CN116334068A - Plasma mutation breeding method - Google Patents
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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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
- A01H1/08—Methods for producing changes in chromosome number
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- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
The invention discloses a plasma mutation breeding method, which comprises the following steps: firstly, starting a plasma mutation breeding device, and setting disinfection and sterilization parameters, environment humidity parameters and plasma treatment parameters on a control panel; secondly, starting a disinfection and sterilization device to disinfect and sterilize the clean working bin; thirdly, placing the animal or plant sample to be treated on a movable workbench in a clean working bin; fourthly, processing the sample based on the environmental humidity parameter and the plasma processing parameter; and fifthly, taking out the processed sample, starting a disinfection and sterilization device, and disinfecting and sterilizing the clean working bin again for the next use. The invention can improve the mutagenic treatment activity of different samples of plants and animals and meet the mutagenic breeding requirements of different samples.
Description
Technical Field
The invention belongs to the technical field of plasma breeding, and particularly relates to a plasma mutation breeding method.
Background
The breeding of high-quality germplasm resources is one of the core works of the modern biological industry.
As a new mutation breeding technology, the plasma technology not only overcomes the defects of long period, slow progress and difficult obtaining of high-efficiency variant varieties in conventional breeding, but also has more efficiency and cost advantages than genetic engineering, and particularly has more unique advantages for biological cells with complex metabolic networks and non-GMO mutation methods. The plasma is a fourth state of a substance which coexists with the solid state, the liquid state and the gas state of the substance, and is a substance form which is composed mainly of a large number of free electrons and charged ions. Research shows that a large amount of active particles in the plasma can act on different biological samples, and cause improvement of various target properties, and has good application prospect in the field of biotechnology.
Currently, plasma technology has been applied to biological mutation breeding, and various mutation breeding devices have been prepared, such as: patent document 1 (publication number CN 202297604U) discloses a plasma microorganism mutation breeding device, which uses plasma jet generated under the conditions of atmospheric pressure and low voltage to carry out mutation breeding on biological samples, has high uniformity and low temperature, and avoids the phenomenon that the jet temperature is too high to cause death of a large number of microorganism samples; patent document 2 (publication No. CN 203700376U) discloses a normal pressure room temperature plasma mutation breeding device, which adopts a cooling system to cool a plasma generator, and is equipped with a multifunctional control operation panel to realize automatic monitoring and control functions, so that plasma mutation breeding under normal pressure room temperature environment is completed more efficiently; patent document 3 (publication No. CN 207653229U) discloses a plasma plant mutagenesis apparatus that provides a cold plasma plant mutagenesis apparatus that can generate stable high-energy ion energy for plant mutagenesis at atmospheric pressure without a vacuum environment. Although the above patent documents all relate to plasma mutagenesis apparatuses which are all of an open structure, have no special requirement for a sterile environment, and are applicable to microorganisms, plant seeds, and the like in suspension, the use of animal cells, plant calli, and the like, which require a strict sterile environment, is limited; in view of the above problems, patent document 4 (CN 103911279B) and patent document 5 (CN 103981091 a) disclose a device and method for acting on biological materials by plasma and a novel atmospheric pressure room temperature plasma mutagenesis breeding device, respectively, which perform plasma mutagenesis in a clean working chamber without bioactive contaminants, and at least reserve one sterilizing device installation position on the inner cavity or wall of the clean working chamber; ensures the high-efficiency and quick treatment of the plasmas, can automatically disinfect, and ensures that the mutagenesis process is not polluted by mixed bacteria. However, the clean working bin in the above patent document adopts a sterilizing device to realize aseptic operation, and long-time plasma jet treatment on sample materials can cause surface drying, especially the aseptic air flow provided with an air inlet and an air outlet in the clean working bin can accelerate the drying of the sample materials, thereby influencing the treatment effect and the survival ability; the operation treatment space is small, the single sterile treatment capacity is limited, and the requirements of mutagenizing different samples of plants and animals in a sterile environment cannot be met.
Therefore, it is necessary to provide a plasma mutation breeding method which is sterile, can effectively control the mutation environment and can improve the biological activity of different samples of plants and animals under the condition of mutation treatment.
