CN111528103B - Plant tissue culture reactor and amplification culture method thereof - Google Patents
Plant tissue culture reactor and amplification culture method thereof Download PDFInfo
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- CN111528103B CN111528103B CN202010619353.5A CN202010619353A CN111528103B CN 111528103 B CN111528103 B CN 111528103B CN 202010619353 A CN202010619353 A CN 202010619353A CN 111528103 B CN111528103 B CN 111528103B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention relates to the technical field of bioreactors, and provides a plant tissue culture reactor and an amplification culture method thereof. The plant tissue culture reactor provided by the invention realizes shearing of plant tissues in the reactor and sterile transfer between the reactors, does not need human intervention, reduces the risk of pollution, and improves the production efficiency.
Description
Technical Field
The invention relates to the field of bioreactors, in particular to a plant tissue culture reactor and an amplification culture method thereof.
Background
The plant tissue culture method mainly comprises the steps of taking spliced plant organs or tissues as explants, inoculating the explants in an artificially prepared culture medium, matching with good nutritional conditions, hormone conditions, temperature conditions, illumination conditions and the like, carrying out dedifferentiation on the explants by means of aseptic culture to form callus, carrying out redifferentiation to form tissues or organs, and carrying out culture and development to form a complete plant.
The bioreactor is a core device for large-scale cell suspension culture, provides a suitable environment for cell growth, and can effectively increase the culture density of cell unit volume. Suspension culture is beneficial to the plant cells to uniformly receive nutrient substances, gas, illumination and the like.
At present, in the artificial plant tissue culture process, all adopt artifical shearing repacking lower level to enlarge the cultivation, whole process needs artificial intervention, has greatly increased the risk of culture medium liquid pollution, and its production efficiency is low, and is unfavorable for automated control, consequently, has the space of improvement in addition.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide the plant tissue culture reactor, which has the advantages of realizing the shearing of plant tissues in the reactor and the sterile transfer between the reactors, avoiding human intervention, reducing the pollution risk and improving the production efficiency.
In order to achieve the purpose, the invention provides the following technical scheme:
a plant tissue culture reactor is characterized by comprising a seeding tank (1), a first-stage amplification culture tank (2) and a second-stage amplification culture tank (3), the seeding tank (1) is communicated with the first-stage amplification culture tank (2) through a sterile pipeline, the first-stage amplification culture tank (2) is communicated with the second-stage amplification culture tank (3) through a sterile pipeline, the top parts of the seeding tank (1), the first-stage amplification culture tank (2) and the second-stage amplification culture tank (3) are respectively provided with a seeding tank stirring device (4), a first-stage amplification stirring device (5) and a second-stage amplification stirring device (7), the bottom of the first-stage amplification culture tank (2) is rotationally connected with a shearing device (6), the shearing device (6) is provided with a first-stage amplification shearing paddle (10), and a distance is reserved between the first-stage amplification shearing paddle (10) and the first-stage amplification stirring device (5).
The invention is further configured to: the primary amplification shearing paddle (10) comprises a back surface (12) and a front surface knife edge (13), when the stirring device (4) of the seeding tank rotates forwards, the back surface (12) rotates clockwise, and when the stirring device (4) of the seeding tank rotates backwards, the front surface knife edge (13) rotates anticlockwise.
The seeding tank integrates plant tissue culture and plant tissue shearing, and the stirring device of the seeding tank rotates forwards during plant tissue culture, so that the back surface rotates, the stirring is more uniform, and the plant tissue culture effect is better; when the stirring device of the seeding tank rotates reversely, the front edge of a knife cannot damage plant tissues, the stirring is rapidly rotated reversely when the plant tissues are sheared, the plant tissues are cut off at a high speed by the front edge of the first-stage amplification shearing paddle, the production efficiency is improved, the first-stage amplification shearing paddle is uniformly distributed with three layers of paddles, and the first-stage amplification shearing paddle is highly layered and is favorable for improving the shearing efficiency of the plant tissues.
The method is divided into three-stage amplification culture of a seeding tank, a first-stage amplification culture tank and a second-stage amplification culture tank, the three-stage amplification culture realizes the transfer of the plant tissue sterile pipeline without manually shearing and transferring the plant tissue, the amplification culture efficiency of the plant tissue is greatly improved, and the damage rate of tissue cells and the pollution risk of a culture carrier are reduced.
