JPH0515363A - Sugar-free culture method by liquid culture medium - Google Patents

Abstract

PURPOSE:To promote photosynthesis of a plant body by raising concentration of carbonic acid gas in light period and simultaneously carry out culture in suppressed contamination by using a liquid culture medium containing no saccharide in culture medium composition and feeding a carbonic acid gas having high concentration to the liquid culture medium under intense light. CONSTITUTION:A liquid culture medium containing no saccharide is packed in a culture tank 1 and a carbonic acid gas having high concentration is controlled to prescribed concentration by a box 3 for controlling concentration of carbonic acid and mixed with air and the mixed gas is fed under pressure and intense light from an aeration apparatus 10 equipped on the bottom of a culture tank 1 through a discharge outlet to a liquid culture medium to culture a plant body in the culture tank.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sugar-free culturing method in a liquid medium, and more specifically, in culturing a plant in a liquid medium, the liquid medium does not contain sucrose as a main carbon source for plant growth. And by supplying a high concentration of carbon dioxide to the liquid medium under strong light, the concentration of carbon dioxide in the light period is increased to promote photosynthesis of the plant, and contamination (miscellaneous bacteria in the culture tank The present invention relates to a sugar-free culturing method using a liquid medium capable of suppressing the growth of plants and proliferating plants without the risk of contamination by various bacteria.

[0002]

2. Description of the Related Art The conventional tissue culture method is a so-called heterotrophic culture method in which MS medium as a basic medium contains about 2% of sucrose which is a main carbon source for plant growth. It was in culture.

However, when the above-mentioned medium tissue is used, sucrose in the medium induces contamination, and various bacteria infiltrate into the medium to contaminate the plant body or make it impossible to culture. There was Therefore, in the conventional tissue culture method, complete sterilization of the medium is indispensable in order to avoid contamination by miscellaneous bacteria, and in order to suppress the loss of the plant body due to contamination, the degree of sealing is high, and the volume is high. A small culture tank had to be used.

However, when a small-capacity culture tank is used, an abnormal environment in the culture tank occurs in which relative humidity and carbon dioxide concentration in the dark period increase and carbon dioxide concentration decreases in the light period. However, there is a problem that the growth of plants is not allowed to be suppressed and it is difficult to control the environment and automate the multiplication work.

[0005]

In view of the above situation, the present invention was devised based on a completely new concept in order to eliminate the problems of conventional tissue culture, and particularly in the medium composition. By using a liquid medium that does not contain sucrose and supplying a high concentration of carbon dioxide to the liquid medium under strong light, the concentration of carbon dioxide in the light period is increased to promote photosynthesis of the plant body and to prevent contamination. It is possible to suppress the nation, eliminate the risk of contamination of plants with various bacteria, simplify the process by eliminating the need to sterilize the medium, enable the use of large-capacity culture tanks, and promote automation of growth. It is an object of the present invention to provide a sugar-free culture method using a liquid medium that can be used.

[0006]

In order to solve the above-mentioned problems, the technical means adopted by the present invention is to apply a liquid culture apparatus of aeration type to cultivate plants.
A culture medium is filled with a liquid medium containing no sucrose, and the plant medium in the culture tank is cultured by supplying a high concentration of carbon dioxide gas to the liquid medium under strong light. .

[0007]

According to the present invention, therefore, since a high concentration of carbon dioxide is supplied to the liquid medium under strong light, the concentration of carbon dioxide in the light period is increased, and photosynthesis is promoted in combination with intense light irradiation. And can be cultured in an environment suitable for plant growth. Moreover, since the liquid medium does not contain sucrose, contamination during the culture is suppressed, and loss of plants due to contamination with various bacteria is also prevented.

[0008]

Embodiments of the present invention will be described in detail with reference to the accompanying drawings. FIG. 1 is a configuration diagram for supplying a high concentration of carbon dioxide gas to a liquid medium filled in a culture tank under strong light. A is an aeration-type culture device, and B is a carbon dioxide gas supply device. Are fixed by a support frame 1a, and the inside thereof is filled with a liquid medium 2 containing no sucrose in an MS medium conventionally used as a basic medium.

3 is a carbon dioxide concentration control box,
A carbon dioxide gas cylinder 4 arranged outside in the box 3
A gas supply port 5 connected to the carbon dioxide concentration meter 7 is provided. The gas supply port 5 is equipped with an electromagnetic valve 6 and is connected to a carbon dioxide concentration meter 7. Reference numeral 8 denotes an air pump or an air compressor, and a flexible tube body 11 is provided between a discharge port 9 of the air pump (air compressor) 8 and an aeration device 10 provided at the bottom of the culture tank 1.
Connected via Reference numeral 12 denotes an outside air inlet formed in the carbon dioxide concentration adjusting box 3, and 13 denotes a gas outlet formed in the closed lid 1b of the culture tank 1. The gas outlet 13 and the carbon dioxide concentration adjusting box 3 are provided. A carbon dioxide gas circulation path C composed of the flexible pipe body 14 may be formed between and, to prevent waste of carbon dioxide gas.

