Agrobacterium infected plant callus device

Abstract

The utility model belongs to the technical field of biology, and discloses an agrobacterium infected plant callus device which comprises an infection bottle body, the top of the infection bottle body is in threaded connection with an infection bottle cap, a through hole is formed in the center of the top of the infection bottle cap, a breathable film is arranged at the position of the through hole, and a containing cylinder is integrally formed in the center of the bottom of the infection bottle cap. According to the device, agrobacterium tumefaciens-mediated genetic transformation infection is simply and quickly realized, the problem of damage caused by repeated movement of calluses in the conventional agrobacterium tumefaciens-mediated genetic transformation infection process is solved, the problem of waste caused by easy discarding of the calluses after infection is also solved, the operation process is reduced, and the cost is reduced. The method has the advantages that the method is simple and convenient to operate, the consistency of the infection time of the calluses can be guaranteed, the agrobacterium-mediated genetic transformation is simpler and more convenient, the movement of the calluses in the operation process is reduced, the pollution possibility is reduced, and meanwhile, the method is suitable for large-scale infection of the calluses and has wide applicability.

Description

Technical Field

[0001] The utility model relates to the field of biotechnology, in particular to a device for infecting plant callus with Agrobacterium. Background Art

[0002] Agrobacterium-mediated genetic transformation technology is currently the most commonly used transgenic method, with the advantages of simple operation, low cost, efficient transformation, and genetic stability. Infection is a very critical step in genetic transformation. During conventional infection, the callus tissue is placed in a triangular pyramidal flask, and then the suspension is poured in for infection for a period of time. After the infection is completed, the infection solution is poured out, and then the callus tissue is transferred to sterile filter paper with tweezers, the bacterial solution on the surface of the callus tissue is blotted dry, and the bacterial solution on the surface of the callus tissue is briefly blown dry in the clean bench, and then inoculated into the co-culture medium for a period of time. After the co-culture is completed, sterilization treatment is carried out, and the callus tissue is placed in a triangular pyramidal flask again, washed several times with sterile water added with antibiotics, or soaked in a suspension medium added with antibiotics for a period of time to achieve an antibacterial effect, and then the washing solution is poured out, and the callus tissue is transferred to sterile filter paper with tweezers to absorb the surface moisture. All operations are performed in a clean bench.

[0003] The main disadvantages are: 1. During the infection process and the co-culture sterilization process, the callus tissue is moved many times with tweezers, which is easy to damage the callus tissue cells; 2. When the waste liquid is poured out after the infection is completed and during the co-culture sterilization and cleaning process, the callus tissue is easily poured out of the vessel, resulting in waste of experimental materials; 3. After the infection is completed and after the co-culture sterilization, the callus tissue needs to be transferred from the triangular flask to sterile filter paper and blown dry on an ultra-clean workbench. The transfer operation process is cumbersome and increases the possibility of contamination. Utility Model Content

[0004] In view of the above problems in the prior art, the utility model designs an Agrobacterium-infected plant callus device, which can process a large amount of callus tissue at one time, avoiding the damage caused by using tweezers to move the callus tissue multiple times during the operation, and avoiding the waste of materials and the increase of the probability of pollution.

[0005] The utility model is achieved in this way:

[0006] A device for infecting plant callus with Agrobacterium comprises an infection bottle body, the top of the infection bottle body is threadedly connected with an infection bottle cap, a through hole is opened in the center of the top of the infection bottle cap, a breathable membrane is arranged at the through hole, a containing cylinder is integrally formed in the center of the bottom of the infection bottle cap, the containing cylinder is located in the upper part of the infection bottle body, the bottom of the containing cylinder is threadedly connected with the opening part at the top of the infection cylinder, the bottom of the containing cylinder is clamped with the bottom of the inner wall of the infection bottle body, a bacterial liquid discharge outlet is arranged on one side of the bottom of the infection bottle body, and a manual valve is arranged at the bacterial liquid discharge outlet.

[0007] In one embodiment of the utility model, a circle of annular protrusions is arranged on the top and near the periphery of the infection bottle cap, and filter paper is sandwiched between the annular protrusions and the top surface of the infection bottle cap.

