JPH0420280A - Gaseous phase cultivation apparatus - Google Patents

Abstract

PURPOSE:To provide a gaseous phase cultivation apparatus easy to sterilize the inner space and enabling long-term cultivation by using a perforated holding device to hold a cultivation object and a cultivation tank placed below the holding device, furnished with a cultivation-liquid holding means and an atomizing means and capable of keeping the inside in air-tight state. CONSTITUTION:A thermal sterilization and temperature-controlling means 11 is placed outside a cultivation tank 1 to enable the thermal sterilization and the temperature control in the tank 1. An implantation hole 100 for charging a cultivation object 14 and an aeration hole 101 are formed at the upper part of the cultivation tank 1 and the bottom of the tank 1 is provided with a cultivation liquid holding part 12. One or more stages of holding devices 13 are placed in the gaseous phase above the holding part to enable the stable cultivation of a plant tissue, etc., over a long period.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a gas phase culture apparatus for culturing a cultured object such as a plant tissue, a plant cell, or a regenerated plant body in a gas phase.

(Prior Art) Mass culture of plant tissue, plant cells, etc. has conventionally been carried out by liquid culture according to the fermentation method using a tank such as a jar fermenter. In liquid culture, stirring and mixing using stirring blades, air, etc. are essential, so there is a risk that plant tissues and the like may be destroyed or damaged during this stirring. Such damage is particularly likely to occur in differentiated plant tissue systems. For this reason, it has not been easy to mass culture plant tissues and the like by liquid culture.

Recently, instead of such liquid culture, methods and devices capable of culturing plant tissues etc. in a gas phase have been developed, for example, in Japanese Patent Publication No. 60
It is disclosed in Japanese Patent No.-4713. As shown in FIG. 2, the apparatus disclosed here includes a closed culture tank IA and a holder 1 consisting of a perforated plate in which a cultured object 14A is held.
3A, a culture solution storage section 12A provided below the holder 13A, a nozzle 15A arranged above the holder 13A for showering the culture solution onto the cultured body 14A of the holder 13A, It includes a liquid feeding section 16A that connects the nozzle 15A and the culture solution storage section 12A, and a circulation pump 17A that is connected to the liquid feeding section 16A and supplies the culture solution 3 in the culture solution storage section 12A to the nozzle 15A. It is configured as follows.

In such a device, the culture solution 3 in the culture solution storage section 12A is transferred to the nozzle section 15A by driving the circulation pump 17A.
The plant tissues 14A, etc. on the retainer 13A are showered from the nozzle part 15A, and then the retainer 13A is assembled.
The culture solution 3 that has fallen from the tank is stored again in the culture solution storage section 12A. Therefore, when culturing the plant tissue 14A in a vapor phase using such a device, there is no need to stir the plant tissue, etc., so there is less physical damage to the plant tissue, etc., and furthermore, the energy required for stirring is reduced. This has the advantage that it is not necessary.

(Problems to be Solved by the Invention) However, due to its structure, the gas phase culture device disclosed in the above publication has problems such as maintaining a sterile state for a long period of time and supplying a stable culture solution for a long period of time. It still wasn't good enough.

That is, the circulation pump 17A may destroy the sterile state, making it difficult to maintain the sterile state for a long period of time. Particularly in plant tissue culture, long-term culture may be required for 4 to 6 weeks, and it is difficult to sufficiently maintain sterile conditions during such long-term culture.

In addition, since the amount of culture solution 3 supplied to the plant tissue 14A etc. depends on the supply capacity of the circulation pump 17A, the amount of the culture solution 3 supplied to the plant tissue 14A etc.
Depending on the capacity of 7A, the liquid feeding flow rate may decrease over time during long-term operation. In addition, a part of the plant tissue 14A or its fragments may be dropped from the top of the holder 13A into the culture solution storage section 1.
If the plant tissue pieces fall to 2A, there is a risk that the plant tissue pieces may clog the liquid supply pipe 16A, the nozzle 15A1, or the circulation pump 17A. As a result, the culture solution is mixed into one plant tissue.
There is a possibility that it will be difficult to regularly supply to 4A etc.

The present invention is intended to particularly solve the above-mentioned problems, and its purpose is to provide a gas phase culture apparatus that can stably continue culturing plant tissues and the like for a long period of time. Another object of the present invention is to provide a vapor phase culture device that can uniformly supply a culture solution to plant tissues and the like without damaging them.

(Means for Solving the Problems) The gas phase culture device of the present invention is a gas phase culture device for culturing a cultured object to be cultured in a gas phase, and is capable of maintaining the inside in an airtight state. a tank, a holder disposed in the culture tank and having a through hole through which a cultured body is held, a culture solution storage part provided below the holder, and a culture solution storage part arranged in the culture solution storage part. The above-mentioned object is achieved by atomizing means for exciting the culture solution in the storage part into the culture tank in the form of a mist.

An example of the gas phase culture apparatus of the present invention is shown in FIG.

Heat sterilization and temperature control means 1 are provided outside the culture tank 1.
1 is provided, and is configured so that the culture tank 1 can be appropriately heat sterilized or the temperature inside thereof can be adjusted.

The top of the culture tank 1 is provided with an implant port 100 and a vent 101 for introducing the cultured body 14, and the bottom of the culture tank 1 is provided with a culture solution storage section 12. At least one stage of retainer 13 is provided in the upper gas phase of this culture solution storage section 12.
is installed.