Disclosure of Invention
The invention provides a plasma mutation breeding method aiming at the problems in the prior art, which can effectively preserve moisture, can improve the activity after treatment in the mutation treatment process of different samples of plants and animals, and meets the mutation breeding requirements of the different samples.
The technical scheme of the invention is as follows:
a method of plasma mutagenesis breeding comprising the steps of:
firstly, starting a plasma mutation breeding device, and setting disinfection and sterilization parameters, environment humidity parameters and plasma treatment parameters on a control panel;
secondly, starting a disinfection and sterilization device to disinfect and sterilize the clean working bin;
thirdly, placing the animal or plant sample to be treated on a movable workbench in a clean working bin;
fourthly, processing the sample based on the environmental humidity parameter and the plasma processing parameter;
fifthly, taking out the processed sample, starting a disinfection and sterilization device, and disinfecting and sterilizing the clean working bin again for the next use;
the adopted plasma mutation breeding device comprises a sample processing system, a cooling system, a detection system and a control system, wherein the sample processing system comprises a clean working bin without bioactive pollutants, a plasma generator, a radio frequency power supply module connected with the plasma generator, a movable working platform below the plasma generator and used for carrying samples, and a spraying device for humidification; the cooling system is used for cooling the plasma generator; the detection system comprises a gas flow controller for detecting the flow of working gas for generating plasma jet, a temperature sensor for detecting the temperature for generating plasma jet, a humidity sensor for detecting the humidity in a clean working bin and a position sensor for positioning a movable working platform; the control system comprises an operation panel and a controller, wherein the operation panel and the controller are used for controlling the operation of the mutation breeding device, and the controller is respectively connected with the radio frequency power supply module, the movable working platform, the gas flow controller, the temperature sensor, the humidity sensor, the cooling system and the operation panel.
Preferably, the movable table in the third step is moved in a vertical lifting and/or horizontal sliding manner. Further preferably, the vertical lifting is vertical rotation lifting, the horizontal sliding is horizontal back and forth sliding through a guide rail, and vertical lifting and/or horizontal sliding is controlled by positioning of a position sensor on the movable workbench.
Preferably, in the fourth step, the environmental humidity parameter is realized based on a humidity sensor and a spraying device for humidification. Preferably, the environmental humidity parameter is controlled to be 55% -95%; further preferred environmental humidity parameters are 65% -85%
Preferably, in the fourth step, the plasma processing parameters include that the flow of the working gas is 0-30SLM, the power of the radio frequency power supply is 0-1000W, the plasma processing time is 15s-60min, and the temperature of the plasma jet is controlled below 40 ℃. Further preferably, the flow rate of the working gas is 15-30SLM, the power of the radio frequency power supply is 0-600W, the plasma treatment time is 3-45min, and the temperature of the plasma jet is controlled below 37 ℃.
Preferably, the atomizing nozzle of the spraying device is arranged on the inner wall of the clean working bin or hung on the top surface of the clean working bin.
Preferably, the liquid sprayed by the spraying device is sterile water and/or 75% ethanol. In the second and fifth steps, the sterilizing device is started, and the 75% ethanol spraying device is started, so that the two sterilizing modes are parallel, and the sterile state in the clean working bin is maintained.
Preferably, in the fourth step, the humidity in the clean bin is controlled using sterile water or sterile water plus 75% ethanol.
Preferably, the clean working bin is connected with a sterile air device, and a lighting device and/or a disinfection and sterilization device are arranged in the clean working bin. Further, the lighting device is a fluorescent lamp or an illuminating lamp, and the disinfection and sterilization device is an ultraviolet lamp.
Preferably, in the fourth step, under the action of the controller of the plasma mutation breeding device, animal or plant samples are processed under the set parameter conditions based on the control of the radio frequency power supply module, the movable working platform, the gas flow controller, the temperature sensor, the humidity sensor and the cooling system.
Preferably, the movable working platform is provided with an objective table which accommodates 6000-10000 mm 2 Area capacity of biological sample.
Further preferably, the emission surface area of the corresponding plasma generator above the movable working platform is 6000-10000 mm 2 Is a plasma of (a).