The invention is further configured to: the back surface (12) is in a concave blunt shape, and the front surface knife edge (13) is in a sharp shape.
The back surface of the primary amplification shearing paddle is concave and blunt, and the back surface is blunt, so that the damage of plant cells caused by the back surface of the primary amplification shearing paddle is reduced; the front edge of the blade is sharp through the first-stage amplification shearing, so that the plant tissue can be cut off rapidly, and the production efficiency is improved.
The invention is further configured to: the first-stage amplification stirring device (5) comprises a first-stage amplification stirring paddle (9) and a first-stage amplification stirring shaft.
When plant tissues are cultured in the first-stage amplification culture tank, the first-stage amplification stirring paddle and the first-stage amplification shearing paddle operate separately, so that the first-stage amplification stirring paddle can be used for culturing normal tissue cells and sinking the plant tissues during shearing, and the first-stage amplification shearing paddle is located at the bottom of the first-stage amplification culture tank and is favorable for improving the shearing efficiency.
The invention is further configured to: the second-stage amplification stirring device (7) comprises a second-stage amplification stirring blade (11) and a second-stage amplification stirring shaft.
The invention also provides a plant tissue amplification culture method, which is characterized by comprising the following steps:
s1, placing cultured plant tissue cells into a seeding tank (1) for automatic culture until plant tissues with the length of 7-10 CM are cultured, wherein the parameters of the seeding tank (1) are set to 35-37 ℃, the rotating speed is 200-600 rpm/min, the dissolved oxygen is 40-60%, the pH value is 6.2-7.2, the air and oxygen flow is 0.2-0.4L/min and 0.05-0.14L/min;
s2, when the length of the plant tissue is cultured to 7-10 CM, starting a seed tank stirring device (4) to reversely rotate at a high speed, wherein the stirring speed is 1000-3000 rpm/min, and the stirring time is 5-10 min, so that the length of the plant tissue is 1-2 CM, then transferring the plant tissue to a first-stage amplification culture tank (2) through a sterile pipeline for automatic culture, and culturing the plant tissue until the length of the plant tissue is 7-10 CM, wherein the parameters of the first-stage amplification culture tank (2) are set to 35-37 ℃, the rotation speed is 400-600 rpm/min, the dissolved oxygen content is 40-50%, the pH value is 6.8-7.4, the air and oxygen flow is 0.2-0.4L/min, and the pH value is 0.05-0.14L/min;
and S3, after the plant tissues are automatically cultured in the first-stage amplification culture tank (2), starting the shearing device (6), stirring and sinking the plant tissues by the first-stage amplification stirring paddle (9), stirring for 10-15 min at a stirring speed of 100-300 rpm/min to enable a front knife edge (13) on the first-stage amplification shearing paddle (10) to shear the plant tissues, wherein the length of the sheared plant tissues is 1-2 CM, and then the plant tissues enter the second-stage amplification culture tank (3) containing the culture medium liquid through a sterile pipeline to be amplified and cultured into finished plants.
The invention increases aeration (namely faster gas exchange exists), particularly increases oxygen and air, can improve the growth efficiency of cells in culture, provides gas exchange for the air and oxygen flow of 0.2-0.4L/min and 0.05-0.14L/min at the inlet of the bioreactor for plant tissue culture, improves the production efficiency, and improves the quality of metabolites and reduces the damage rate of tissue cells when the pH values of a seeding tank and a first-stage amplification culture tank are respectively 6.2-7.2 and 6.8-7.4.
The invention is further configured to: the culture medium liquid in the step S3 comprises the following raw materials in percentage by mass: 10-15% of cane sugar and FeSO 4 5％～10％、NH4NO310% -15% of lysine 25 percent, 10 to 15 percent of aspartic acid, 4 to 9 percent of L-methionyl glycinate hydrochloride and 10 to 30 percent of deionized water.
After the plant cells are subjected to multiple passages, the activity of the plant cells is reduced, and the improved culture medium liquid is added into the secondary amplification culture tank, so that the biological activity of the plant cells entering the secondary amplification culture tank is enhanced, and the effect of the cultured plant cells is better.
The invention is further configured to: the culture medium liquid in the step S3 comprises the following raw materials in percentage by mass: 12% of sucrose and FeSO4 6％、NH4NO312%, lysine 3%, aspartic acid 14%, L-methionyl glycine hydrochloride 5% and deionized water 28%.