[0008] In an embodiment of the utility model, a handle is provided on one side of the surrounding surface of the infection bottle body, and anti-slip patterns are provided on the surrounding surface of the infection bottle cap.

[0009] In one embodiment of the utility model, the upper part of the infection cylinder is surrounded by stainless steel plates, the middle part is surrounded by stainless steel filter screens, and the bottom is a circular stainless steel plate.

[0010] In an embodiment of the utility model, a circle of clamping grooves is arranged in the center of the bottom of the inner wall of the infection bottle, and the edge of the circular stainless steel plate at the bottom of the infection cylinder is clamped with the clamping grooves.

[0011] Compared with the prior art, the beneficial effects of the utility model are:

[0012] The device realizes Agrobacterium-mediated genetic transformation infection simply and quickly, solves the problem of damage caused by multiple callsus movement during conventional Agrobacterium-mediated genetic transformation infection, and solves the problem of waste caused by discarding the callus after the infection is completed. It reduces the operation process and can ensure the consistency of callus infection time, making Agrobacterium-mediated genetic transformation simpler and more convenient. The movement of callus is reduced during the operation, reducing the possibility of contamination. It is also suitable for large-scale callus infection. The Agrobacterium-infected plant callus device can also be used for the infection of other plant explants and has a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solution of the implementation mode of the utility model, the drawings required for use in the implementation mode will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the utility model and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying creative work.

[0014] Figure 1 It is a schematic diagram of the overall structure of the utility model;

[0015] Figure 2 It is a schematic diagram of the structure of the infected bottle cap;

[0016] Figure 3 It is a schematic diagram of the structure of the infection bottle;

[0017] Figure 4 It is a schematic diagram of the structure of the infection tube.

[0018] Explanation of the accompanying drawings: 1. infection bottle cap; 2. through hole; 3. breathable membrane; 4. annular protrusion; 5. accommodating cylinder; 6. infection cylinder; 7. infection bottle body; 8. slot; 9. bacterial liquid discharge port; 10. handle. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of the implementation of the utility model clearer, the technical solution in the implementation of the utility model will be clearly and completely described below in conjunction with the drawings in the implementation of the utility model. Obviously, the described implementation is a part of the implementation of the utility model, not all of the implementations. Based on the implementation of the utility model, all other implementations obtained by ordinary technicians in this field without creative work are within the scope of protection of the utility model.

[0020] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0021] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0022] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present invention.

[0023] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present utility model, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0024] Example

[0025] See attached Figure 1-4 As shown, the utility model provides an Agrobacterium-infected plant callus device, including an infection bottle body 7, the top of the infection bottle body 7 is threadedly connected with an infection bottle cap 1, a through hole 2 is opened in the center of the top of the infection bottle cap 1, a breathable membrane 3 is arranged at the through hole 2, a accommodating cylinder 5 is integrally formed in the center of the bottom of the infection bottle cap 1, the accommodating cylinder 5 is located in the upper part of the infection bottle body 7, the bottom of the accommodating cylinder 5 is threadedly connected with the opening part of the top of the infection cylinder 6, the bottom of the accommodating cylinder 5 is clamped with the bottom of the inner wall of the infection bottle body 7, a bacteria liquid discharge outlet 9 is arranged on one side of the bottom of the infection bottle body 7, and a manual valve is arranged at the bacteria liquid discharge outlet 9.

[0026] In an embodiment of the utility model, further, a circle of annular protrusions 4 are arranged on the top and near the periphery of the infection bottle cap 1 , and filter paper is sandwiched between the annular protrusions 4 and the top surface of the infection bottle cap 1 .

[0027] In an embodiment of the present invention, further, a handle 10 is provided on one side of the surrounding surface of the infection bottle body 7, and anti-slip patterns are provided on the surrounding surface of the infection bottle cap 1.

[0028] In an embodiment of the present invention, further, the upper part of the infection cylinder 6 is surrounded by stainless steel plates, the middle part is surrounded by stainless steel filter screens, and the bottom is a circular stainless steel plate.

