Plant cell drift culture method and device

Abstract

The invention relates to a biotechnology, in particular to a plant cell drift culture method and device. A plant cell drifting culture device comprises an explant floating culture bottle, a plant cell suspension culture bottle and a parameter detector, a culture cage is placed in the explant floating culture bottle through a supporting table, and the culture cage is controlled to ascend and descend through magnetic force. The explant floating culture bottle is communicated with the plant cell suspension culture bottle through a culture bottle connecting pipe, a stirrer is arranged in the plant cell suspension culture bottle, plant callus culture and cell suspension culture are integrated, the treatment link from callus to plant single cells is reduced, and the treatment efficiency is improved. The in-situ cell suspension culture of the callus is realized, the culture process of plant cells is simplified, and the bacterial contamination probability in the culture process is reduced; and parameters such as soaking and floating frequency and time length of plant tissues or cells can be automatically controlled according to the growth characteristics of the cultured different plant cells.

Description

TECHNICAL FIELD

[0001] The present application relates to biotechnology, and particularly to a plant cell drift culture method and device. BACKGROUND

[0002] In the field of botanical research and industrial production, plant tissue culture and cell separation technology are indispensable key links. However, the existing technology has long been faced with the dual challenges of efficiency bottleneck and low survival rate. Traditional culture methods, such as plant callus culture and cell suspension culture, not only have strict technical requirements for operators, making it difficult to achieve standardized mass production, but also result in high culture costs. In addition, traditional culture systems are single-functioned, focusing on either plant tissue culture or cell suspension culture, with a complicated and lengthy process, which greatly limits the improvement of production efficiency. SUMMARY

[0003] In view of the deficiencies of the prior art, one of the purposes of the present application is to provide a plant cell drift culture device, and the second purpose of the present application is to provide a plant cell drift culture method.

[0004] One of the purposes of the present application is achieved by the following technical solutions: A plant cell drift culture device comprises an explant floating culture bottle, a plant cell suspension culture bottle and a parameter detector. A culture cage is placed in the explant floating culture bottle through a support table. The culture cage has a mesh aperture of 0.2-1 cm. The explant floating culture bottle is connected to the plant cell suspension culture bottle through a culture bottle connecting pipe. A stirrer is arranged in the plant cell suspension culture bottle. A motor is arranged at the upper part of the plant cell suspension culture bottle. The motor provides power for the stirrer. The plant cell suspension culture bottle is connected to a plant cell suspension culture liquid sampling pipe at the upper part of the parameter detector through a culture bottle and detector connecting pipe. A sampling port of the explant floating culture bottle is connected to an explant floating culture liquid sampling pipe of the parameter detector, and the explant floating culture liquid sampling pipe is provided with a filter membrane. The plant cell suspension culture liquid sampling pipe and the explant floating culture liquid sampling pipe are jointly connected to a sample liquid detection inlet.

[0005] Preferably, an air compressor connecting port is arranged on the culture bottle and detector connecting pipe. Since the plant cell suspension culture liquid sampling pipe is normally closed in the non-sampling state of the parameter detector, the culture bottle and detector connecting pipe can pressurize the plant cell suspension culture bottle by connecting compressed air, so that the plant cell suspension culture liquid flows to the explant floating culture bottle. Conversely, by venting the plant cell suspension culture bottle to release pressure, the explant floating culture liquid in the explant floating culture bottle can flow to the plant cell suspension culture bottle.

[0006] Preferably, the culture bottle is in communication with a plant cell suspension liquid supplementing port connected with a detector instrument pipe.

[0007] Preferably, the explant floating culture bottle is provided with a supplement bottle connecting port for supplementing the explant floating culture liquid.