The invention is further configured to: the plant tissue is selected from the group consisting of root cells, celery cells, algal cells, carrot cells and tobacco cells.
The invention is further configured to: the plant tissue is selected from algal cells.
Compared with the prior art, the invention has the following beneficial effects:
1. according to the plant tissue culture reactor provided by the invention, the shearing of plant tissues in the reactor and the sterile transfer between the reactors are realized, no manual operation is needed, the production cost is reduced, and the production efficiency is improved;
2. according to the plant tissue amplification culture method provided by the invention, the damage degree of plant cells is reduced through setting parameters such as temperature, rotating speed, dissolved oxygen, pH value, air flow and oxygen flow;
3. according to the plant tissue amplification culture method provided by the invention, the culture medium liquid can improve the biological activity of plant cells, so that the effect of the cultured plant cells is better.
Drawings
FIG. 1 is a schematic view of a plant tissue culture reactor according to the present invention;
FIG. 2 is a schematic view of a one-stage enlarged shear blade according to the present invention.
In the figure: 1. a seed tank; 2. a first-stage amplification culture tank; 3. a second-stage amplification culture tank; 4. a seeding tank stirring device; 5. a first-stage amplification stirring device; 6. a shearing device; 7. a second-stage amplification stirring device; 8. stirring blades of a seeding tank; 9. a first-stage amplification stirring blade; 10. first-stage amplification shearing paddle; 11. a second-stage amplification stirring blade; 12. a back side; 13. a front edge.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
In the following examples and comparative examples, the starting materials were commercially available.
Example 1 plant tissue culture reactor of the present invention
As shown in figure 1, the plant tissue culture reactor comprises a seeding tank 1, a first-stage amplification culture tank 2 and a second-stage amplification culture tank 3, wherein the bottom of the seeding tank 1 is communicated with the side edge of the first-stage amplification culture tank 2 through a sterile pipeline, the bottom of the first-stage amplification culture tank 2 is communicated with the side edge of the second-stage amplification culture tank 3 through a sterile pipeline, the tops of the seeding tank 1, the first-stage amplification culture tank 2 and the second-stage amplification culture tank 3 are respectively provided with a seeding tank stirring device 4, a first-stage amplification stirring device 5 and a second-stage amplification stirring device 7, and the bottom of the first-stage amplification culture tank 2 is rotatably connected with a shearing device 6.
According to fig. 1 and fig. 2, a first-stage amplification shearing blade 10 is arranged on the shearing device 6, a distance is reserved between the first-stage amplification shearing blade 10 and the first-stage amplification stirring device 5, the extending direction of the first-stage amplification shearing blade 10 and the extending direction of the first-stage amplification stirring device 5 form an included angle, the first-stage amplification shearing blade 10 comprises a back 12 and a front 13, the back 12 is in a concave blunt shape, the front 13 is in a sharp shape, when the seed tank stirring device 4 rotates forwards, the back 12 rotates clockwise, when the seed tank stirring device 4 rotates backwards, the front 13 rotates anticlockwise, and plant tissues are cut off at high speed.
The setting directions of the seed tank stirring device 4, the first-stage amplification stirring device 5 and the second-stage amplification stirring device 7 are parallel to each other, the seed tank 1, the first-stage amplification culture tank 2 and the second-stage amplification culture tank 3 are vertically extended respectively, the seed tank stirring device 4 comprises a seed tank stirring blade 8 and a seed tank stirring shaft, the first-stage amplification stirring device 5 comprises a first-stage amplification stirring blade 9 and a first-stage amplification stirring shaft, and the second-stage amplification stirring device 7 comprises a second-stage amplification stirring blade 11 and a second-stage amplification stirring shaft.
The side wall of the plant tissue culture reactor is provided with an opening, so that solvents such as deionized water and the like can be added, the pH value can be adjusted, and the parameters such as air, oxygen flow, dissolved oxygen and the like can be adjusted.
