Culture mold and its application in tissue culture

Abstract

The application relates to a culture mold and application thereof in tissue culture. The culture mold comprises a lower culture dish comprising a first lower bottom wall and a first side wall connected with the first lower bottom wall; and an upper culture dish comprising a second lower bottom wall, a second side wall and an upper top cover, the second lower bottom wall comprises a porous material layer, at least part of the porous material layer is porous material, the second lower bottom wall and the second side wall are connected, and the upper top cover is adjustably connected with the second side wall; wherein the upper culture dish is adapted to be arranged on the top of the lower culture dish, and the upper culture dish is adapted to be sealingly connected with the lower culture dish. The culture mold has the advantages of short operation period, high efficiency, effectively reduced tissue pollution, reduced culture cost and the like in culture of tissues.

Description

Technical Field

[0001] This application belongs to the field of biotechnology. Specifically, this application relates to culture molds and their application in tissue culture. More specifically, this application relates to culture molds and tissue culture methods. Background Technology

[0002] Plant genetic transformation and gene editing, as key technologies in the field of bioscience, are of great significance for improving plant characteristics, increasing crop yields, and addressing environmental challenges. However, these technologies have faced many challenges in their long-term development, such as long cycles, low efficiency, and high pollution rates. These challenges mainly stem from the dependence on explant culture and subculture operations, which have historically been primarily performed manually.

[0003] In traditional explant culture and subculture, manually picking up and transferring explants to the culture medium is a repetitive and time-consuming task. This process needs to be repeated multiple times throughout the entire genetic transformation and gene editing process, requiring precise manual operation each time. Especially in the subculture stage, the use of forceps to pick up and transfer explants to fresh culture medium is a crucial step, but it is also the most time-consuming and contamination-prone part of the entire process. This manual operation leads to serious problems such as long operation cycles, low efficiency, and high contamination rates, hindering the further development and application of plant genetic transformation and gene editing.

[0004] Therefore, current methods for subculturing explants still need improvement. Summary of the Invention

[0005] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application proposes a culture mold that effectively reduces the manual operation cycle of tissue culture, improves operational efficiency, and reduces contamination rates.

[0006] In a first aspect, this application proposes a culture mold. According to an embodiment of this application, the culture mold includes: a lower culture dish comprising a first lower bottom wall and a first side wall connected together; and an upper culture dish comprising a second lower bottom wall, a second side wall, and a top cover, wherein the second lower bottom wall comprises a porous material layer, at least a portion of which is a porous material; the second lower bottom wall and the second side wall are connected together; and the top cover is adjustablely connected to the second side wall; wherein the upper culture dish is adapted to be disposed on top of the lower culture dish, and the upper culture dish is adapted to be sealed to the lower culture dish. In some examples of this application, the culture mold has advantages such as short operation cycle, high efficiency, effective reduction of tissue contamination, and reduced culture costs in tissue culture.

[0007] In a second aspect, this application proposes a tissue culture method. According to embodiments of this application, the tissue to be cultured is cultured under suitable conditions for tissue growth using the culture mold described in the first aspect of this application; wherein the tissue to be cultured is disposed on the upper surface of the second lower bottom wall of the upper culture dish of the culture mold, the lower culture dish of the culture mold contains tissue culture medium, and the upper culture dish is disposed on top of the lower culture dish. In some examples of this application, using this method for tissue culture can significantly shorten the operation cycle, improve operation efficiency, effectively reduce tissue contamination, and lower tissue culture costs, among other advantages.