[0008] The second object of the present application is achieved by the following technical solutions. A plant cell floating culture method comprises the following steps: Step one, the explant is sterilized and stored; the plant cell floating culture device is used for culture; Step two, the explant is placed in a culture cage, and the liquid level is adjusted so that the explant is soaked in the explant floating culture liquid or floats above the explant floating culture liquid (by connecting a compressed air to the plant cell suspension culture bottle to increase the pressure, so that the plant cell suspension culture liquid flows to the explant floating culture bottle, on the contrary, by discharging the plant cell suspension culture bottle to release the pressure, the explant floating culture liquid in the explant floating culture bottle flows to the plant cell suspension culture bottle); Step three, a small amount of culture liquid is sucked through the explant floating culture liquid sampling pipe to detect the temperature and pH through a parameter detector, and the soaking and floating time interval is set according to the oxygen demand and water demand of the explant type; Step four, the explant floating culture liquid is supplemented through the supplement bottle connecting port regularly; Step six, when the callus is formed, the liquid level is switched between soaking and floating, the loose callus cells flow into the plant cell suspension culture bottle with the explant floating culture liquid, and the plant cell suspension liquid is obtained by shearing and stirring through the stirrer; when the plant cell growth enters the platform period, the culture is stopped, and the plant cell is obtained by centrifuging the culture liquid.

[0009] Preferably, the plant explant is taken from the bud or root of the plant, the obtained explant is washed under flowing clean water for 30 min, the explant is sterilized by soaking in 75% alcohol for 30 s in an ultra-clean bench, then sterilized by 0.1% mercury solution for 10 min, and the explant is washed with sterile water for 4 times after sterilization.

[0010] The present application has the following beneficial effects: The plant callus culture and cell suspension culture are integrated, the processing link from callus to plant single cell is reduced, the callus in situ cell suspension culture is realized, the plant cell culture process is simplified, and the probability of contamination in the culture process is reduced; the plant source compounds can be synthesized efficiently, and the plant source compounds can partially replace plant planting to avoid yield loss caused by uncontrollable factors in planting. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1This is a schematic diagram of the structure of the present invention.

[0012] In the diagram: 1. Explant floating culture bottle; 2. Plant cell suspension culture bottle; 3. Parameter detector; 4. Culture cage; 5. Support platform; 6. Stirrer; 7. Air compressor connection port; 8. Parameter display and control panel; 9. Plant cell suspension feed port; 10. Sample solution detection inlet; 11. Filter membrane; 12. Plant cell suspension culture sample tube; 13. Motor; 14. Feed bottle connection port; 15. Explant floating culture sample tube; 16. Culture bottle connection tube; 17. Culture bottle and detector connection tube. Detailed Implementation

[0013] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings: Example

[0014] The structure of a plant cell drift culture device is as follows: like Figure 1 As shown, the apparatus includes an explant floating culture bottle 1, a plant cell suspension culture bottle 2, and a parameter detector 3. A culture cage 4 is placed inside the explant floating culture bottle 1 via a support platform 5. The explant floating culture bottle 1 is connected to the plant cell suspension culture bottle 2 via a culture bottle connecting pipe 16. A stirrer 6 is installed inside the plant cell suspension culture bottle 2, and a motor 13 is installed at the top of the plant cell suspension culture bottle 2, providing power to the stirrer 6. The plant cell suspension culture bottle 2 is connected to the plant cell suspension culture solution sampling tube 12 at the top of the parameter detector 3 via a culture bottle-to-detector connecting pipe 17. The sampling port of the explant floating culture bottle is connected to the explant floating culture solution sampling tube 15 of the parameter detector, and the explant floating culture solution sampling tube 15 is equipped with a filter membrane 11. The plant cell suspension culture solution sampling tube 12 and the explant floating culture solution sampling tube 15 are both connected to the sample solution detection inlet 10. An air compressor connection port 7 is provided on the culture bottle-to-detector connecting pipe 17. The culture flask and the detector are connected by a plant cell suspension feeding port 9. The explant floating culture flask 1 is provided with a feeding bottle connection port 14.

[0015] In this embodiment: (1) Floating culture bottle for explants: The bottle is made of high borosilicate glass, which is not only resistant to chemical corrosion, but also has good light transmittance, making it convenient to observe the growth status of plant explants. The culture cage inside is made of 316L stainless steel or plastic material, with uniformly distributed mesh and a pore diameter of 0.5cm, which can ensure the free flow of culture medium and prevent explants from falling off.