The invention has the following technical effects:
before a sample is subjected to plasma mutagenesis treatment, the method starts the sterilizing device to sterilize, maintains the sterile environment in the bin, controls the spraying device to perform spraying operation through the controller or the parameter setting of the controller based on the humidity sensor, maintains the air humidity of the clean working bin, enables the biological material to maintain the surface humidity under the working environment of the ion jet or the sterile wind and the ion jet, and avoids the influence of the activity of the biological material caused by the humidity problem.
The method is realized by adopting a plasma mutation breeding device, wherein a movable workbench capable of vertically lifting and/or horizontally sliding is arranged in the device, biological materials on the movable workbench can be immediately far away from a plasma generator and/or a sterile air outlet after being processed by vertical lifting and/or horizontal sliding, so that the action intensity and action time of air flow on the materials are reduced, and the drying of the biological materials is reduced.
Drawings
FIG. 1 is a schematic diagram of the steps of a plasma mutagenesis breeding method of the present invention;
FIG. 2 is a schematic diagram of the overall structure of an embodiment of a plasma mutation breeding device used in the method of the invention;
FIG. 3 is a perspective view of one embodiment of a plasma mutagenic breeding apparatus for use in the method of the present invention;
FIG. 4 is a perspective view of another embodiment of a plasma mutagenic breeding apparatus for use in the method of the present invention.
1. A sample processing system; 2. a cooling system; 3. a detection system; 4. a control system; 5. a clean working bin; 6. a plasma generator; 7. a radio frequency power supply module; 8. a plasma jet port; 9. a movable work platform; 10. a spraying device; 11. a gas flow controller; 12. a temperature sensor; 13. a humidity sensor; 14. a position sensor; 15 an operation panel; a 16 controller; 17. a disinfection and sterilization device; 18. a lighting device; 19. an operation room door; 20. a sterile air guide port; 21. an air inlet window; 22. a heat dissipation window; 23. a power interface; 24. a stepping motor; 25. a housing.
Detailed Description
Specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
It should be noted that certain terms are used throughout the description and claims to refer to particular components. Those of skill in the art will understand that a person may refer to the same component by different names. The description and claims do not identify differences in terms of components, but rather differences in terms of the functionality of the components. As used throughout the specification and claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description hereinafter sets forth a preferred embodiment for practicing the invention, but is not intended to limit the scope of the invention, as the description proceeds with reference to the general principles of the description. The scope of the invention is defined by the appended claims.
For the purpose of facilitating an understanding of the embodiments of the invention, reference will now be made to the drawings of several embodiments illustrated in the drawings, and the accompanying drawings are not to be taken as limiting the embodiments of the invention.
A plasma mutation breeding method, as shown in figure 1, comprises the following steps:
firstly, starting a plasma mutation breeding device, and setting disinfection and sterilization parameters, environment humidity parameters and plasma treatment parameters on a control panel;
secondly, starting a disinfection and sterilization device to disinfect and sterilize the clean working bin;
thirdly, placing the animal or plant sample to be treated on a movable workbench in a clean working bin;
fourthly, processing the sample based on the environmental humidity parameter and the plasma processing parameter;
and fifthly, taking out the processed sample, starting a disinfection and sterilization device, and disinfecting and sterilizing the clean working bin again for the next use.
The plasma mutation breeding device is shown in fig. 2, and the plasma technology is used for carrying out mutation treatment on biological materials which need to be strictly sterilized under the condition of normal pressure and room temperature. The plasma mutation breeding device comprises a sample processing system (1), a cooling system (2), a detection system (3) and a control system (4); the sample processing system (1) comprises a clean working bin (5) without bioactive pollutants, a plasma generator (6), a radio frequency power supply module (7) connected with the plasma generator, a movable working platform (9) for carrying samples below the plasma generator, and a spraying device (10) for humidifying; the cooling system (2) is used for cooling the plasma generator; the detection system (3) comprises a gas flow controller (11) for detecting the flow of working gas for generating plasma jet, a temperature sensor (12) for detecting the temperature for generating plasma jet, a humidity sensor (13) for detecting the humidity in a clean working bin and a position sensor (14) for positioning a movable working platform; the control system (4) comprises an operation panel (15) and a controller (16) for controlling the operation of the mutation breeding device, wherein the controller is respectively connected with the radio frequency power supply module (7), the movable working platform (9), the gas flow controller (11), the temperature sensor (12), the humidity sensor (13), the position sensor (14), the cooling system (2) and the operation panel (15).