[0008] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0009] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0010] Figure 1 This is a schematic diagram of the lower culture dish structure of the culture mold according to one embodiment of this application;

[0011] Figure 2 This is a schematic diagram of the upper culture dish (including a second bottom wall, a second side wall, and a support layer) of the culture mold according to one embodiment of this application;

[0012] Figure 3 This is a schematic diagram of the top cover structure of the culture mold according to one embodiment of this application;

[0013] Figure 4 This is a schematic diagram of the sealing ring structure of the culture mold according to one embodiment of this application;

[0014] Figure 5 This is a schematic diagram of the culture mold structure according to an embodiment of this application; wherein, A is a structural diagram of the top cover of the upper culture dish; B is a structural diagram of the upper culture dish (including the second bottom wall, the second side wall and the support layer); C is a structural diagram of the sealing ring; D is a structural diagram of the lower culture dish; E is a structural diagram of the main body of the culture mold;

[0015] Figure 6This diagram illustrates a comparison of the state of Arabidopsis callus subcultured using a conventional method (ordinary petri dish) and the method of this application (culture mold) according to one embodiment of this application. A shows the callus just placed in an ordinary petri dish containing culture medium; B shows the callus just placed in a culture mold containing culture medium; C shows the callus after 56 days of subculture in an ordinary petri dish; D shows the callus after 56 days of subculture in a culture mold; E shows the callus after 145 days of subculture in an ordinary petri dish; F shows the callus after 145 days of subculture in a culture mold.

[0016] Figure 7 This is a schematic diagram showing the comparison of manual operation time for subculturing Arabidopsis callus using the method described in this application and conventional methods, according to one embodiment of this application.

[0017] Figure label:

[0018] 101: First lower bottom wall; 102: First side wall; 103: Second lower bottom wall; 104: Second side wall; 105: Top cover; 106: Sealing ring; 107: Support layer. Detailed Implementation

[0019] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0020] In this document, unless otherwise specified, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0021] Unless otherwise specified herein, the terms "comprising" or "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0022] Unless otherwise specified herein, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0023] Unless otherwise specified herein, the term "explant" refers to a portion of tissue or cells taken from a plant that, under appropriate culture conditions, is capable of cell differentiation and regeneration, and has the potential to form a new plant. In some examples of this application, explants are commonly used in plant tissue culture, regeneration, and plant genetic improvement processes, serving as starting material for plant genetic transformation, gene editing, and propagation.

[0024] Traditional explant culture subculturing typically involves manual handling under aseptic conditions. Explants are individually picked up with forceps and placed into culture dishes containing solid culture medium. The dishes are then sealed and incubated for two weeks. Afterward, the dishes are removed and placed in a clean bench under aseptic conditions. The membrane is removed, and the explants are transferred from the old dishes to new ones (subculturing). This process is repeated 5-7 times throughout the genetic transformation and gene editing process. Traditional methods are inefficient and cumbersome because explants must be picked up one by one, and the culture dishes need to be sealed and unsealed repeatedly. Furthermore, the manual operation is time-consuming, labor-intensive, and prone to introducing contaminants.

[0025] To overcome the problems faced by traditional methods, this application proposes a culture mold. This culture mold is used for the overall subculture transfer of batches of explants. This culture mold greatly improves the subculture efficiency of explants, reduces human intervention, and is adaptable to automated equipment, making it widely applicable in the fields of plant genetic transformation and gene editing.

[0026] In some examples of this application, the culture mold includes:

[0027] Lower petri dish ( Figure 1 The lower culture dish includes a first lower bottom wall (101) and a first side wall (102), which are connected. In some examples of this application, the lower culture dish is used to contain culture medium.

[0028] Upper petri dish ( Figure 2 A and Figure 3 The upper culture dish includes a second lower bottom wall (103), a second side wall (104), and a top cover (105). The second lower bottom wall includes a porous material layer, at least a portion of which is a porous material. The second lower bottom wall and the second side wall are connected, and the top cover is adjustablely connected to the second side wall. In some examples of this application, the upper culture dish is used to place tissues, which are placed on the porous material layer.

[0029] The upper culture dish is adapted to be placed on top of the lower culture dish, and the upper culture dish is adapted to be sealed to the lower culture dish. In some examples of this application, the upper culture dish and the lower culture dish can form a sealed structure to prevent tissue contamination during the culture process. Using the above-described culture mold for tissue culture results in shorter manual operation time, higher efficiency, and lower cost.

[0030] In some examples of this application, the culture mold further includes: a sealing ring ( Figure 4 The sealing ring (106) is disposed on the outer surface of the second sidewall, and the upper culture dish is disposed on top of the lower culture dish. The upper culture dish and the lower culture dish are sealed together by the sealing ring. The sealing ring seals the upper and lower culture dishes to prevent tissue contamination.