In the above structure, in order to supply a high concentration of carbon dioxide gas to the liquid medium 2 filled in the culture tank 1 under strong light, first, a carbon dioxide concentration meter 7 is used to supply a predetermined gas concentration ( (1,000 to 2,000 ppm) is set, but when the gas concentration in the carbon dioxide concentration adjusting box 3 is less than the set concentration, the electromagnetic valve 6 automatically opens and carbon dioxide gas is supplied from the carbon dioxide cylinder 4. It is supplied to the box 3 via the mouth 5. When the carbon dioxide gas in the box 3 satisfies the set concentration, the electromagnetic valve 6 is automatically closed according to the instruction of the carbon dioxide concentration meter 7, and the supply of carbon dioxide gas is stopped.

Then, when carbon dioxide gas having a set concentration mixed with air is pressure-fed to the liquid medium 2 from the aeration device 10 provided at the bottom of the culture tank 1 through the discharge port 9 by the air pump (air compressor) 8, the air is discharged. While being dissolved in the liquid medium 2 in the form of fine bubbles to increase dissolved oxygen, the concentration of carbon dioxide gas in the light period increases in the liquid medium 2, and photosynthesis is promoted in combination with intense light irradiation.
The growth rate of the cultured plant was equal to or higher than that of the conventional tissue culture method (conventional nutrient culture method).

Moreover, when carbon dioxide gas is supplied to the liquid medium 2, not only the concentration of carbon dioxide gas in the light period and the promotion of photosynthesis but also the air flow in the culture tank 1 are promoted as described above.
Since relative humidity is reduced, it is confirmed that proper control of these phenomena can suppress transpiration, nutrient absorption, and vitrification (a condition in which foliage becomes water-immersive and vulnerable to water stress). Was done. Moreover,
Since sucrose was not contained in the liquid medium 2, contamination during the culture was suppressed, and the loss of plants due to contamination with various bacteria could be prevented. The culture is performed by dividing the day into a light period and a dark period, but the carbon dioxide gas is supplied only during the light period. In this case, the light irradiation illuminance is the light irradiation illuminance (5 klx) in the conventional tissue culture method. ) About 2 to 3 times, that is, 1
It was set to 0 klx or more.

Plants cultured by the above method are
Although divided and transferred to the acclimation step, in the present invention, the plant is divided in an aseptic state before transferring to the acclimation step,
1-2 in a medium containing an appropriate amount of auxin plant hormones
After supplementing the culture for about a week, the wounds were healed at the time of division, stress was removed, and root-promoting treatment was performed.
As a result, as in the conventional method, the cultured plants were divided and transplanted to a rooting medium, and the rooted plants were transferred to acclimation or, after rooting, the divided plants were immediately acclimated. Rooting during habituation was carried out more smoothly and the survival rate could be improved compared to the one transferred to.

FIG. 2 shows a configuration diagram of the culture tank 1 and the liquid culture medium 2 disinfection device D, which can be detached from the culture tank 1 except during the disinfection process of the culture tank 1. In addition to being configured to improve the efficiency of the sterilization work, the sterilization of the device itself is also possible.

That is, in the piping system, two sterilization tanks 15 and 16 are communicatively connected via a three-way valve 21, and the three-way valve 21 is communicatively connected to the culture tank 1 via three-way valves 22, 23 and 26. The culture tank 1 is connected to a sterilization tank 16 via a three-way valve 25 and is connected to the sterilization tank 15 via a three-way valve 24 connected to the three-way valve 25. A pump 17 and an ejector 18 are provided in communication between the three-way valves 24 and 23, an ozone gas generator 19 is connected to the ejector 18, and a filter member is provided between the three-way valves 22 and 26. A sterilizing filter 20 in which F and F are connected in parallel is connected in a communicating manner. 27 and 28 are sterilization treatment devices D in the culture tank 1.
It is a joint member for attaching and detaching.

By the way, in order to perform the sterilization treatment of the culture tank 1 and the liquid medium 2 by using the sterilization treatment apparatus D configured as described above, the following steps are performed. (1) The liquid medium in the culture tank 1 is transferred to the sterilization tank 15. The flow path is as follows: culture tank 1 → joint 27 → three-way valve 25 → three-way valve 24 → pump 17 → ejector 18 → three-way valve 23 → three-way valve 22 → three-way valve 2.
Transfer via 1 to sterilization tank 15. (2) The sterilizing solution filled in the sterilization tank 16 is transferred to the culture tank 1. The flow path is sterilization tank 16 → three-way valve 2
5 → Three-way valve 24 → Pump 17 → Ejector 18 → Three-way valve 23 → Three-way valve 26 → Transfer to culture tank 1 via joint 28. (3) The sterilizing liquid in the culture tank 1 is returned to the sterilizing tank 16. The flow path is culture tank 1 → joint 27 → three-way valve 25 → three-way valve 24 → pump 17 → ejector 1
8-> 3-way valve 23-> 3-way valve 22-> 3-way valve 21
And return to the sterilization tank 16. (4) The liquid culture medium 2 in the sterilization tank 15 is added to the culture tank 1
Return to. The flow path is a sterilization tank 15 → a three-way valve 24.
→ Pump 17 → Ejector 18 → Three-way valve 23 → Three-way valve 22 → Filter 20 → Three-way valve 26 → Return to the culture tank 1 via the joint 28.