Example 2 plant tissue amplification culture method of the present invention
The plant tissue amplification culture method comprises the following steps:
s1, shearing artificially cultured celery plants to a length of 1-2 CM, placing the celery plants in a seeding tank (1) for automatic culture, and culturing the celery plants to a plant tissue with a length of 7CM, wherein the parameters of the seeding tank (1) are set to 35-37 ℃, the rotating speed is 200rpm/min, the dissolved oxygen content is 40%, the pH value is 6.2, and the air and oxygen flow is 0.2L/min and 0.05L/min;
s2, when the length of the plant tissue is cultured to 7CM, starting a seed tank stirring device (4) to reversely rotate at a high speed, wherein the stirring speed is 1000rpm/min, the stirring time is 5min, the length of the plant tissue is 1-2 CM, then transferring the plant tissue to a first-stage amplification culture tank (2) through a sterile pipeline for automatic culture, and culturing the plant tissue until the length of the plant tissue is 8CM, wherein the parameters of the first-stage amplification culture tank (2) are set to 35-37 ℃, the rotation speed is 400rpm/min, the dissolved oxygen content is 50%, the pH value is 6.8, the air and oxygen flow is 0.2L/min and 0.1L/min;
s3, after the plant tissue is automatically cultured in the first-stage amplification culture tank (2), starting a shearing device (6), stirring and sinking the plant tissue by a first-stage amplification stirring paddle (9), stirring for 10min at a stirring speed of 100rpm/min to enable a front knife edge (13) on the first-stage amplification shearing paddle (10) to shear the plant tissue, wherein the length of the sheared plant tissue is 1-2 CM, and then the plant tissue enters the second-stage amplification culture tank (3) containing a culture medium liquid through a sterile pipeline to be amplified and cultured into a finished plant;
wherein the culture medium liquid in the step S3 comprises the following raw materials in percentage by mass: 10% of sucrose and FeSO 4 5％、NH4NO310%, lysine 2%, asparagus10% of amino acid, 4% of L-methionyl glycinate hydrochloride and 10% of deionized water.
Example 3 plant tissue amplification culture method of the present invention
The plant tissue amplification culture method comprises the following steps:
s1, shearing an artificially cultured algae plant to a length of 1-2 CM, placing the algae plant in a seeding tank (1) for automatic culture, and culturing to plant tissues with a length of 8CM, wherein the parameters of the seeding tank (1) are set to 35-37 ℃, the rotating speed is 400rpm/min, the dissolved oxygen content is 50%, the pH value is 6.6, and the air and oxygen flow rates are 0.3L/min and 0.1L/min;
s2, when the length of the plant tissue is cultured to 8CM, starting a seed tank stirring device (4) to reversely rotate at a high speed, wherein the stirring speed is 2000rpm/min, the stirring time is 8min, the length of the plant tissue is 1-2 CM, then transferring the plant tissue to a first-stage amplification culture tank (2) through a sterile pipeline for automatic culture, and culturing the plant tissue until the length of the plant tissue is 8CM, wherein the parameters of the first-stage amplification culture tank (2) are set to 35-37 ℃, the rotation speed is 500rpm/min, the dissolved oxygen content is 45%, the pH value is 7.0, and the air and oxygen flow is 0.3L/min and 0.05L/min;
s3, after the plant tissue is automatically cultured in the first-stage amplification culture tank (2), starting a shearing device (6), stirring and sinking the plant tissue by a first-stage amplification stirring paddle (9), stirring for 15min at a stirring speed of 200rpm/min to enable a front knife edge (13) on the first-stage amplification shearing paddle (10) to shear the plant tissue, wherein the length of the sheared plant tissue is 1-2 CM, and then the plant tissue enters the second-stage amplification culture tank (3) containing a culture medium liquid through a sterile pipeline to be amplified and cultured into a finished plant;
wherein the culture medium liquid in the step S3 comprises the following raw materials in percentage by mass: 12% of sucrose and FeSO4 7％、NH4NO313%, lysine 4%, aspartic acid 11%, L-methionyl glycine hydrochloride 7% and deionized water 25%.
