Method for improving efficiency of agrobacterium-mediated transformation of tea plant and device used

Abstract

The present application belongs to the field of plant genetic engineering and biotechnology, and particularly relates to a method and device for plant tissue culture and genetic transformation. The present application discloses a device for improving Agrobacterium-mediated transformation efficiency of tea plants, which comprises a vacuum infection device and an air sterilization and purification comprehensive device. The air sterilization and purification comprehensive device comprises an air sterilization device and an air purification device connected in series. The air sterilization device, the air purification device and the vacuum infection device form a sealed system. The present application also provides a method for improving Agrobacterium-mediated transformation efficiency of tea plants by using the above device. The present application can realize efficient and stable genetic transformation of tea plant explants.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and biotechnology, specifically relating to a method and apparatus for plant tissue culture and genetic transformation. Background Technology

[0002] tea tree( Camellia sinensis (L.) Kuntze is an important economic crop, and tea made from its new shoots has extremely high economic value. With consumers' increasing demands for tea quality, flavor, and health benefits, higher requirements are being placed on the breeding of superior tea varieties. However, as a perennial woody plant, the tea tree faces many inherent biological limitations in traditional hybridization breeding methods. These factors collectively lead to long breeding cycles and low efficiency in traditional methods, making it difficult to quickly and specifically integrate superior traits such as disease and pest resistance, high quality, high yield, and specific metabolite content into a single variety (References 1, 2).

[0003] To overcome the bottlenecks of traditional breeding, modern biotechnology, especially plant genetic transformation technology, has provided a new and efficient approach for the genetic improvement of tea plants. Through genetic transformation, target genes can be precisely introduced into the tea plant genome, thereby achieving targeted improvement of specific traits in a relatively short time. Currently, the main genetic transformation methods applied to tea plants include Agrobacterium-mediated transformation and gene gun transformation. Among them, Agrobacterium-mediated transformation has become the most commonly used method in plant genetic transformation due to its relatively simple operation, relatively complete integration of T-DNA fragments, and high single-copy insertion rate (References 3 and 4).

[0004] Building upon traditional Agrobacterium co-culture techniques, Agrobacterium-mediated negative pressure transformation technology has been applied to various plant materials, including tea plants, with promising results. During the formation of negative pressure equilibrium, air is extracted from the intercellular spaces, causing elastic deformation of the cell structure due to pressure changes. Upon re-pressure, as the elastic deformation recovers, the Agrobacterium culture and air re-enter the tissue, ensuring thorough contact between Agrobacterium and internal cells, in addition to surface cells, thus improving transformation efficiency. However, during re-pressure, external air carrying a large number of microorganisms can enter the sterile transformation device, leading to microbial contamination of the material.

[0005] Therefore, removing airborne microorganisms is one of the key measures to reduce pollution, improve infection efficiency and transformation stability, and has important application value.

[0006] The cited references are as follows: 1. Agrobacterium transformation of tea plants (Camellia sinensis (L.)KUNTZE): A small experiment with great prospects. - Plants , 2024, 13 (5), 675; 2. Research progress on in vitro regeneration and genetic transformation of tea trees. Journal of Anhui Agricultural University, 2021, 48(5), 738; 3. Effects of catechins on Agrobacterium-mediated genetic transformation of Camellia sinensis, Plant Cell, Tissue and Organ Culture, 2014, 119, 27; 4. Agrobacterium rhizogenes: paving the road to research and breeding for woody plants, Front in Plant Science, 2023, 14, 1196561. https: / / doi.org / 10.3389 / fpls.2023.1196561. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method and apparatus for preventing and reducing fungal contamination during the Agrobacterium tumefaciens permeation transformation of tea plants.

