JPH0555094B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a tissue culture method with good workability that allows tissue culture seedlings to be efficiently mass-produced in a simple manner, and a tissue culture device using the same.

[Conventional technology]

In the case of so-called tissue culture, in which organs, tissues, and cells of a plant are separated from the plant and cultured under appropriate culture conditions in a sterile manner to regenerate a fully functional plant, 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. Conventionally, the openings of these containers used were cotton plugs made of hardened cotton fibers, and so-called aluminum caps, which were covered with aluminum foil and then closed by twisting the protruding part of the container by hand to create wrinkles. This creates a winding passageway between the outside air and the inside of the container around the mouth of the container, and when air passes through this passageway, it prevents bacteria from settling in the passageway and causing contamination.

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. They take advantage of the fact that they are difficult to move and keep them sterile.

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 in the light season, the carbon dioxide concentration drops to less than 100 ppm. At such a low concentration, sufficient photosynthesis cannot take place, and unless sugars such as sucrose and glucose are added. It didn't happen. Moreover, these seedlings were very weak and had a very low survival rate during acclimatization.

The present inventor disclosed in Japanese Patent Publication No. 11717/1987,
A breathable cap sheet to which a porous sheet is attached is disclosed. Furthermore, patent application No. 61-243058 and patent application No.
In No. 61-243059, 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.
Moreover, he proposed a technology that supplies sufficient carbon dioxide gas to quickly grow strong seedlings.

Plant tissue culture seedlings are largely manual and the majority of production costs are due to labor costs, so mechanization,
There was a need for less labor and faster growth.

A method has been achieved to some extent by using a microporous film, preferably a single-layer microporous film, to prevent contamination by microorganisms, supply sufficient carbon dioxide gas, and accelerate the growth of seedlings.

[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 was a need for a simple means to prevent the medium from drying out to an appropriate extent that would not adversely affect the growth of the plants during that period.

Therefore, in order to improve productivity and further expand the types of plants that can use tissue culture seedlings, we can eliminate the use of clean rooms by providing sufficient ventilation for growing, without the risk of drying the medium or contaminating microorganisms. Tissue culture equipment was in demand.

[Means of problem solving]

The present invention aims to solve the above problems,
Its structure consists of a container with a wide entrance into which a culture medium for growing callus or small plants is charged, and a water source capable of supplying water are housed in the same sealed chamber, and ventilation holes made of microporous film are provided in the sealed chamber. The method is characterized in that it eliminates contamination by microorganisms and supplies sufficient carbon dioxide gas, and at the same time, water vapor volatilized from the microporous film is replenished from a water source in the sealed chamber to prevent the culture medium from becoming extremely dry.

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 preferred for implanting plant tissue into a culture medium, both manually and mechanically. Furthermore, it is preferable that the sides of the container above the medium be shallow enough to prevent the culture 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, the bag is preferably transparent, and does not need to be entirely transparent, as long as 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. Alternatively, a clip can be used for sealing as shown in FIG. If clips are used, the bag can be reused.

The microporous film used in the present invention is 0.02μ
As mentioned above, the pore preferably has a size of 0.01μ or more that microorganisms cannot pass through, and when the difference between the major axis and the minor axis is small, the minor axis of the pore is 0.1μ or less, preferably 0.05μ.
It is as follows. In addition, in the case of a drainboard-like elongated hole, the maximum short axis is 0.1μ or less, preferably 0.05μ or less,
More preferably, if the diameter is 0.02μ or less, passage of microorganisms can be prevented even if the major axis is large. The porosity is 50% or less, preferably 40% 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 many layers with large pores, forming a bottleneck. In the case of such a microporous film having a multilayer structure, ventilation resistance inevitably becomes large, which tends to impede the 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, fluorine resin, or the like. Since microorganisms mainly move with water, it is difficult for microorganisms to pass through even if the pores are the same size if the microporous film material is water repellent. In addition, drying of the medium is due to the evaporation of water contained in the 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. Although it has sufficient air permeability, it has some effect of preventing medium drying.

In order to use a microporous film in 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 airtightly sealed using a lid, as shown in FIG. 3, and the above-mentioned 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 bonded to the perforations, but as shown in FIG. A flat ring made of a rigid plate that can be attached and detached in an airtight manner is installed in this opening.
A reinforced microporous film is pasted or fused to the wide central opening of this flat ring. In this way, the flat ring can be airtightly attached and detached via the packing, 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 provided with a microporous film, or a part of the container itself is made of a microporous film, so that the inside of the bag or container itself can be used as a small clean room. form, allowing free air circulation but preventing microorganisms from entering. Therefore, there is no need to add sugars to the culture medium just by placing it in a normal bright room.
Sturdy seedlings grow quickly and can be directly planted without 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 was flat, had a wide entrance, and had short sides, making it easier to plant and collect seedlings, and it also became possible to mechanize the process.