Example 4 plant tissue amplification culture method of the present invention
The plant tissue amplification culture method comprises the following steps:
s1, cutting the artificially cultured carrots to the length of 1-2 CM, placing the carrots in a seeding tank (1) for automatic culture, and culturing the carrots to plant tissues with the length of 10CM, wherein the parameters of the seeding tank (1) are set to 35-37 ℃, the rotating speed is 600rpm/min, the dissolved oxygen is 60%, the pH value is 7.2, and the air and oxygen flow is 0.4L/min and 0.14L/min;
s2, when the length of the plant tissue is cultured to 10CM, starting a seed tank stirring device (4) to reversely rotate at a high speed, wherein the stirring speed is 3000rpm/min, the stirring time is 10min, so that the length of the plant tissue is 1-2 CM, then transferring the plant tissue to a first-stage amplification culture tank (2) through a sterile pipeline for automatic culture, and culturing the plant tissue until the length of the plant tissue is 8CM, wherein the parameters of the first-stage amplification culture tank (2) are set to 35-37 ℃, the rotation speed is 600rpm/min, the dissolved oxygen content is 50%, the pH value is 7.4, the air and oxygen flow is 0.4L/min and 0.14L/min;
s3, after the plant tissue is automatically cultured in the first-stage amplification culture tank (2), starting a shearing device (6), stirring and sinking the plant tissue by a first-stage amplification stirring paddle (9), stirring for 15min at a stirring speed of 300rpm/min to enable a front knife edge (13) on the first-stage amplification shearing paddle (10) to shear the plant tissue, wherein the length of the sheared plant tissue is 1-2 CM, and then the plant tissue enters the second-stage amplification culture tank (3) containing a culture medium liquid through a sterile pipeline to be amplified and cultured into a finished plant;
wherein the culture medium liquid in the step S3 comprises the following raw materials in percentage by mass: 15% of sucrose and FeSO 410％、NH4NO315%, lysine 5%, aspartic acid 15%, L-methionyl glycine hydrochloride 9% and deionized water 30%.
Example 5 plant tissue amplification culture method of the present invention
The plant tissue amplification culture method comprises the following steps:
s1, shearing an artificially cultured algae plant to a length of 1-2 CM, placing the algae plant in a seeding tank (1) for automatic culture, and culturing to a plant tissue with a length of 7CM, wherein the parameters of the seeding tank (1) are set to 35-37 ℃, the rotating speed is 550rpm/min, the dissolved oxygen content is 45%, the pH value is 6.8, and the air and oxygen flow rates are 0.35L/min and 0.12L/min;
s2, when the length of the plant tissue is cultured to 7CM, starting a seed tank stirring device (4) to reversely rotate at a high speed, wherein the stirring speed is 2500rpm/min, the stirring time is 8min, the length of the plant tissue is 1-2 CM, then transferring the plant tissue to a first-stage amplification culture tank (2) through a sterile pipeline for automatic culture, and culturing the plant tissue until the length of the plant tissue is 7CM, wherein the parameters of the first-stage amplification culture tank (2) are set to 35-37 ℃, the rotation speed is 480rpm/min, the dissolved oxygen content is 48%, the pH value is 7.2, the air and oxygen flow rate is 0.3L/min, and 0.08L/min;
s3, after the plant tissue is automatically cultured in the first-stage amplification culture tank (2), starting a shearing device (6), stirring and sinking the plant tissue by a first-stage amplification stirring paddle (9), stirring for 15min at a stirring speed of 270rpm/min to enable a front knife edge (13) on the first-stage amplification shearing paddle (10) to shear the plant tissue, wherein the length of the sheared plant tissue is 1-2 CM, and then the plant tissue enters the second-stage amplification culture tank (3) containing a culture medium liquid through a sterile pipeline to be amplified and cultured into a finished plant;
wherein the culture medium liquid in the step S3 comprises the following raw materials in percentage by mass: 12% of sucrose and FeSO4 6％、NH4NO312%, lysine 3%, aspartic acid 14%, L-methionyl glycine hydrochloride 5% and deionized water 28%.
Example 6 plant tissue amplification culture method of the present invention
The plant tissue amplification culture method comprises the following steps:
s1, shearing artificially cultured tobacco cells to a length of 1-2 CM, placing the tobacco cells in a seeding tank (1) for automatic culture, and culturing to plant tissues with a length of 7CM, wherein the parameters of the seeding tank (1) are set to 35-37 ℃, the rotating speed is 550rpm/min, the dissolved oxygen content is 45%, the pH value is 6.8, and the air and oxygen flow rates are 0.35L/min and 0.12L/min;
s2, when the length of the plant tissue is cultured to 7CM, starting a seed tank stirring device (4) to reversely rotate at a high speed, wherein the stirring speed is 2500rpm/min, the stirring time is 8min, the length of the plant tissue is 1-2 CM, then transferring the plant tissue to a first-stage amplification culture tank (2) through a sterile pipeline for automatic culture, and culturing the plant tissue until the length of the plant tissue is 7CM, wherein the parameters of the first-stage amplification culture tank (2) are set to 35-37 ℃, the rotation speed is 480rpm/min, the dissolved oxygen content is 48%, the pH value is 7.2, the air and oxygen flow rate is 0.3L/min, and 0.08L/min;
s3, after the plant tissue is automatically cultured in the first-stage amplification culture tank (2), starting a shearing device (6), stirring and sinking the plant tissue by a first-stage amplification stirring paddle (9), stirring for 15min at a stirring speed of 270rpm/min to enable a front knife edge (13) on the first-stage amplification shearing paddle (10) to shear the plant tissue, wherein the length of the sheared plant tissue is 1-2 CM, and then the plant tissue enters the second-stage amplification culture tank (3) containing a culture medium liquid through a sterile pipeline to be amplified and cultured into a finished plant;
wherein the culture medium liquid in the step S3 comprises the following raw materials in percentage by mass: 12% of sucrose and FeSO4 6％、NH4NO312%, lysine 3%, aspartic acid 14%, L-methionyl glycine hydrochloride 5% and deionized water 28%.
