JPH05268947A - Method for culture in vapor phase and apparatus therefor - Google Patents

Abstract

PURPOSE:To carry out mass culture of various kinds of plant tissues by placing a plant tissue on a grooved plate and feeding a culture solution thereto via fine pipes projected from the grooved plate. CONSTITUTION:A plant tissue 23 is placed on a grooved plate 2 set in a culture tank 1 so as to allow a culture solution flow evenly throughout the tissue. Then, the culture solution is supplied to fine pipes 21 projected from the grooved plate using a feed pump 5; thereby, the culture solution can be fed into the cell aggregates.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for efficiently culturing a large number of plant cells in the gas phase, and an apparatus therefor.

[0002]

2. Description of the Related Art For mass-culturing plant cells, "Biotechnology Encyclopedia" CMC Co., Ltd. (published on October 9, 1986) p. As is clear from 559 to 560, there are methods such as solid culture, submerged dispersion culture, and submerged culture. When culturing plant cells in the gas phase, the culture medium is supplied by agar or the like. There are known a method for culturing plant cells on a solid medium, and a method for culturing plant cells on a perforated plate by supplying a culture solution from an upper nozzle.

[0003]

Among the conventionally known plant cell culturing methods described above, in liquid culture not only energy is required for stirring but also the cells are damaged by the propeller, and in the culture using a solid medium, Large-scale culture cannot be performed using large-scale equipment. Further, in the culture in which the culture solution is supplied from the upper nozzle, it becomes difficult to supply the culture solution into the cell mass when the cells grow into a large cell mass. Therefore, in this method, it is necessary to spread the plants thinly, a very large space is required for large-scale culture, and this method cannot be used for the large-scale culture method, together with the decrease in cell growth rate.

[0004]

The present invention has been made for the purpose of solving the drawbacks of the above-mentioned conventional methods and newly developing a system for efficiently mass-cultivating plant tissues.
As a result of diligent research from each direction, the plant cells on the groove cutting plate,
By supplying a culture solution from a small-diameter tube protruding from a grooved plate and performing culture, the first successful large-scale culture of various plant tissues efficiently in the gas phase, which has led to the completion of the present invention Is.

The present invention will be described in detail below.

The culturing method of the present invention comprises sterilizing in advance a culturing tank in which a grooved plate having a protruding small diameter tube is placed,
A step of inoculating plant tissue on the grooved plate of the culture tank as a seed, and a step of culturing by supplying a pre-sterilized culture solution to the plant tissue on the grooved plate for a predetermined period from a protruding small diameter tube in the grooved plate Includes and.

As shown in FIGS. 1 and 2, the culture device to be used is composed of a culture tank 1 and is provided with a sterilization and temperature control device 3. On the upper surface or the side surface of the culture tank 1, an implantation recovery port 11 for inoculating a plant tissue as a seed and collecting it after culturing and a ventilation port 12 are provided. Culture tank 1
Inside, the at least one-stage grooved plate 2 for holding the plant tissue is arranged. The grooves of the grooving plate 2 are provided so that the plant tissues 23 on the grooving plate 2 do not flow and become uneven, and are spread evenly without slipping, and the shape thereof is not limited. The grooved plate 2 is provided with a projecting small-diameter tube 21 for supplying the culture solution and a stop plate 22 for preventing the plant tissue 23 from falling from the edge. The stop plate 22 has a porous or net-like structure so that the culture solution is not excessively accumulated on the grooved plate 2. The small-diameter pipe 21 is connected to the bottom of the culture tank via the liquid feeding pipe 4, and the culture liquid accumulated in the lower portion of the culture tank is fed by the liquid feeding pump 5 through the liquid feeding pipe 4. The culture solution is supplied from the small diameter tube 21 continuously or intermittently, and the flow rate thereof is arbitrarily set.