Next, the sterilization treatment of the decontamination treatment device D itself is as follows.
The order is as follows. (1) The culture tank 1 is detached and a sterilization tank (not shown) is separately connected to the disinfection processing apparatus D via the joints 27 and 28. (2) The sterilization liquid filled in the sterilization tank 16 is circulated in the next flow path to sterilize the sterilization tank 16. That is, the sterilization tank 16 → three-way valve 25 → three-way valve 24 → pump 17 → ejector 18 → three-way valve 23 → three-way valve 22.
→ The three-way valve 21 is circulated to sterilize the sterilization tank 16. (3) The sterilization liquid in the sterilization tank 16 is circulated to the next flow path to sterilize the sterilization tank 15. That is, the sterilization tank 16 → three-way valve 25 → three-way valve 24 → pump 17 →
Ejector 18-> 3-way valve 23-> 3-way valve 22-> 3-way valve 21-> Sterilization tank 15-> 3-way valve 24-> Pump 17-> Ejector 18-> 3-way valve 23-> 3-way valve 22
→ The three-way valve 21 is circulated to sterilize the sterilization tank 15. (4) The sterilizing liquid in the sterilization tank 15 is circulated to the next flow path to sterilize the piping system. That is, sterilization tank 15 → three-way valve 24 → pump 17 → ejector 18 → three-way valve 23 → three-way valve 26 → joint 28 → sterilization tank → joint 27 → three-way valve 25 → sterilization tank 16 →
Three-way valve 24 → pump 17 → ejector 18 → three-way valve 23 → three-way valve 26 → joint 28 → sterilization tank → joint 27 → three-way valve 25 → sterilization tank 16
To sterilize the piping system. (5) The sterilizing solution in the sterilization tank 16 is used to backwash the sterilizing filter 20. The flow path is drained through the sterilization tank 16, the three-way valve 25, the three-way valve 24, the pump 17, the ejector 18, the three-way valve 23, the three-way valve 26, and the filter members F and F. The sterilizing liquid used for the sterilization treatment of the culture tank 1 and the liquid medium 2 and the sterilization treatment of the decontamination treatment device D itself performed in the above-described order may be either chemicals or ozone water.

Further, in the above-mentioned decontamination processing device D, a filter 20 is provided in the middle of the piping line to remove dust, bacteria and the like mixed in the liquid medium 2 by the filter 20, In view of the fact that there is a limit to the filtration capacity and it causes clogging and deterioration over time, a required number of hollow disk-shaped members 30 are provided in the middle of the pipe 29 as shown in FIG. The circular center portion 32 of the separation pipe 31 which is connected to each other may be provided in communication with the pipe 29, and the separation pipe 31 may be coupled to the motor 33 via the gear transmission mechanism 34 and rotated. In this case, dust, bacteria and the like mixed in the liquid medium 2 are accumulated in the circumferential portion of the separation tube 31 by the centrifugal force, so that the clean liquid medium 2 can be obtained.
Will pass through the central portion 32.

[0019]

In summary, the present invention is a method for cultivating a plant body by utilizing a liquid culture device of aeration system, which is filled in a tank for culturing a sucrose-free liquid medium, The plant tissue in the culture tank was cultivated by supplying a high concentration of carbon dioxide gas to the culture site under strong light. Therefore, the conventional tissue culture method (heterotrophic culture method) was used.
The following remarkable effects are obtained as compared with the culture by. (1) Since the carbon dioxide concentration in the light period is increased and photosynthesis is promoted in combination with intense light irradiation, the plant can be cultured in an environment suitable for growth. (2) Since the air flow in the culture tank is promoted and the relative humidity is reduced in association with the increase of carbon dioxide concentration in the light period and the promotion of photosynthesis, the transpiration is promoted by appropriately controlling these phenomena. In addition to promoting nutrient absorption, vitrification is suppressed and plant growth is homogenized. (3) Since sucrose is not contained in the liquid culture medium, contamination during culture can be suppressed and loss of plants due to contamination with various bacteria can be prevented.

[0020]

[Brief description of drawings]

FIG. 1 is a configuration diagram for supplying a high concentration of carbon dioxide gas to a liquid culture medium in a culture tank under strong light.

FIG. 2 is a configuration diagram of a culture tank and a liquid culture medium disinfection device.

FIG. 3 is a front view of a separation tube.

[Explanation of symbols]

 1 Culture Tank 2 Liquid Culture Area 3 Carbon Dioxide Concentration Adjustment Box 5 Carbon Dioxide Cylinder 8 Air Pump 9 Gas Discharge Port 10 Aeration Device

Claims (1)

Claim: What is claimed is: 1. A method of culturing a plant by applying an aeration type liquid culture device, comprising filling a culture tank with a sucrose-free liquid medium, and adding a strong medium to the liquid medium. A sugar-free culture method using a liquid medium, which comprises culturing a plant in a culture tank by supplying a high concentration of carbon dioxide under light.