[0031] In some examples of this application, the adjustable connection includes a detachable connection or a movable connection. A detachable connection includes a top cover that is separable from the second sidewall in the upper culture dish. A movable connection includes a top cover that is movably connected to the connected second sidewall in the upper culture dish, such as via a movable connecting shaft or via a rotary thread.

[0032] In some examples of this application, the porous material is nylon, and the pore size of the porous material is 100–200 μm. In some preferred examples of this application, the pore size of the porous material is 150 μm.

[0033] In one specific example of this application, the porous material is selected from nylon mesh.

[0034] In some examples of this application, the second lower bottom wall further includes a support layer ( Figure 2 B), the support layer (107) is disposed on the lower surface of the porous material layer. In some examples of this application, the support layer is used to fix the aforementioned nylon material.

[0035] In some examples of this application, the support layer consists of a plurality of radially arranged crossbeams and circular beams, the circular beams being disposed in the middle of the support layer, and the plurality of radially arranged crossbeams being connected to the circular beams, with equal spacing between the plurality of radially arranged crossbeams. In some examples of this application, the support layer can also be used to divide nylon material into different regions to prevent contact between tissues.

[0036] To facilitate understanding, the structure of the above-mentioned culture mold is illustrated below with a specific example.

[0037] In a specific example of this application, reference is made to Figure 5The culture mold comprises four parts: A, B, C, and D. Part A is the top cover, which fits into part B (connected to the second lower bottom wall and second side wall). The bottom of part B is made of nylon material (such as nylon mesh) and can hold tissues (such as explants). Culture medium is injected into part D, bringing it into contact with the nylon material in part B, providing the nutrients needed for tissue growth. Parts B and D are sealed together by a sealing ring C.

[0038] At the start of the experiment, tissue (such as callus) was placed in sterile part B, and then sterile culture medium was poured into part D. Subsequently, ABCD were combined and sealed for culture.

[0039] When subculture is required, simply inject fresh sterile culture medium into part D, and then separate the ABCD cultured for two weeks into two parts: A+B+C and D. Next, combine part A+B+C with part D containing fresh sterile culture medium to complete the subculture. Repeat this step to perform multiple subcultures.

[0040] In another aspect of this application, a tissue culture method is proposed. The method includes: culturing the tissue to be cultured under conditions suitable for tissue growth using a culture mold as described above. The tissue to be cultured is disposed on the upper surface of the second lower bottom wall of the upper culture dish of the culture mold, the lower culture dish of the culture mold contains tissue culture medium, and the upper culture dish is disposed on top of the lower culture dish.

[0041] In some examples of this application, the culture mold and the tissue culture medium are further sterilized before the culture process. This application does not specifically limit the sterilization method; commonly used laboratory sterilization methods are applicable, such as autoclaving. Sterilizing the culture mold and culture medium effectively prevents tissue contamination during the culture process.

[0042] In some tissue subculture processes, the culture medium needs to be changed multiple times. In some examples of this application, after the tissue to be cultured has been cultured for a predetermined time, only the lower culture dish is replaced, and the replaced lower culture dish contains fresh tissue culture medium. Before replacing the lower culture dish, the lower culture dish and the fresh tissue culture medium are further sterilized.

[0043] In some examples of this application, the tissue includes plant explants or animal tissue.

[0044] In some preferred embodiments of this application, the tissue is selected from plant explants, which are selected from at least one of callus, root, stem, leaf, and embryo. In a specific embodiment of this application, the tissue is selected from callus.

[0045] During the subculture of callus tissue, the culture medium comprises: 4.4 g / L MS medium, 30 g / L sucrose, 3 g / L plant gel, 1 mg / L 2,4-D, and 0.05 mg / L 6-BA. In each subculture, the callus tissue is cultured in the culture mold at 21–25°C in the dark.

[0046] In some examples of this application, the temperature of the culture treatment is 22°C.