One or a plurality of small diameter tubes 21 may be provided on the grooving plate 2, and the tip end thereof may be a nozzle shape or a shower outlet shape in addition to opening the pipe as it is.
The culture solution is made into various shapes and structures so that the culture solution is uniformly supplied into the plant tissue mass. The height of the small-diameter tube may be the same, but the height of the small-diameter tube may be adjusted to various heights by making the height of the small-diameter tube different so that the culture solution is uniformly supplied. It may be fixed or may be configured to be freely controlled. Further, the outlet of the culture medium of the small diameter tube is not limited to the top portion, and one or more outlets may be provided on the side surface of the tube.

Depending on the plant cell, for example, the culture may be promoted by irradiating it with light. In such a case, it is possible to install a large number of light sources and to rotate the groove plate.

FIG. 2 shows another embodiment of the device of the present invention, showing a culture tank 1 in which grooved plates 2 are provided in multiple stages. In FIG. 2, the grooving plate 2 is provided in five stages, but the number of stages is not limited to five, and an appropriate number can be installed. By thus providing the grooved plates 2 in multiple stages, not only can a large amount of plant cells be efficiently cultured in a small space, but also different types of plant cells can be simultaneously and efficiently cultured in a large amount. .. It was impossible to realize such culture with the conventional culture system.

Examples of gas phase culture according to the present invention will be shown below.

[0012]

Example 1 (1) Sesame (Sesamum indicum L.)
The seed was prepared, immersed in a 75% ethanol solution for several seconds, and then washed twice with sterilized distilled water. This was immersed for 2 minutes in a 0.1% benzalkonium chloride solution (manufactured by Amata Chemical Co., Ltd.). After washing 3 times with sterilized distilled water, soak in a germicide solution containing 1% sodium hypochlorite (Wako Pure Chemical) 0.1% Tween 20 (Wako Pure Chemical) for 30 minutes, and wash with sterile water. Then, sterilized sesame seeds were prepared.

Sterilized water and sterilized gauze were placed in a wide-mouth container for culturing plants (commercial plastic product), and sesame seeds sterilized in advance were seeded on the sterilized water. 20 in a constant temperature room at 30 ° C
When left for 2 weeks under the light of a watt fluorescent lamp, sesame seedlings having a length of 5 to 7 cm were obtained.

(2) MS medium (Murashige-S)
koog medium) in the formulated medium added in combination kinetin 1 × 10- ⁵ M and 2,4-dichloro-phenoxyacetic acid ⁸ × 10- 8 M. Gellan gum 0.2% or agar 0.8% was added to this as a solidifying agent to adjust the pH to 5.7, and then it was dispensed into a Petri dish commonly used for culturing microorganisms and solidified. Into this, a fragment of sesame seedlings aseptically prepared by (1) as described above was transplanted, and in a thermostatic chamber or thermostatic box at a temperature of 28 to 30 ° C,
When culturing was performed in the dark, after 2-3 weeks of culturing, the cells proliferated from the cut end of the cut pieces of sesame seeds and formed a mass to form a callus.

(3) The proliferative cell mass (callus) derived from sesame seedlings obtained as described above is used as described in (2) above.
4 cells were transplanted per Petri dish containing the same medium as the above. Culturing was carried out for 2 weeks in a plant cell culture device at 33 to 36 ° C. and a light of 12,000 lux. A cell clump having good proliferative property was selected and used as a seed cell for 33 to 3
Subculture was repeated 4 times at 6 ° C. Thus, cultured cells that stably grow at high temperature were grown.

(4) The sesame proliferating cells obtained above were subjected to gas phase culture in the culture apparatus shown in FIG.

That is, as a culture solution, kinetin 1
× 10- ⁵ M and 2,4-dichloro-phenoxyacetic acid 8 × 10
Using MS medium containing ⁸ M, this was put in the bottom of the culture tank of the culture tank 1 and sterilized. This was inoculated with the seed tissue consisting of sesame proliferating cells obtained in (3) on the grooving plate 2 from the implantation recovery port 11.