[0016] (2) Plant cell suspension culture flasks: The suspension culture flasks are made of food-grade engineering plastics to ensure they are non-toxic and harmless. The magnetic stirrer has a three-bladed spiral impeller made of polyetheretherketone (PEEK), which has high strength, wear resistance, and chemical corrosion resistance. The angle and shape of the impeller are optimized to create a good liquid circulation pattern at different speeds, ensuring that the culture medium has a mixing uniformity of over 98%. The speed of the magnetic stirrer can be steplessly adjusted within the range of 50~500 rpm to meet the needs of different culture stages.

[0017] (3) Parameter detector: The pipes are made of silicone rubber, which has the advantages of high elasticity, aging resistance and chemical corrosion resistance. Quick-connect fittings are used at the pipe connection points for easy installation and disassembly. The intelligent timer control switch is equipped with a high-performance microcontroller with a built-in real-time clock chip, and the timing accuracy can reach ±1s. Through the touch screen (display and control panel 8), users can intuitively set parameters such as immersion and floating time and frequency, and can view the operating status of the device in real time.

[0018] (4) Air compressor: The air compressor is a variable frequency oil-free scroll compressor, which operates stably and with low noise. Through feedback from pressure and flow sensors, the control system can accurately adjust the air flow and pressure to provide stable upward power for the culture medium, ensuring that the circulation flow of the culture medium is between 0.5 and 2 L / min. An air filter membrane can be used at the connection port. A polyvinylidene fluoride nanofiber membrane with a pore size of 0.01 μm is selected, which has a filtration efficiency of up to 99.999% for bacteria, fungi and viruses.

[0019] Konjac callus cells were cultured using a plant cell drift culture device, and glucomannan was prepared. (1) Select healthy konjac explants, rinse with clean water to remove surface impurities, and then disinfect them by soaking in 75% alcohol and 0.1% mercuric chloride solution respectively. Rinse the explants with sterile water and set aside for use; (2) Inoculate the disinfected explants into the culture cages in the sterilized floating culture bottles. The konjac explants are cultured in a plant floating tank with a specific culture medium formula. The frequency and duration of floating and soaking are set according to the oxygen and water content required for konjac cell growth, and the temperature, pH value and light conditions in the floating tank are controlled; (3) According to The growth status of konjac callus tissue, replace / replenish the explant floating culture medium to form more callus tissue; (4) the loose callus tissue cells are moved to the suspension culture bottle with magnetic stirrer by the power generated by the flow of the explant floating culture medium. After a certain period of culture, a certain concentration of plant cell suspension is formed, and glucomannan gradually accumulates in the suspension; (6) after a period of culture, the reaction is terminated, the cell suspension is centrifuged, the precipitate is recovered to obtain colloidal glucomannan, and after low temperature drying, powdered glucomannan is obtained.

[0020] The konjac explants were taken from the terminal buds of konjac tubers. The obtained explants were rinsed under running water for 30 minutes to initially remove surface impurities. On a clean bench, the explants were disinfected by soaking in 75% alcohol for 30 seconds, followed by disinfection with 0.1% mercuric chloride solution for 10 minutes. After disinfection, the explants were rinsed with sterile water 4 times to ensure that there was no residual disinfectant on the surface of the explants.

[0021] The konjac explants are placed in the culture cage of a floating culture bottle. The culture cage is fixed by a support frame at a height of 10 cm from the bottom of the bottle. An air compressor is turned on to push the explant culture medium in the bottle to rise. When the air compressor is turned off, the explant culture medium sinks accordingly, so that the explants are either soaked or floating in the culture medium. The soaking frequency is about 10 minutes every 3 hours. The culture temperature is controlled at 26℃, the pH value is adjusted in the range of 6.5~8.5, and the light intensity is controlled in the range of 3000~9000 lx.

[0022] The culture medium in the plant floating tank needs to be changed three times according to the growth status of the konjac callus. The culture medium is MS + NAA 1 mg / L + 6-BA 0.5 mg / L. Potassium ions 0.5 mg / L and magnesium ions 0.2 mg / L are added to promote the rapid division and germination of the konjac explants. The culture cycle is 6 days.

