the upper box and lower box, and the upper box and the lid. A method is used to ensure that the door is closed securely.

The microporous film is provided on the top side of the lid or top box. In order to provide a microporous film, perforations may be provided and the microporous film may be directly fused or adhered to the perforations q, but as shown in Figure 3, openings may be made in the lid. A flat ring made of a rigid plate that can be attached and detached in an airtight manner is provided in this opening, and a reinforced microporous film is pasted or fused to the wide (opened) center part of this flat ring.

In this way, the flat ring can be airtightly attached and detached via the gasket, and the container itself can be used repeatedly by simply replacing the flat ring.

[Effect]

In the present invention, the tissue culture container itself is sealed with a plastic bag partially covered with a microporous film, or a part of the container itself is made of a microporous film, so that a small clean room can be created inside the bag or container itself. form, allowing free air circulation but preventing microorganisms from entering. Therefore, by simply placing the seedlings in a normal bright room, there is no need to add tin to the culture medium, and strong seedlings grow in a short period of time, and can be planted as is without the need for acclimatization.

Sufficient supply of carbon dioxide gas will result in the drying of the culture medium, but since a water source is provided inside the sealed room for drying the culture medium, the moisture evaporating from this water source will increase the humidity in the small clean room mentioned above, causing drying of the culture medium. can be prevented.

In addition, the upper box is flat, has a wide entrance, and has short sides, making it easier to plant and collect seedlings, and it also allows for mechanization.

〔Example〕

FIG. 1 is a cross-sectional view of the present invention. 1 is a lower box containing water 2, and a plurality of water vapor discharge holes 3 are provided above the water surface. The upper layer 4 having a depth of about 1.5 cm was placed on top of the lower box 1. Place the agar medium in the upper box 4 so that the medium 10 does not overflow by approximately 1 cm.
It was poured into a thick layer and hardened. Plant callus was planted in the culture medium in this shallow upper box, and a transparent polypropylene frame 5 was placed thereon.

The containers stacked 3N in this way were inserted into a transparent polypropylene bag 6 with a thickness of 55 μm, and the inlet was heat-sealed.

9 is a heat-sealed portion of the bag. The bag 6 had two openings 7, and a microporous film 8 was heat-sealed to the openings 7.

In this example, Duraguard (trade name) manufactured by Celanese, Inc., USA was used as the microporous film. 11 is a growing small plant.

In the present invention, the upper box 4 is made shallow and the transparent frame 5 is used as the protruding body, making the work easier and improving the work efficiency. In addition, one or more partition plates may be provided as the protruding body, or a rod or a tube may be protruded. In this example, the lower box was made black to absorb light, raise the water temperature, and promote the evaporation of water vapor.

FIG. 2 is a perspective view of another embodiment. In this embodiment, the upper box is made deeper and the protrusion is omitted. Further, a plurality of notches 12 were cut into the upper end of the lower box 1 as water vapor discharge holes. Evaporated water vapor is discharged from this notch 12, and since the container is sealed with a bag, the humidity inside the bag increases and the medium dries out during the necessary growth period even though the supply of carbon dioxide gas is sufficient. I didn't.

FIG. 3 is a sectional view of an embodiment in which no bag is used.

A tubular vent hole 13 was provided as a passage for water vapor in the lower box 1, projecting from the lower end of the bottom of the upper box 4 to the upper surface of the culture medium 10. Since this vent hole 13 can directly introduce water vapor into the upper box without leaving the container, it can of course be used in the embodiments described above, and can also be used in the lower box 1 and the upper box without using a bag as in this embodiment. It can also be used for a culture container of the type in which a flange is provided with 4 and is held airtight with a clip 16 via a gasket 15.

17 is a lid, which is similarly sealed by an upper box, a flange, a packing, and a clip. 7° is an opening provided in the lid, and a thin groove 18 is carved around this opening 7°. 19 is a rigid ring that airtightly fits into the groove 18, and a microporous film 8 is attached to the center thereof. This ring 19 can be airtightly attached to and detached from the opening 7 of the lid.
It was possible to exchange the container if it was to be used again.

FIG. 4 shows another method of fusing the bag. Various methods other than fusion can be used as long as the bag can be kept airtight during the culture period. 20 is a stopper, which consists of a female body 21 and a male body 22. When the male body 22 is fitted into the female body 21 through the end of the bag 6, the inside of the bag can be kept airtight due to the packing effect of the bag material. 23 is a microporous film reinforced with nonwoven fabric. The bag shown in Fig. 4 has intermittent perforations 24 in the film constituting the bag, as shown in Fig. 5.
A strip-shaped microporous film was placed on top of this, and both ends were heat-sealed. 25 is a fused portion.

FIG. 6 is a sectional view of another embodiment. Opaque lower box 1
and a shallow upper box 4 are integrally formed, and an engaging portion 26 extends inward from the inner surface. A frame 5 was placed on the upper end of the upper box. In order to ensure the fit between the two, the upper end of the upper box was cut diagonally so that the outside was sharp, and the lower end of the frame 5 was cut diagonally so that the inside was sharp.

A support plate 27 made of a rigid plastic net and having the same size as the inner diameter of the upper box is placed on the engaging portion 26, and a porous film 28 slightly larger than the support plate is placed on the support plate 27.
was installed. Medium 10 was forced on top of this. Porous films allow water vapor to pass through, but not moisture or culture medium.

When the container of this example was sealed and stored in the bag described in the example of FIGS. 1 and 2, the same effect was obtained.

〔effect〕

The present invention allows free air circulation and supplies a large amount of carbon dioxide while preventing the culture medium from drying out and improving workability.
It has become possible to supply tissue culture seedlings in large quantities. Tissue culture seedlings obtained using the device of the present invention are durable and do not require acclimation.
Tissue culture seedlings can now be used for direct pricing, expanding the variety of plants for which tissue culture seedlings can be used.