Because the animal or plant samples treated by the method are biological materials such as fish eggs, plant calluses and the like, the water on the surfaces of the animal or plant samples is more easily taken away in the plasma irradiation treatment process, so that the mutagenesis effect is affected, and even the sample materials are not deactivated due to the action of plasma.
In the third step of the invention, the movable workbench in the third step is vertically lifted and/or horizontally slid, and the animal or plant sample is moved to the position under the plasma generator (6) to be treated.
The vertical lifting is vertical rotation lifting, and the horizontal sliding is horizontal back and forth sliding through a guide rail.
In one embodiment, a movable working platform (9) is arranged in the clean working bin (5), and a biological sample is placed on the upper end of the movable working platform (9). As shown in figure 3, the movable working platform (9) is a vertical lifting movable working platform, and the upper end of the vertical lifting movable working platform is provided with a carrying tray which accommodates 6000-10000 mm 2 An area capacity of biological sample; the emission surface area of the corresponding plasma generator above the movable workbench is 6000-10000 mm 2 Is a plasma of (a); the average temperature of the plasma is 40 ℃ or lower. The carrying disc is supported by the shaft rod, a stepping motor capable of accurately controlling vertical lifting is arranged below the clean working bin (5), and the carrying disc further comprises a motor capable of controlling axial rotation. When the movable working platform (9) is a vertically lifting movable working platform, the shape of the jet nozzle of the plasma generator is circular, so that the highest utilization rate of the plasma jet is ensured.
In one embodiment, the movable working platform (9), as shown in FIG. 4, is a horizontally sliding movable working platform, which accommodates 6000-8000 mm 2 An area capacity of biological sample; the emission surface area of the corresponding plasma generator above the movable workbench is 6000-8000 mm 2 Is a plasma of (a); the average temperature of the plasma is 40 ℃ or lower. The horizontal sliding movable workbench horizontally slides back and forth through a stepping motor (24), and the highest utilization rate of plasma jet flow is guaranteed.
In one embodiment, vertical lift and/or horizontal slide is controlled by a position sensor positioning on the movable table.
In one embodiment, in the fourth step, the humidity of the working environment during plasma treatment is controlled, and in the third step, the humidity is controlled to be 55% -95% by a humidity sensor and a spraying device according to set humidity parameters. Further controlling the content to 65-85%.
The method is characterized in that a humidity sensor (13) and a spraying device (10) are arranged in a clean working bin (5) of a plasma mutation breeding device, and humidity parameters in the clean working bin (5) are fed back to a controller (16) through the humidity sensor (13) to realize humidity control in the clean working bin (5).
In a specific embodiment, the spraying device (10) is a miniature spraying device, and comprises an atomizer, a liquid adding pipeline and a control unit. The liquid adding pipeline can be one or more, and sterile water and/or 75% ethanol are added. When 75% ethanol is in the liquid pipeline, in the operation of the second step and the fifth step, the sterilizing device is started, and the sterilizing device is started, so that the two sterilizing modes are parallel, and the sterile state in the clean working bin is maintained.
The number of atomizers is not limited and may be one or more. In the case of one atomizer, a plurality of atomizer heads may be provided. In a specific embodiment, the atomizing nozzle is an ultrasonic atomizing nozzle for atomizing water into mist by ultrasonic waves. The atomizing time and the water mist generation amount are automatically controlled by a control unit and a controller (16). In one implementation, the ultrasonic atomizing nozzle is arranged on the inner wall of the clean working cabin or hung on the cabin top surface of the clean working cabin (5). The atomizing nozzle sprays water mist in the clean working bin (5) to enable the water mist to be ultrafine particles to float in the clean working bin (5), so that the air humidity is improved. The control system controls the humidity in the clean working bin to be 55% -95% through controlling the humidity sensor and the humidifying module; further preferably 65% -85%.