〔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. An upper box 4 with a depth of about 1.5 cm was placed on top of the lower box 1. An agar medium was poured into the upper box 4 to a thickness of about 1 cm without overflowing the medium 10, and the agar medium was solidified. Plant callus was planted in the culture medium in this shallow upper box, and a transparent polypropylene frame 5 was placed thereon. The container thus stacked in three layers was inserted into a transparent polypropylene bag 6 having a thickness of 55 μm, and the inlet was heat-sealed. 9
is the heat-sealed part of the bag. The bag 6 has two openings 7.
was provided, and a microporous film 8 was heat-sealed to this opening 7. In this example, Zyuraguard (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 embodiment, 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. The 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 allows water vapor to be directly introduced 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 in a culture container of the type in which a flange is provided with 4 and is held airtight with a clip 16 via a packing 15. 1
7 is a lid, which is similarly sealed by an upper box, a flange, a packing, and a clip. Reference numeral 7' denotes an opening provided in the lid, and a thin groove was carved around this opening 7'. Reference numeral 19 denotes a rigid ring that airtightly fits into the groove, and a microporous film 8 is attached to the center part. This ring 19 is connected to the opening 7' of the lid.
The container can be attached and detached in an airtight manner, and can be replaced when the container is 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, and the female body 2
1 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 perforations 24 intermittently formed in the film constituting the bag, as shown in FIG.
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. An opaque lower box 1 and a shallow upper box 4 are integrally formed, and an engaging part 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 fitting 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 having the same size as the inner diameter of the upper box was placed on the engaging portion 26, and a porous film 28 slightly larger than the support plate was placed on the support plate 27. A layer of double layer 10 was laid 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〕

According to the present invention, it is possible to prevent the medium from drying out, improve workability, and supply a large amount of tissue culture seedlings while freely circulating air and supplying a large amount of carbon dioxide gas. Tissue culture seedlings obtained using the device of the present invention are durable, do not require acclimatization, and can be directly planted as is, expanding the types of plants for which tissue culture seedlings can be used.

[Brief explanation of the drawing]

The drawings show embodiments of the present invention, and FIGS. 1, 3, and 6 are cross-sectional views, FIG. 2 is a perspective view, FIG. 4 is a perspective view showing bag fusing means, and FIG. 5 is a cross-sectional view. FIG. 3 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 and 23 are microporous films, and 9 is a thermal melting film. 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 grip, 17 is a lid, 19 is a ring,
20 is a stopper, 24 is a hole, 25 is a fused portion, 26 is an engaging portion, 27 is a support plate, and 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, allowing microorganisms to pass through. This is a culture method that provides sufficient carbon dioxide gas by using a microporous film that does not allow water to evaporate from the culture medium. A method for culturing plant tissue, characterized by storing the tissue. 2. When growing plant tissue using a container containing a culture medium, the container containing the culture medium itself is sealed except for a part, and air and water vapor are allowed to pass through this part.
This is a culture method that provides sufficient carbon dioxide gas by using a microporous film that does not allow microorganisms to pass through, and in which water is placed in a container sealed with the microporous film as a culture medium to replenish the water vapor volatilized from the culture medium. A method for cultivating plant tissue, which is characterized by storing the tissue independently. 2. It consists 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 is air-tight. and tissue culture equipment, which is a microporous film that allows water vapor to pass through but not microorganisms. 4. 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 small plants, with a water vapor exhaust hole that allows communication with the outside air when the upper box is stacked on the lower box. A tissue culture device consisting of a container and 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. 5. A support plate consisting of a lower box and an upper box placed on the lower box, where the upper box has a bottomless frame shape, and which is made of rigid bodies arranged with a space from the lower end of the upper box. The engaging part on which the
It consists of a container containing a medium for cultivating callus or small plants on the support plate through a porous film that allows water vapor to pass through but not water, and a plastic bag that can house and seal the container. , a tissue culture device in which a portion of the bag is a microporous film that allows air and water vapor to pass through but does not allow microorganisms to pass through. 6 Consisting of a lower box and an upper box placed on the lower box, the upper box forms a rigid support plate arranged at the bottom with space maintained, and on the support plate, water vapor is passed through. It consists of a container containing a medium for cultivating callus or small plants through a porous film that does not allow water to pass through, and a plastic bag that can house and seal the container, with a portion of the bag being air- and air-tight. A tissue culture device that is a microporous film that allows water vapor to pass through but not microorganisms. 7. An engaging part on which a support plate made of a rigid body can be placed extends from the inner side surface of the box, and a porous film that allows water vapor to pass through but does not allow water to pass is laid on the support plate, and a It consists of a container containing a medium for cultivating callus or plantlets, and a plastic bag that can house and seal the container, with a part of the bag made of a microporous film that allows water vapor to pass through but does not allow microorganisms to pass through. tissue culture equipment. 8 A container consisting of a lower box for holding water, an upper box for cultivating callus or plantlets by stacking it on top of the lower box, and a protrusion provided on the upper box, and a plastic bag for storing and sealing the container. It has a water vapor exhaust hole that allows the lower box to communicate 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 medium, and the protrusion is provided so that the plantlets growing in the medium on the upper box do not come into contact with the plastic bag and hinder their growth. . A tissue culture device, wherein the plastic bag is partially a microporous film that allows water vapor to pass through but does not allow microorganisms to pass through. 9. 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. 10. The tissue culture container according to claim 7, wherein the protrusion is one or more rods or tubes. 11. 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. 12. 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. 13 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,
Either the upper box and the lower box are airtightly attached, or the upper box and the lower box are formed integrally, and a device is provided between the lower box and the upper box that allows water vapor to freely pass through, and the bottom of the upper box is free from callus or In addition to providing a container containing a medium for cultivating small plants, a transparent lid is attached airtight to the upper box, and a part of the lid is equipped with a microporous film that allows air to pass through but does not allow microorganisms to pass through. Characteristic tissue culture equipment.