[Brief explanation of the drawing]

The drawings show embodiments of the invention, FIGS. 1, 3 and 6.
FIG. 2 is a sectional view, FIG. 2 is a perspective view, FIG. 4 is a perspective view showing means for fusing the bag, and FIG. 5 is a perspective view showing a state in which a microporous film is attached. In the drawings, 1 is a lower box, 2 is water, 3 is a steam exhaust hole, 4 is an upper box, 5 is a frame, 6 is a bag, 7 is an opening, 8.23 is a microporous film, and 9 is a thermal melt 10 is a medium, 11 is a small plant, 12 is a notch, 13 is a ventilation hole, 14
is a flange, 15 is a gasket, 16 is a clip, 17 is a lid, 18 is a groove, 19 is a ring, 20 is a stopper, 24 is a hole, 25 is a fusion part, 26 is an engaging part, 27 is a support plate, 28 is a porous film.

Claims (1)

[Scope of Claims] (1) When growing plant tissue using a container containing a culture medium, the container containing the culture medium is stored and sealed in a bag, and a portion of the bag is allowed to pass through air and water vapor. By using a microporous film that does not allow the passage of microorganisms, or by sealing a part of the container itself and making it a microporous film that allows air and water vapor to pass through but does not allow microorganisms to pass through, carbon dioxide gas can be sufficiently removed. A method for culturing tissue culture of plants, characterized in that water for replenishing water vapor volatilized from the culture medium is stored independently from the culture medium in a container or bag sealed with the above-mentioned microporous film. Method. (2) Consisting of a container in which the culture medium and water are separated from each other, and a plastic bag that can store and seal these containers with free air circulation between the water surface and the surface of the culture medium, and a part of the bag A tissue culture device that is a microporous film that allows air and water vapor to pass through but does not allow microorganisms to pass through. (3) Consisting of a lower box and an upper box that is stacked on top of the lower box and contains a culture medium to culture callus or plantlets, and has a water vapor exhaust hole that allows communication with the outside air when the upper box is stacked on the lower box. a container provided with
A tissue culture device comprising a plastic bag that can house and seal the container, and a part of the bag is a microporous film that allows air and water vapor to pass through but does not allow microorganisms to pass through. (4) Consisting of a lower box and an upper box placed on top of the lower box, the upper box has a bottomless frame shape, and consists of rigid bodies arranged with a space from the lower end of the upper box. A container in which an engaging part on which a support plate can be placed extends inward, and a medium for cultivating callus or plantlets is placed on the support plate through a porous film that allows water vapor to pass through but does not allow water to pass through. and a plastic bag capable of storing and sealing the container, a part of which is a microporous film that allows air and water vapor to pass through but does not allow microorganisms to pass through. (5) Consisting of a lower box and an upper box placed on the lower box, the upper box forms a rigid support plate arranged with space at the bottom, and the water vapor is placed on the support plate. It consists of a container containing a culture medium for callus or small plants through a porous film that allows water to pass through but does not allow water to pass through, and a plastic bag that can house and seal the container. A tissue culture device that is a microporous film that allows air and water vapor to pass through, but does not allow microorganisms to pass through. (6) An engaging part on which a support plate made of a rigid body can be placed is extended on the inner side surface of the box, a porous film that allows water vapor to pass through but not water is laid on the support plate, and the porous film It consists of a container containing a medium for cultivating callus or plantlets on a film, and a plastic bag that can house and seal the container, with a part of the bag having microporous holes that allow water vapor to pass through but not microorganisms. Tissue culture equipment that is a sexual film. (7) A container consisting of a lower box for containing water, an upper box for culturing callus or plantlets by stacking it on top of the lower box, and a protrusion provided on the upper box, and storing and sealing the container. The lower box has a water vapor discharge hole that allows communication with the outside air when the upper box is stacked, the upper box is a culture medium container with a depth that does not overflow the culture medium, and the protrusion is located on the top of the upper box. The plantlets growing in the medium are provided so that their growth is not hindered by contact with the plastic bag, and the plastic bag is partially made of a microporous film that allows water vapor to pass through but does not allow microorganisms to pass through. Some tissue culture equipment. (8) The tissue culture container according to claim 7, wherein the protruding body is a frame body placed on the outer frame of the upper box. (9) The tissue culture container according to claim 7, wherein the protruding body is one or more rods or tubes. (10) The tissue culture container according to claim 7, wherein the protrusion is one or more partition plates placed on the bottom of the culture medium container. (11) The tissue culture container according to any one of claims 7 to 10, wherein the water vapor discharge hole is one or more notches formed in the upper end of the lower box. (12) The tissue culture container according to any one of claims 7 to 11, wherein at least the portion above the medium is transparent. (13) The tissue culture device according to any one of claims 7 to 12, wherein the plastic bag is transparent at least in a portion above the surface of the medium when the container is housed and sealed. (14) The tissue culture device according to any one of claims 7 to 13, wherein the microporous film is a single layer film having a fine pore size that does not allow microorganisms to pass through. (15) A container consisting of a lower box and an upper box in which a culture medium is placed on top of the lower box to cultivate callus or plantlets, and the upper box and lower box are either airtightly attached, or the upper box and the lower box are The box is formed integrally, and a device is provided between the lower box and the upper box to allow water vapor to freely pass through, a container containing a medium for cultivating callus or plantlets is provided at the bottom of the upper box, and a A tissue culture device characterized by having a transparent lid that is airtightly attached to the lid, and a microporous film that allows air to pass through but does not allow microorganisms to pass through the lid.