In one embodiment, in the third step, the humidity in the clean working bin is controlled by using sterile water or sterile water plus 75% ethanol, wherein the humidity in the clean working bin is detected by a humidity sensor, and the sterile water spray and the spraying amount thereof are selected according to the requirement.
In an embodiment, in the fourth step, the plasma processing parameters include that the flow of the working gas is 0-30SLM, the power of the radio frequency power supply is 0-1000W, the plasma processing time is 15s-60min, and the plasma jet temperature is controlled below 40 ℃. It is further preferable that the flow rate of the working gas is 15-30SLM, the power of the radio frequency power supply is 0-600W, the plasma treatment time is 3-45min, and the temperature of the plasma jet is controlled below 37 ℃.
In one embodiment, the clean working cabin (5) is internally provided with a lighting device and/or a disinfection and sterilization device. The disinfection and sterilization device is any one of an ultraviolet disinfection device and a chemical disinfection device. In a specific embodiment, the disinfection and sterilization devices (17) are ultraviolet lamps, more than 2 disinfection and sterilization devices are respectively arranged on the inner side walls of the clean working bin (5). The lighting device (18) may be a lighting device that provides a good lighting function, such as a lighting lamp. The lighting device and/or the disinfection and sterilization device can realize the control of the switch and the time on the operation panel (15) through the controller (16). In the operation process, the clean working bin (5) is sterilized in advance, ventilation is carried out after sterilization is finished, and then biological materials are put into the clean working bin for plasma irradiation treatment. The cleaning grade in the cleaning work bin (5) reaches 10 ten thousand grades.
In one embodiment, as shown in fig. 3 and 4, the plasma mutation breeding device adopted by the invention comprises a shell (25), wherein the left side of the shell is a clean working bin (5), and the right side of the shell is an operation panel (15). An air inlet window (21) of the sterile air device is arranged on a shell (25) above the clean working bin (5), a fan is connected with the clean working bin (5) through an air duct, and air entering from the air inlet window (21) is processed through a filter and blown in from the top or the back of the sterile working bin (5). The operation panel (15) is arranged on the outer surface of the shell (25), and the power interface (23) is arranged on the outer side wall of the shell (25), and more preferably on the left outer side wall. A heat dissipation window (22) is arranged on the right outer side wall of the shell (25), so that heat dissipation of the radio frequency power supply module (7), the controller (16) and the spraying device (10) is facilitated.
The plasma generator (6) of the plasma mutation breeding device is connected with the radio frequency power supply module (7), the working gas pipeline and the cooling system (2) to generate normal-pressure room-temperature plasma for mutation breeding. One end of a jet port of the plasma generator (6), namely a plasma jet port (8), a movable working platform (9) and a spraying device (10), is arranged in the clean working bin (5); further, the plasma generator (6) is arranged at the top end of the clean working bin (5), the movable working platform (9) is supported by the bottom surface of the clean working bin (5) and is arranged in the clean working bin (5), and the spraying device (10) is arranged at a mounting position reserved in the inner cavity or the wall of the clean working bin (5).
According to the invention, the cooling system (2) is a water cooling system or an air cooling system, and is connected with the plasma generator through a pipeline to realize internal circulation cooling.
In one embodiment, a gas flow controller (11) is connected with the plasma generator (6) and feeds back gas flow information to a controller (16) to control the flow of the working gas of the plasma generator on line in real time. The temperature sensor (12) is used for detecting the temperature of the cooling system and feeding back temperature information to the controller (16) for temperature centralized control and real-time monitoring. The humidity sensor (13) is arranged on the inner cavity or the wall of the clean working bin (5) and is used for detecting the humidity in the clean working bin (5) and feeding back humidity information to the controller (15) so that the controller can realize on-line control of the humidity through the spraying device (10).
In one embodiment, the operating panel (15) sets processing parameters such as gas flow, radio frequency power supply, cooling system temperature, plasma processing time, humidity in the clean room, etc., and the controller controls the emission of the plasma generator (6) and its processing of the biological sample based on the processing parameter settings and signals from the gas flow controller (11), radio frequency power supply module (7) power sensor, temperature sensor (12), humidity sensor (13), etc.