[0029] In an embodiment of the present utility model, further, a circle of clamping grooves 8 are provided at the center of the bottom of the inner wall of the infection bottle body 7 , and the edge of the circular stainless steel plate at the bottom of the infection tube 6 is clamped with the clamping grooves 8 .

[0030] Specifically, the infection bottle cap 1 and the accommodating cylinder 5 integrally formed at the bottom thereof are supported by PP material, the infection bottle body 7 is made of high-temperature transparent glass (high white glass), and the infection cylinder 6 is made of a stainless steel filter screen and a stainless steel plate welded together, and all parts can be sterilized by high temperature and high pressure;

[0031] During use, the callus tissue is placed in the infection tube 6 and its opening is threadedly connected to the bottom of the accommodating tube 5 at the bottom of the infection bottle cap 1, and then the whole is placed in the infection bottle body 7. The Agrobacterium infection liquid is poured into the infection bottle body 7 along the edge in advance, and the Agrobacterium infection liquid enters through the stainless steel mesh of the infection tube 6 to soak the callus tissue. The infection bottle cap 1 is threadedly connected and tightly closed with the top of the infection bottle body 7. The bottom of the infection tube 6 is engaged with the card groove 8 set at the bottom of the inner wall of the infection bottle body 7 to increase the overall stability of the device. The air permeable membrane 3 is set at the through hole 2 on the top of the infection bottle cap 1 to increase the air permeability in the device, and bacteria and dust can be isolated. After the soaking is completed, the Agrobacterium infection liquid can be discharged through the bacterial liquid discharge port 9 set at the bottom of one side of the infection bottle body 7, which is convenient and quick, and effectively prevents the callus tissue inside the device from being led out to cause operation accidents and waste of materials.

[0032] In addition, by providing a circle of annular protrusions 4 around the top of the infection bottle cap 1 and by clamping a sterile filter paper in the annular protrusion 4, after the callus tissue is soaked, by inverting the device, the callus tissue can smoothly enter the containing tube 5 without temporary storage, so that the Agrobacterium infection liquid remaining on the surface of the callus tissue can be absorbed by the sterile filter paper through the breathable membrane 3, and there is no need to clamp the callus tissue multiple times with tweezers, which will increase the risk of tissue damage and infection.

[0033] In summary, the device realizes Agrobacterium-mediated genetic transformation infection simply and quickly, solves the damage problem caused by multiple callsus movement during conventional Agrobacterium-mediated genetic transformation infection, and solves the problem of waste caused by discarding the callus after the infection is completed. It reduces the operation process and can ensure the consistency of callus infection time, making Agrobacterium-mediated genetic transformation simpler and more convenient. The movement of callus is reduced during the operation, reducing the possibility of contamination. At the same time, it is also suitable for large-scale callus infection. The Agrobacterium-infected plant callus device can also be used for the infection of other plant explants and has a wide range of applications.

[0034] In the embodiment of the utility model, the infection bottle body 7 is 120±0.1mm high, the bottom diameter of the infection bottle body 7 is 140±0.1mm, the infection bottle cap 1 has a diameter of 132±0.1mm, the infection bottle cap 1 has an outer diameter of 134±0.1mm, an inner diameter of 130±0.1mm, and a height of 10±0.1mm. The through hole 2 has a diameter of 50±0.1mm, the air permeable membrane 3 has a diameter of 49±0.1mm, and a pore size of 0.22μm. The annular protrusion 4 is 3±0.1mm wide, and the distance between the annular protrusion 4 and the infection bottle cap 1 is 6±0.1mm. The accommodating tube 5 has a diameter of 102±0.1mm and a height of 30±0.1mm. The infection tube 6 has a diameter of 100±0.1mm and a height of 84±0. 1mm, including an upper stainless steel plate with a height of 10±0.1mm, a middle stainless steel filter with a height of 74±0.1mm, a stainless steel filter with a pore size of 3±0.1mm, a bottom slot 8 with a height of 2+0.1mm, a distance from the infection bottle body 7 of 19+0.1mm, a width of 1+0.1mm, a bacterial liquid discharge outlet 9 with a diameter of 8+0.1mm, a distance from the bottom of 2±0.1mm, a handle 10 on the outside of the infection bottle body 7 with a thickness of 8±0.1mm, a height of 70±0.1mm, and an inner diameter of 50±0.1mm from the infection bottle body 7, the thread height involved in the embodiments is 9+0.1mm, the bottle cap thickness is 1+0.1mm, and the bottle bottom thickness is 5+0.1mm.