Comparative example 1 plant tissue amplification culture method of the present invention
As in example 5, only the difference from example 5 is: the medium liquid in step S3 does not include L-methionyl glycinate hydrochloride, and other parameters are similar to those in example 5.
Comparative example 2 plant tissue amplification culture method of the present invention
As in example 5, only the difference from example 5 is: the seeding tank and the first-stage amplification culture tank have pH values of 6.0 and 6.5, respectively, and other parameters are similar to those of example 5.
Comparative example 3 plant tissue amplification culture method of the present invention
As in example 5, only the difference from example 5 is: the seeding tank and the first-stage amplification culture tank have pH values of 7.5 and 7.6, respectively, and other parameters are similar to those of example 5.
Test example I measurement of algal cell Activity
The method comprises the steps of taking algae cells which are subjected to amplification culture in a seeding tank, a first-stage amplification culture tank and a second-stage amplification culture tank, digesting logarithmic phase cells by pancreatin, inoculating the cells into a 96-well plate, centrifugally collecting after termination to prepare cell suspension, counting the cells, and adjusting the concentration to 6 x 104After overnight incubation and adherence, 1ml of the medium of examples 2-6 and comparative examples 1-3 was added to the well plate, 6 wells were set, after 12h, 10 μ l of MTT (5mg/ml, i.e. 0.5% MTT) was added, after incubation at 37 ℃ for 4h, the medium and MTT were aspirated, DMSO was added, the reaction was carried out on a shaker at room temperature for 30min, then absorbance was measured at 570nm wavelength with a chemiluminescence apparatus, and a blank control (deionized water was added) was set for this experiment, with the results shown in Table 1.
TABLE 1
| Group of | OD value | Survival rate |
| Example 2 | 0.614 | 0.952 |
| Example 3 | 0.620 | 0.950 |
| Example 4 | 0.618 | 0.959 |
| Example 5 | 0.625 | 0.961 |
| Example 6 | 0.616 | 0.954 |
| Comparative example 1 | 0.548 | 0.654 |
| Comparative example 2 | 0.606 | 0.943 |
| Comparative example 3 | 0.609 | 0.942 |
| Blank control group | 0.515 | 0.591 |
As is clear from the data in Table 1, the culture medium solutions of examples 2 to 5 to which L-methionylglycine hydrochloride was added had higher OD values and higher survival rates of plant cells than those of comparative examples 1 to 3, and the survival rate of algal cells was the highest and was as high as 96.1%;
compared with example 5, comparative example 1, in which the culture medium solution without addition of L-methionyl glycine hydrochloride was used, had a lower OD value and survival rate of algal cells, but still had a higher survival rate than the blank control group, indicating that L-methionyl glycine hydrochloride can increase the OD value and survival rate of plant cells;
compared with example 5, the pH value of the seeding tank and the pH value of the first-stage amplification culture tank in the comparative example 2 or the comparative example 3 are reduced or increased, which affects the OD value and the survival rate of plant cells, and shows that the pH value range plays a crucial role in the plant tissue amplification culture method.
The embodiments of the present invention are preferred embodiments of the present invention, and the scope of the present invention is not limited by these embodiments, so: all equivalent changes made according to the structure, shape and principle of the invention are covered by the protection scope of the invention.