As shown in fig. 2 and 3, the plasma mutation breeding device used in the present invention ensures the working environment of the clean working cabin (5) by a sterile wind device and a disinfection and sterilization device.
In one embodiment, the clean working bin (5) is connected with a sterile wind device, and the sterile wind system at least comprises a fan for generating sterile wind, a filter element and a pumping unit. Besides the operation room door (19), the other three surfaces of the clean operation room (5) are provided with the baffle plates which are connected with each other, and the baffle plates above the clean operation room (5) or the baffle plates of the back of the body, which are opposite to the operation table, are provided with the sterile air guide ports (20), so that the sterile air enters the clean operation room (5) through the sterile air guide ports (20).
In one embodiment, the operation room door (19) is a door which is opened and closed by pushing and pulling up and down, and the operation room door (19) comprises a window which is provided with a transparent and convenient for observation.
In one embodiment, the vertical lift movable stage and the horizontal slide movable stage are each controllable by a controller (16) to position the load tray or stage by a stepper motor (24) based on a position sensor (14) signal.
Before a sample is subjected to plasma mutagenesis treatment, the method starts the sterilizing device to sterilize, maintains the sterile environment in the bin, controls the spraying device to perform spraying operation through the controller or the parameter setting of the controller based on the humidity sensor, maintains the air humidity of the clean working bin, enables the biological material to maintain the surface humidity under the working environment of the ion jet or the sterile wind and the ion jet, and avoids the influence of the activity of the biological material caused by the humidity problem.
The method is realized by adopting a plasma mutation breeding device, wherein a movable workbench capable of vertically lifting and/or horizontally sliding is arranged in the device, biological materials on the movable workbench can be immediately far away from a plasma generator and/or a sterile air outlet after being processed by vertical lifting and/or horizontal sliding, so that the action intensity and action time of air flow on the materials are reduced, and the drying of the biological materials is reduced.
Examples
The process according to the invention is further illustrated below with reference to examples.
Example 1
In an embodiment, the crucian fertilized egg is processed using a plasma mutation breeding device as shown in fig. 3. The method comprises the following specific steps:
(1) Selecting active healthy male and female crucian with weight of more than 300g without diseases as a parent, and carrying out artificial insemination by using oxytocin to prepare fertilized eggs;
(2) The first, second and third steps are operated according to the invention;
(3) Fertilized eggs were divided into 12 parts (80 fertilized eggs per part), and plasma jet irradiation was performed in batches according to the fourth step, and the treatment conditions were:
control group: 6 fertilized eggs, without any treatment, were set to have humidity of 45%, 55%, 65%, 75%, 85%, 95% in the clean working bin.
Treatment group: 6 fertilized eggs, helium as working gas, 15 SLM as working gas flow, 400W as radio frequency power supply, 15 min as treatment time, 28-30deg.C as plasma jet temperature controlled by a water cooling system, 45%, 55%, 65%, 75%, 85% and 95% as humidity in the set clean working bin.
(4) And a fifth step of incubating the irradiated fertilized eggs.
The results show that: the humidity in the clean working bin reaches 95%, the plasma generator still discharges normally, but water drops appear on the wall of the bin; in the control group, the humidity is 45 percent and 55 percent, the surface of fertilized eggs slightly lose gloss, the emergence rate of crucian is slightly lower than that of the control group with the humidity of 55 percent to 95 percent, but the control group is not obvious; in the treatment group, the surface of the fertilized eggs is slightly dehydrated and atrophic, the humidity is 55%, the surface of the fertilized eggs is tarnished, and the emergence rate of the treatment group with the humidity of 45% and the 55% after hatching is obviously lower than that of the treatment group with the humidity of 65-95%; the emergence rates of 85% of the treatment groups and 95% of the treatment groups were not very good; and the emergence rate of fertilized eggs under the same humidity condition is lower than that of a control group.
Example 2
In an example, maize callus was treated using the plasma mutation breeding device shown in fig. 4. The method comprises the following specific steps:
(1) Selecting 360 calli with almost the same shape and size;
(2) The first, second and third steps according to the invention;
(3) Corn callus was divided into 12 parts (30 per part) and plasma jet irradiated in batches according to the fourth step described above, treatment conditions:
control group: 6 parts of corn callus, without any treatment, the humidity in the set clean working bin is 45%, 55%, 65%, 75%, 85% and 95% in sequence.