[0023] The culture medium in the suspension culture medium with magnetic stirrer will be in a flowing state under the stirring action. The fluid dynamics generated will break up the konjac callus cells in the floating tank and gradually proliferate in the tank. The conditions are temperature 26℃, pH 7, and culture period of 20 days.

[0024] The konjac callus cells were cultured in suspension and then centrifuged at a low speed of 5000 rpm. The precipitate was recovered and then crushed by grinding or ultrasonication and centrifuged again at a low speed. Glucomannan with a viscosity of about 3000 was obtained from the supernatant.

[0025] The glucomannan is dried at low temperature to obtain powdered glucomannan. The drying temperature is controlled at 60℃, and color-protecting agents such as sodium isoascorbate are added to prevent browning of the glucomannan powder. The powder was tested using gas chromatography (DB43 / T477-2009) for the determination of glucomannan in the konjac food industry. The glucomannan purity was found to be 59.3%, which also indirectly confirms the feasibility of this technical solution.

[0026] The embodiments and descriptions above are merely illustrative of the principles and preferred embodiments of the present invention. Various changes and modifications may be made to the present invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. A plant cell drift culture device, characterized by, The plant cell drift culture device comprises an explant floating culture bottle, a plant cell suspension culture bottle and a parameter detector, a culture cage is placed in the explant floating culture bottle through a support table, the culture cage has a mesh size of 0.2-1 cm, the culture cage is lifted and lowered through magnetic force control, the explant floating culture bottle is connected with the plant cell suspension culture bottle through a culture bottle connecting pipe, a stirrer is arranged in the plant cell suspension culture bottle, a motor is arranged at the upper portion of the plant cell suspension culture bottle, the motor provides power for the stirrer, the plant cell suspension culture bottle is connected with a plant cell suspension culture liquid sampling pipe arranged at the upper portion of the parameter detector through a culture bottle and detector connecting pipe, a sampling port of the explant floating culture bottle is connected with an explant floating culture liquid sampling pipe of the parameter detector, and the explant floating culture liquid sampling pipe is provided with a filter membrane, and the plant cell suspension culture liquid sampling pipe and the explant floating culture liquid sampling pipe are connected with a sample liquid detection inlet.

2. The plant cell drift culture device according to claim 1, characterized by: An air compressor connecting port is arranged on the culture bottle and detector connecting pipe.

3. The plant cell drift culture device of claim 1, wherein: The culture bottle and detector connecting pipe is connected with a plant cell suspension liquid supplementing port.

4. The plant cell drift culture device of claim 1, wherein: The explant floating culture bottle is provided with a supplementing bottle connecting port.

5. A plant cell drift culture method, characterized by, The method comprises the following steps: Step one, the explant is sterilized and stored; Step two, the explant is placed in the culture cage, and the liquid level is adjusted so that the explant is soaked in or floated above the explant floating culture liquid; Step three, a small amount of culture liquid is sucked through the explant floating culture liquid sampling pipe and detected by the parameter detector for temperature and pH, and the soaking and floating time interval is set according to the oxygen demand and water demand of the explant type; Step four, the explant floating culture liquid is supplemented through the supplementing bottle connecting port at regular time intervals; Step five, when the callus is formed, the liquid level is switched between soaking and floating, the loose callus cells flow into the plant cell suspension culture bottle with the explant floating culture liquid, the plant cell suspension liquid is obtained through shearing and stirring of the stirrer, and the culture is stopped when the plant cells grow into the platform period, and the plant cells are obtained by centrifuging the culture liquid.

6. The plant cell drift culture method according to claim 5, characterized by: The plant explant is taken from the bud or root of a plant, the obtained explant is washed under flowing clean water for 30 min, the explant is sterilized by soaking in 75% alcohol for 30 s in an ultra-clean workbench, and then sterilized by soaking in 0.1% mercury solution for 10 min, and the explant is washed with sterile water for 4 times after sterilization.

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