[0035] The working principle of the device for infecting plant callus with Agrobacterium:

[0036] 1. Sterilize the entire device in a high-pressure and high-temperature autoclave, take it out and open it in a clean bench;

[0037] 2. Take several sterile filter papers and place them inside the annular protrusion 4, place the callus tissue in the infection tube 6, and pour the Agrobacterium infection solution from the edge of the infection bottle 7;

[0038] 3. Screw the infection bottle cap 1 and the infection bottle body 7 together through threads, and at the same time screw the containing tube 5 and the infection tube 6 together through threads to start infection;

[0039] 4. After the infection is completed, open the bacterial liquid discharge port 9 to discharge the bacterial liquid. If there is any residual bacterial liquid, the whole device can be slightly tilted to facilitate the discharge of the residual bacterial liquid;

[0040] 5. Invert the whole set of equipment so that the callus tissue can be transferred into the containing tube 5. Use the sterile filter paper placed between the annular protrusion 4 and the infection bottle cap 11 to absorb the bacterial liquid on the surface of the callus tissue. Unscrew the infection bottle cap 1, take it out together with the containing tube 5 and the infection tube 6, and blow it with sterile air for 3-8 minutes to fully remove the Agrobacterium infection liquid.

[0041] 6. Transfer the callus to the co-culture medium and culture in the dark for 2-3 days;

[0042] 7. After the co-cultivation is completed, use the device to wash or soak;

[0043] 8. After washing or soaking, dry the water and blow dry in the same way as step 5;

[0044] 9. Place the callus tissue in screening medium for screening.

[0045] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may be modified and varied in various ways. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A device for infecting plant callus with Agrobacterium, characterized in that: The invention comprises an infection bottle body (7), the top of the infection bottle body (7) is threadedly connected with an infection bottle cap (1), a through hole (2) is provided at the center of the top of the infection bottle cap (1), a breathable membrane (3) is provided at the through hole (2), a receiving tube (5) is integrally formed at the center of the bottom of the infection bottle cap (1), the receiving tube (5) is located at the upper part of the infection bottle body (7), the bottom of the receiving tube (5) is threadedly connected with the opening at the top of the infection tube (6), the bottom of the receiving tube (5) is clamped with the bottom of the inner wall of the infection bottle body (7), a bacterial liquid discharge port (9) is provided at one side of the bottom of the infection bottle body (7), and a manual valve is provided at the bacterial liquid discharge port (9).

2. The device for infecting plant callus with Agrobacterium according to claim 1, characterized in that: A circle of annular protrusions (4) are arranged on the top and near the periphery of the infection bottle cap (1), and filter paper is sandwiched between the annular protrusions (4) and the top surface of the infection bottle cap (1).

3. The device for infecting plant callus with Agrobacterium according to claim 2, characterized in that: A handle (10) is provided on one side of the surrounding surface of the infection bottle body (7), and anti-slip patterns are provided on the surrounding surface of the infection bottle cap (1).

4. The device for infecting plant callus with Agrobacterium according to claim 3, characterized in that: The infection cylinder (6) is surrounded by stainless steel plates on the top, stainless steel filter screens on the middle, and a round stainless steel plate on the bottom.

5. The device for infecting plant callus with Agrobacterium according to claim 4, characterized in that: A circle of clamping grooves (8) is arranged at the center of the bottom of the inner wall of the infection bottle body (7), and the edge of the circular stainless steel plate at the bottom of the infection cylinder (6) is clamped with the clamping grooves (8).

Images