Treatment group: 6 parts of corn callus, helium as working gas, 20 SLM as working gas flow, 400W as radio frequency power supply, 30min as treatment time, 28-30deg.C as plasma jet temperature controlled by a water cooling system, and 45%, 55%, 65%, 75%, 85% and 95% of humidity in a set clean working bin.
(4) The fifth step was performed and the culture was further induced after the treatment.
The result shows that the humidity in the clean working bin reaches 95%, the plasma generator still discharges normally, but water drops appear on the bin wall; in the control group, the humidity is 45%, the surfaces of the corn calli slightly lose gloss, and the corn calli recover after 2 days of induction culture; in the treatment groups, the humidity of 45% and 55% of the treatment groups is slightly dehydrated, the surface of the corn callus loses gloss, after 2 days of induction culture, the browning of the humidity of 45% and 55% of the treatment groups is obviously higher than that of 65-95% of the control group, and the browning of the callus of 75% of the treatment groups is not much higher than that of 85% and 95% of the treatment groups; corn calli under the same humidity conditions, the browning of the treated group was higher than that of the control group.
Claims (10)
1. A method of plasma mutagenesis breeding comprising the steps of:
firstly, starting a plasma mutation breeding device, and setting disinfection and sterilization parameters, environment humidity parameters and plasma treatment parameters on a control panel;
secondly, starting a disinfection and sterilization device to disinfect and sterilize the clean working bin;
thirdly, placing the animal or plant sample to be treated on a movable workbench in a clean working bin;
fourthly, processing the sample based on the environmental humidity parameter and the plasma processing parameter;
fifthly, taking out the processed sample, starting a disinfection and sterilization device, and disinfecting and sterilizing the clean working bin again for the next use;
the adopted plasma mutation breeding device comprises a sample processing system, a cooling system, a detection system and a control system, wherein the sample processing system comprises a clean working bin without bioactive pollutants, a plasma generator, a radio frequency power supply module connected with the plasma generator, a movable working platform below the plasma generator and used for carrying samples, and a spraying device for humidification; the cooling system is used for cooling the plasma generator; the detection system comprises a gas flow controller for detecting the flow of working gas for generating plasma jet, a temperature sensor for detecting the temperature for generating plasma jet, a humidity sensor for detecting the humidity in a clean working bin and a position sensor for positioning a movable working platform; the control system comprises an operation panel and a controller, wherein the operation panel and the controller are used for controlling the operation of the mutation breeding device, and the controller is respectively connected with the radio frequency power supply module, the movable working platform, the gas flow controller, the temperature sensor, the humidity sensor, the cooling system and the operation panel.
2. The method of claim 1, wherein the movable stage in the third step is moved in a manner of vertical lifting and/or horizontal sliding.
3. The method of claim 2, wherein the vertical lift is a vertical rotary lift, the horizontal slide is a horizontal back and forth slide via a rail, and vertical lift and/or horizontal slide are controlled by a position sensor on the movable stage.
4. The method of claim 1, wherein in the fourth step, the environmental humidity parameter is realized based on a humidity sensor and a humidifying spraying device.
5. The method of claim 4, wherein the environmental humidity parameter is controlled to be 55% -95%.
6. The method of claim 5, wherein the environmental humidity parameter is 65% -85%.
7. The method of claim 1, wherein in the fourth step, the plasma processing parameters include a working gas flow rate of 0-30SLM, a power of a radio frequency power source of 0-1000W, a plasma processing time of 15s-60min, and a plasma jet temperature of 40 ℃ or lower.
8. The method of claim 1, wherein the atomizing nozzle of the atomizer is disposed on the inner wall of the clean room or hung on the top surface of the clean room.
9. The method of claim 8, wherein the liquid sprayed by the spraying device is sterile water and/or 75% ethanol.
10. The plasma mutation breeding method according to claim 1, wherein the clean working bin is connected with a sterile wind device, and a lighting device and/or a disinfection and sterilization device are arranged in the clean working bin.
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