After sealing the implantation recovery port 11, the liquid feed pump 5 is driven to continuously or intermittently flow the culture liquid at the bottom of the culture tank from the small diameter pipe 21 through the liquid feed pipe 4. It was supplied to the plant tissue 23 and the culture was started. A necessary amount of the supplied culture solution is contained in the plant tissue 23,
Were sufficiently contacted with air to perform culture in the gas phase.
The remaining culture solution flowed down to the bottom of the culture tank from the porous stop plate 22 provided at the edge of the grooved plate 2. The flowed-down culture solution was again supplied to the plant tissue 23 from the small diameter pipe 21 through the liquid feed pipe 4 by the liquid feed pump 5.

The culture solution was circulated by repeating such a cycle. Meanwhile, the pH of the culture solution is maintained at 5.6 to 5.8, and the temperature is adjusted to 33 to 36 by the sterilization / temperature control device 3.
Irradiation of 8,000 to 15,000 lux was performed from a light source (not shown) while controlling the temperature. Further, the ventilation hole 12 was adjusted to supply a slight amount of air, and the cells were cultured for 10 days. The relationship between the elapsed culture time and the cell growth (wet weight g) is shown in FIG.

(5) On the other hand, as a comparative example, a method of spraying from above the sesame plant tissue using a nozzle, which is a gas phase method (gas phase method (nozzle)), and culturing by a conventionally known method using a solid medium. I went. The culture conditions were exactly the same as the above-described method of the present invention (gas phase method (small diameter tube)) (however, in the latter case, 0.2% gellan gum was added to the culture solution.
The added solid medium was used).

The results obtained are shown in FIG. 3. As is clear from these results, the culture system according to the present invention has a solid-phase culture method and a gas-phase method using a nozzle, which is a conventionally known gas-phase culture system. Also proved to be much more efficient. This point is further enhanced by the multistage system shown in FIG.

[0022]

EFFECTS OF THE INVENTION According to the present invention, even when cells proliferate into a large mass, the culture solution is supplied from the small diameter tube protruding from the groove plate, so that the culture solution can be sufficiently supplied inside the cell mass. You can Also, since it does not wet the surface of the cell mass,
The cells can be made to breathe sufficiently. Therefore, according to the present invention, the plant cells can be proliferated and cultured very efficiently.

Since the system of the present invention can be widely applied to all plants, not only sesame,
Since it is possible to obtain a large amount of the target plant cells by an industrial method without cultivating the plant which is labor-intensive and easily affected by the weather, it becomes possible to efficiently produce various useful substances derived from these plants. Therefore, the present invention makes a great contribution to various technical fields such as food and drink, pharmaceuticals, cosmetics, and industrial chemicals.

[Brief description of drawings]

FIG. 1 shows a single culture device which is an embodiment of the device according to the present invention.

FIG. 2 shows a multistage culture device which is another embodiment of the device according to the present invention.

FIG. 3 is a comparison diagram between the method according to the present invention (gas phase method (small diameter tube)) and conventionally known methods (gas phase method (nozzle) and solid culture method).

[Explanation of symbols]

 1: Culture tank 2: Grooved plate 21: Small diameter tube

Claims (4)

[Claims] 1. A method for vapor phase culture of plant tissue, which comprises culturing by placing plant cells on a grooving plate and supplying a culture solution to the plant cells from a small diameter tube protruding from the grooving plate. .. 2. A pre-sterilized grooved plate of a culture tank in which a grooved plate having a small diameter tube protruding inside is arranged,
Inoculating the plant tissue as a seed, and
(B) a step of supplying a culture solution sterilized in advance to a plant tissue on the grooved plate through a small-diameter tube protruding from the grooved plate, and culturing, Gas phase culture method. 3. A culture vessel provided with a sterilization and temperature control device, the culture vessel comprising (A) at least one step grooved plate disposed therein for holding a plant tissue, and (B) each of the above. A small-diameter tube protruding from the groove plate, (C) a liquid-feeding tube connecting the small-diameter tube to the bottom of the culture tank, and (D) a culture solution collected in the lower part of the culture tank connected to the liquid-feeding tube. And a liquid feed pump for feeding the small diameter pipe through a liquid pipe. 4. The gas phase culture according to claim 3, wherein one or more small-diameter tubes are provided so as to project, and the heights of the plurality of small-diameter tubes are the same or different. apparatus.