[0047] In some examples of this application, the culture treatment time is 13 to 20 days. This culture treatment time refers to the time of one subculture. The culture time can optionally be 13, 14, 15, 16, 17, 18, 19, or 20 days. In some preferred examples of this application, the culture treatment time is 14 days.

[0048] To facilitate understanding, we will use plant tissue as an example to explain the tissue subculture process in detail through a specific example.

[0049] a. Dispensing of culture medium

[0050] First, prepare a culture medium suitable for plant material growth, and autoclave it and the culture mold to ensure sterility. In a laminar flow hood, introduce the sterilized culture mold (… Figure 5 Add sterile culture medium to D in ABCD. After the culture medium cools and solidifies, reassemble ABCD.

[0051] b. Initial placement of plant materials

[0052] During the initial culture, open section A of the culture mold and use tweezers to individually pick up the prepared plant material (such as callus) and place it in section B (placed on a nylon mesh). Then, seal the mold to form section E. Remove the sealed culture mold (containing plant material and culture medium) from the laminar flow hood and place it in an incubator. Adjust the culture conditions (such as temperature, time, and light) to promote plant growth.

[0053] c. Subculture of plant material every two weeks

[0054] When performing subculture every two weeks, prepare a new culture medium suitable for the growth of plant material, and autoclave the new culture medium and part D of the culture mold. In a laminar flow hood, fill the sterilized part D with the new culture medium. After the culture medium has cooled and solidified, remove the plant material (such as callus tissue) that has been cultured for two weeks from the incubator and place it on the laminar flow hood.

[0055] Next, separate part A+B+C from part D containing the old culture medium. Then, combine and seal part A+B+C with part D containing the new culture medium. Remove the sealed culture mold (containing plant material and new culture medium) from the laminar flow hood, place it back in the incubator, and adjust the culture conditions to continue culturing. Repeat the subculture every two weeks until the requirements are met.

[0056] In one example of this application, based on the above method, taking the subculture process of Arabidopsis leaf callus as an example, the time and process of the culture method of this application are compared with the traditional culture method. The specific steps are as follows:

[0057] Traditional procedure: Tear the membrane off a regular culture dish → open the lid → transfer callus tissue piece by piece with tweezers → close the lid → seal the dish.

[0058] The operational procedure of this application is as follows: separate D containing the old culture medium from the transfer mold → combine D containing the new culture medium.

[0059] The results show that, under the condition of 10 callus tissues per dish and 10 subcultures, the average time required for each subculture is 6.3 seconds in the present invention, compared to 46.6 seconds in the traditional method, saving 86.5% of the time cost. On the one hand, the process is greatly simplified; on the other hand, the traditional method requires a new dish for each subculture, while the present invention only requires a new dish per subculture, further reducing culture costs.

[0060] Beneficial effects

[0061] Using the above-mentioned culture mold to culture tissues has the following advantages compared to traditional methods:

[0062] 1) Whole explant transfer: There is no need to individually grasp the explants; the tissue can be transferred in one go. A simple pressing action is used to seal the explants with a sealing ring, avoiding the cumbersome membrane tearing and sealing steps, greatly simplifying the operation process.

[0063] 2) Saves operation time: When multiple subgeneration operations are required in the entire process of genetic transformation and gene editing, the culture mold design can shorten the manual operation time and reduce the input of manpower.

[0064] 3) Reduced contamination risk: Due to its simple operation, the culture mold design can reduce the contamination risk caused by human operation, thus improving the accuracy and reliability of the experiment.

[0065] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0066] Example 1: Plant explant culture

[0067] This embodiment uses both traditional methods and the proposed solution to perform subculture of plant explants, in order to verify the advantages of the proposed solution in tissue subculture.