The holder 13 holds the target cultured body 14, and as the holder 13, a perforated plate such as a mesh-like material such as a stainless steel wire mesh having a large number of holes is preferably used. Although this example shows the case of a single-stage perforated plate, a multi-stage cage 13 may be provided.

The size of the hole provided in the holder 13 is determined by the size of the cultured object 14 to be handled.
It may be selected appropriately depending on the type of etc. In the present invention, as the cultured body 14, for example, one selected from the group consisting of plant tissues, plant cells, and regenerated plants can be used. A body 14 is placed.

An example of the atomizing means 2 used in the present invention is an ultrasonic transmitter. The atomizing means 2 is disposed within the culture solution storage section 12 and can be operated from the outside of the culture tank 1.

When this atomization means 2 is activated, the culture solution storage section 1
The culture solution 3 in 2 is atomized and raised to form a holder 13.
The culture solution 3 can be supplied to the cultured body 14 through the through hole. The remaining amount of the culture solution 3 falls through the through hole of the holder 13 and is stored in the culture solution storage section 12, and is again caused to rise in the tank by the atomization means 2 in the form of a mist. The supply of the culture solution 3 by the atomizing means 2 such as an ultrasonic transmitter is performed continuously or intermittently. The intermittent time and number of transmitting elements are arbitrarily set so that the supply amount is the minimum required amount of coated culture bodies.

(Example) Next, an example in which plant tissues were cultured in the vapor phase culture apparatus will be specifically shown.

Acanthopanax esntico (scientific name: Acanthopanax esntico, a member of the Araliaceae family)
Callus derived from the leaves of A. sus) was used as a seed. Callus induction is disclosed in detail in JP-A-1-228464. 1 ppm of IBA as auxin and 1 ppm of kinetin as cytokinin were added to MS medium with a corrected nitrogen source to prepare a culture solution. Sucrose 3% was used as a sugar source.

Two volumes of this culture solution were added to the single-perforated plate culture tank 1 (capacity t
ox) and sterilized. A predetermined amount of the above-mentioned seed tissue was then inoculated onto the perforated plate 13 through the implant port 100. The amount of inoculation was 105 g for the first time and 150 g for the second time (wet weight in both cases). After sealing the implant port 100, use an ultrasonic transmitter (ultrasonic humidification frequency is approximately 1.7 MH).
z, atomization capacity of approximately 600 cc/h) 2 is activated to raise the culture solution 3 in the culture solution storage section 12 into the culture tank 1, and intermittently (operates for a total of 4 hours a day) onto the seed tissue. supplied.

A necessary amount of the supplied culture solution (mist-like, average particle size 5 to 50 μm) 3 was absorbed by the plant tissue 14, and the remainder fell from the perforated plate 13 into the culture solution storage section 12 in the form of droplets.

In addition, some of the culture solution (in the form of mist) fell on the inner wall of the culture tank 1 into droplets into the culture solution storage section 12 . Fallen culture solution 3
Once again, the ultrasonic transmitter 2 atomizes the mixture into the culture tank 1, where it is absorbed into the plant tissue 14. This cycle was repeated while the ultrasonic transmitter 2 was activated. While the ultrasonic transmitter 2 was not activated and stopped, no mist was generated and the state in which the culture solution 3 was not supplied to the plant tissue 14 was maintained.

Plant tissue 14 was thus cultured at 25°C for 2 weeks. After culturing, the growth rate was measured. Table 1 shows the results.
Shown below.

As a comparative example on a milk-free milk, vapor phase culture was carried out using the conventional vapor phase culture apparatus shown in FIG. A tubing pump was used as a circulation pump, and culture was carried out for two weeks using the same amount of the same culture solution as above.

Table 1 shows the results of measuring the growth rate after culturing.

Using the culture solution and seeds used in Example 1,
Agar culture was performed in a mjZ flask according to a conventional method. Table 1 shows the results of measuring the growth rate after two weeks of culture.

Table 1 Table 1 shows that the productivity of the gas phase culture of the present invention is superior to that of the conventional gas phase culture of the comparative example and the flask culture on agar substrate.

(Effects of the Invention) Since the gas phase culture device of the present invention has a simple structure, the inside can be easily sterilized, and therefore a sterile state can be maintained for a long period of time. Further, since there is no need to circulate the culture solution as in the conventional method, the culture solution can be stably supplied without the flow rate decreasing over time due to blockage of the cultured body in the circulation system. Furthermore, since the atomized culture solution can be supplied to the cultured body, the culture solution can be uniformly supplied to the plant tissue etc. without damaging the plant tissue etc.

4. "No, No Words H" FIG. 1 is a sectional view showing an example of the vapor phase culture apparatus of the present invention, and FIG. 2 is a sectional view of a conventional vapor phase culture apparatus.

■...Culture tank, 2...Atomization means, 3...Culture solution,
12... Culture solution storage part, 13... Holder, 14...
- Cultured object, 100... implantation port, 101... ventilation port.

that's all No. figure

Claims (1)

[Scope of Claims] 1. A gas phase culture device for culturing a cultured body to be cultured in a gas phase, comprising: a culture tank capable of maintaining an airtight interior; a holder having a through hole through which the cultured body is held; a culture solution storage section provided below the holder; A gas phase culture device comprising: atomizing means for exciting and raising a mist into the culture tank.