Claims (7)
1. A method for carrying out plant tissue amplification culture by using a plant tissue culture reactor is characterized by comprising the following steps:
s1, placing cultured plant tissue cells into a seeding tank (1) for automatic culture, and culturing the plant tissue to a length of 7-10 cm, wherein the parameters of the seeding tank (1) are set to 35-37 ℃, the rotating speed is 200-600 rpm/min, the dissolved oxygen is 40-60%, the pH value is 6.2-7.2, the air and oxygen flow is 0.2-0.4L/min and 0.05-0.14L/min;
s2, when the length of the plant tissue is cultured to 7-10 cm, starting a seed tank stirring device (4) to reversely rotate at a high speed, wherein the stirring speed is 1000-3000 rpm/min, and the stirring time is 5-10 min, so that the length of the plant tissue is 1-2 cm, then transferring the plant tissue to a first-stage amplification culture tank (2) through a sterile pipeline for automatic culture, and culturing the plant tissue until the length of the plant tissue is 7-10 cm, wherein the parameters of the first-stage amplification culture tank (2) are set to be 35-37 ℃, the rotation speed is 400-600 rpm/min, the dissolved oxygen content is 40-50%, the pH value is 6.8-7.4, the air and oxygen flow is 0.2-0.4L/min, and the pH value is 0.05-0.14L/min;
s3, after the plant tissue is automatically cultured in the first-stage amplification culture tank (2), starting a shearing device (6), stirring and sinking the plant tissue by a first-stage amplification stirring paddle (9), stirring for 10-15 min at a stirring speed of 100-300 rpm/min to enable a front knife edge (13) on the first-stage amplification shearing paddle (10) to shear the plant tissue, wherein the length of the sheared plant tissue is 1-2 cm, and then the plant tissue enters the second-stage amplification culture tank (3) containing a culture medium liquid through a sterile pipeline to be amplified and cultured into a finished plant;
the plant tissue culture reactor comprises a seeding tank (1), a first-stage amplification culture tank (2) and a second-stage amplification culture tank (3), the seeding tank (1) is communicated with the first-stage amplification culture tank (2) through a sterile pipeline, the first-stage amplification culture tank (2) is communicated with the second-stage amplification culture tank (3) through a sterile pipeline, the top parts of the seeding tank (1), the first-stage amplification culture tank (2) and the second-stage amplification culture tank (3) are respectively provided with a seeding tank stirring device (4), a first-stage amplification stirring device (5) and a second-stage amplification stirring device (7), the bottom of the first-stage amplification culture tank (2) is rotationally connected with a shearing device (6), a primary amplification shearing paddle (10) is arranged on the shearing device (6), and a distance is reserved between the primary amplification shearing paddle (10) and the primary amplification stirring device (5);
the culture medium liquid in the step S3 comprises the following raw materials in percentage by mass: 10-15% of cane sugar and FeSO4 5％～10％、NH4NO310 to 15 percent of lysine, 2 to 5 percent of aspartic acid, 4 to 9 percent of L-methionyl glycinate hydrochloride and 10 to 30 percent of deionized water.
2. The method of claim 1, wherein the primary amplified shearing blade (10) comprises a back side (12) and a front side edge (13), wherein the back side (12) rotates clockwise when the seeding-tank agitation device (4) is rotating forward and the front side edge (13) rotates counterclockwise when the seeding-tank agitation device (4) is rotating backward.
3. The method for plant tissue amplification culture using a plant tissue culture reactor according to claim 2, wherein the back surface (12) is concave and blunt, and the front surface edge (13) is sharp.
4. The method for plant tissue culture amplification according to claim 1, wherein the primary amplification stirring device (5) comprises a primary amplification stirring blade (9) and a primary amplification stirring shaft.
5. The method for plant tissue culture amplification according to claim 1, wherein the secondary amplification stirring device (7) comprises a secondary amplification stirring blade (11) and a secondary amplification stirring shaft.
6. The method according to claim 1, wherein said method comprises culturing plant tissueThe method for carrying out plant tissue amplification culture by using the culture reactor is characterized in that the culture medium liquid in the step S3 comprises the following raw materials in percentage by mass: 12% of sucrose and FeSO4 6％、NH4NO312%, lysine 3%, aspartic acid 14%, L-methionyl glycine hydrochloride 5% and deionized water 28%.
7. The method for plant tissue amplification culture using a plant tissue culture reactor as claimed in claim 1, wherein the plant tissue is derived from celery plants or algae plants.
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