[0068] 1. Experimental steps:

[0069] 1.1 Culture medium dispensing

[0070] First, prepare 1L of culture medium suitable for callus growth induced from Arabidopsis leaves and roots and autoclave it. The culture medium composition is as follows: 4.4g MS (Sigma M5519-50), 30g sucrose (Sigma S112236-500g), 3g plant gel (Sigma G1910-1KG), 1mg / L 2,4-D (Sigma D7299-250g), 0.05mg / L 6-BA (Sigma B3408-1L), and add purified water to a final volume of 1L. Take sterilized ordinary petri dishes and the culture mold used in this application (…). Figure 5 Place 10 of each on a laminar flow hood. Dispense the sterilized culture medium into ordinary petri dishes and the lower petri dishes of the culture mold. Figure 5 D). After the culture medium cools and becomes a solid medium, close the lid of the ordinary petri dish and seal it with a film. Use a sealing ring to seal the culture mold. Figure 5 C) Seal the container.

[0071] 1.2 First insertion of Arabidopsis callus

[0072] Ordinary petri dishes containing solid culture medium and culture molds are placed in a clean bench. According to conventional methods, the ordinary petri dishes are peeled and opened, while the tweezers are sterilized. Callus tissue induced from Arabidopsis leaves and roots is individually picked up with the tweezers and placed on the culture medium, then the lid is closed and the film is sealed. According to the method of this application, the culture mold has its top cover unscrewed (…). Figure 5 A) Using sterilized tweezers, the callus tissue pieces were individually picked up and placed on the nylon mesh. Figure 5 (The porous material layer of the second lower bottom wall in B), then tighten the top cover ( Figure 1Seal the contents. Remove the sealed ordinary culture dish and culture mold containing the callus tissue and culture medium from the laminar flow hood and place them in an incubator. Adjust the temperature to 22°C for dark incubation.

[0073] 1.3 Subculture should be performed every two weeks.

[0074] Prepare a 1L culture medium suitable for callus growth induced from Arabidopsis leaves and roots, and autoclave it. The medium composition is as follows: 4.4g MS (Sigma M5519-50), 30g sucrose (Sigma S112236-500g), 3g plant gel (Sigma G1910-1KG), 1mg / L 2,4-D (Sigma D7299-250g), 0.05mg / L 6-BA (Sigma B3408-1L), and add purified water to a final volume of 1L. Take sterilized ordinary petri dishes and the lower layer of the culture mold used in this application (…). Figure 5 D) Place 10 of each type of callus tissue in a laminar flow hood and fill with culture medium. After the culture medium cools and becomes solid, remove the callus tissue from the incubator after two weeks of culture and place it in the laminar flow hood. Following conventional methods, tear the membrane and open the lid of the ordinary culture dish containing the callus tissue, while simultaneously sterilizing the forceps. Use the forceps to pick up each callus tissue and place it onto the culture medium in the new dish, then close the lid and seal it. According to the method of this application, the base of the culture mold containing the callus tissue is separated from the main body, and then the base containing new culture medium is replaced and sealed with the main body. After sealing, the ordinary culture dish and culture mold containing the callus tissue and new culture medium are removed from the laminar flow hood and placed in an incubator, with the temperature adjusted to 22°C for dark culture. Repeat step 1.3 every two weeks until the callus tissue begins to brown (145 days after the initial placement of Arabidopsis thaliana callus).

[0075] 1.4 Comparison of Operation Time

[0076] During subculturing every two weeks, 10 ordinary culture dishes and culture molds are used for subculturing, with 10 or 20 Arabidopsis callus tissues placed in each dish and mold at a time. According to the traditional method, the ordinary culture dish containing callus tissue is peeled off its membrane as the starting point, then the lid is opened while the forceps are sterilized. Next, the ordinary culture dish containing fresh culture medium is peeled off its membrane and the lid is opened. Then, the callus tissues are individually placed onto the culture medium in the new dish using forceps. Finally, the ordinary culture dish containing callus tissue and fresh culture medium is closed, and the operation ends with sealing the membrane. According to the technical solution of this application, the separation of the base of the culture mold containing callus tissue from the main body is the starting point, and the reassembly of the base containing fresh culture medium from the main body is the ending point. The results showed that when 10 or 20 Arabidopsis callus tissues were placed in each culture dish and culture mold at a time, the time taken by the conventional method was 46.6 ± 2.5 seconds and 75 ± 2.1 seconds, respectively, while the time taken by the method of this invention was 6.3 ± 1.1 seconds and 6 ± 1.1 seconds, respectively. Compared with the conventional method, the method of this application saves 86.5% and 92% of the time, respectively.

[0077] 2. Experimental Results

[0078] 2.1 Callus tissue grows normally in the culture mold

[0079] Callus induced from Arabidopsis roots and leaves was placed on culture medium in ordinary culture dishes and on nylon mesh in culture molds. Figure 6 After culturing for approximately 56 days, the callus tissue in ordinary culture dishes and culture molds was found to be pale yellow and dense in morphology. Figure 6 C and D) are consistent with embryogenic callus characteristics, indicating normal callus growth and no bacterial contamination, proving that the culture mold can normally culture callus tissue. This type of embryogenic callus can be further subjected to genetic transformation or subculture. After approximately 145 days of culture, the callus tissue in both ordinary culture dishes and culture molds gradually begins to brown ( Figure 6 E and F) indicate that the longest time that the culture mold and the ordinary culture dish can continuously culture callus tissue is similar, approximately more than 5 months.

[0080] 2.2 Compared with traditional tissue subculture methods, the proposed method reduces manual operation time by 86.5% or 92% per dish for subculturing 10 or 20 calluses. Figure 7 ).

[0081] 2.3 Traditional methods require replacing the culture dish with a new one for each subculture, while the method described in this application only requires replacing the culture base for each subculture. Figure 5 D) can further reduce costs.

[0082] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0083] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A cultivation mold, characterized in that, include: The lower culture dish includes a first lower bottom wall and a first side wall, which are connected together. as well as The upper culture dish includes a second lower bottom wall, a second side wall, and an upper top cover. The second lower bottom wall includes a porous material layer, at least a portion of which is a porous material. The second lower bottom wall and the second side wall are connected, and the upper top cover is adjustablely connected to the second side wall. The upper culture dish is adapted to be placed on top of the lower culture dish, and the upper culture dish is adapted to be sealed to the lower culture dish.

2. The culture mold according to claim 1, characterized in that, Further includes: A sealing ring is disposed on the outer surface of the second sidewall, and the upper culture dish is disposed on top of the lower culture dish. The upper culture dish and the lower culture dish are sealed together by the sealing ring.

3. The cultivation mold according to claim 1, characterized in that, The adjustable connection includes a detachable connection or a movable connection.

4. The culture mold according to claim 1, characterized in that, The porous material is nylon, and the pore size of the porous material is 100-200 μm.

5. The culture mold according to claim 1, characterized in that, The second lower bottom wall further includes a support layer disposed on the lower surface of the porous material layer.

6. The cultivation mold according to claim 5, characterized in that, The support layer consists of several radially arranged crossbeams and circular beams. The circular beams are located in the middle of the support layer. The several radially arranged crossbeams are connected to the circular beams, and the spacing between the several radially arranged crossbeams is the same.

7. A tissue culture method, characterized in that, include: Using the culture mold according to any one of claims 1 to 6, the tissue to be cultured is cultured under conditions suitable for tissue growth; The tissue to be cultured is disposed on the upper surface of the second lower bottom wall of the upper culture dish of the culture mold. The lower culture dish of the culture mold contains tissue culture medium. The upper culture dish is placed on top of the lower culture dish.

8. The method according to claim 7, characterized in that, After the tissue to be cultured has been cultured for a predetermined time, the lower culture dish is replaced. The replaced lower culture dish contains fresh tissue culture medium. The lower culture dish and the tissue culture medium need to be sterilized.

9. The method according to claim 7, characterized in that, The tissue includes plant explants or animal tissues, preferably, the plant explants are selected from at least one of callus, root, stem, leaf and embryo.

10. The method according to claim 9, characterized in that, The plant explants are callus tissue, and the culture medium includes: 4.4 g / L MS medium, 30 g / L sucrose, 3 g / L plant gel, 1 mg / L 2,4-D and 0.05 mg / L 6-BA; the culture temperature is 21–25 °C. The culture treatment time is 13 to 20 days.

Images