[0008] To address the aforementioned problems, this invention provides a device for improving the permeation conversion efficiency of Agrobacterium tumefaciens in tea, comprising a vacuum infection device and an integrated air sterile treatment device. The air sterilization treatment integrated device includes a connected air sterilization device and an air purification device; The air sterilization and air purification devices have similar structures, both including a sealed container filled with liquid, with an air inlet pipe and an air outlet pipe at the top of the container; the air inlet pipe extends into the liquid inside the container, and an air stone is placed at the bottom of the air inlet pipe (the air stone is used to disperse the airflow and is located near the bottom of the container); after the gas enters through the air inlet pipe, it is dispersed into tiny bubbles by the action of the air stone and then enters the liquid (to increase the surface area and improve the treatment effect); the air treated by the liquid escapes from the liquid and returns to the upper space of the container above the liquid; the bottom of the air outlet pipe is located in the upper space of the container above the liquid (and near the top of the container). The exhaust pipe of the air sterilization device is sealed to the air inlet pipe of the air purification device, the exhaust pipe of the air purification device is sealed to the air inlet valve of the vacuum contamination device, and the air extraction valve of the vacuum contamination device is connected to the vacuum pump, thereby assembling the air sterilization device, the air purification device, and the vacuum contamination device into a sealed system.

[0009] An improvement to the apparatus of the present invention for increasing the permeation conversion efficiency of Agrobacterium tumefaciens in tea: The liquid in the air sterilization device is a water-soluble disinfectant; The liquid in the air purification device is sterile water.

[0010] Note: The purpose of sterile water is to wash away any bactericides that may be present in the air after sterilization, ensuring that the air entering the Agrobacterium infection system does not affect the growth of plant materials and Agrobacterium.

[0011] As a further improvement to the device of the present invention for improving the permeation conversion efficiency of Agrobacterium tumefaciens in tea: The bactericide is any one of the following: sodium hypochlorite, mercuric chloride, strong acid, or strong alkali.

[0012] That is, water-soluble bactericides such as high-concentration sodium hypochlorite (20% sodium hypochlorite), mercuric chloride, or aqueous solutions of strong acids or strong alkalis.

[0013] The present invention also provides a method for improving the permeation transformation efficiency of Agrobacterium tumefaciens in tea using the above-mentioned device, comprising the following steps: 1) Place the container containing both plant material and Agrobacterium in a vacuum inoculation device; 2) First, turn on the vacuum pump to create a vacuum. The original gas (original air) in the system (including the air sterilization device, air purification device, and vacuum contamination device) is discharged by the vacuum pump until the gas in the entire closed system is sterilized and purified air. Then, close the air inlet valve of the vacuum contamination device, evacuate to the set air pressure, and close the vacuum pump and the air extraction valve to maintain the vacuum contamination device in the required negative pressure state (for example, evacuate to a pressure of about 0.08 MPa). After the set contamination time (for example, 8-12 minutes), open (slowly open) the air inlet valve of the vacuum contamination device. The clean air that has been fully sterilized and purified by the air sterilization device and air purification device enters the vacuum contamination device for repressurization (restoring to normal pressure). The Agrobacterium solution also enters the interior of the treated material (plant material) during the repressurization process, restoring the internal and external pressures to equilibrium.

[0014] Agrobacterium-mediated negative pressure osmotic transformation is a significant improvement over the atmospheric pressure transformation method and a crucial measure to address the low efficiency of the atmospheric pressure transformation method. However, during the system's repressurization (restoration to atmospheric pressure), untreated outside air entering the system often contaminates the treated materials due to the microorganisms it carries. This invention adds an integrated air sterilization device before the conventional Agrobacterium infection apparatus. The air undergoes sterilization and purification sequentially, becoming sterile air, which can greatly improve the efficiency, repeatability, and success rate of Agrobacterium transformation.

[0015] That is, by using the present invention, the air entering the system during the repressurization process is pre-sterilized and purified to ensure that the entire process is sterile and prevent microbial contamination, thereby significantly improving the efficiency, stability and reproducibility of genetic transformation.

[0016] In summary, this invention relates to a method for treating tea plants, an important economic crop, using Agrobacterium as a mediator. Camellia sinensis This invention provides a system and method for efficient and stable genetic transformation of explants from tea plants. It offers an important technological platform for functional genomics research, germplasm resource innovation, and molecular breeding in tea trees. Attached Figure Description

[0017] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0018] Figure 1 This is a schematic diagram showing the operational status of the integrated air sterilization treatment device and the vacuum contamination device; the integrated air sterilization treatment device consists of a sterilization device and a purification device.

[0019] Figure 2 Results of Agrobacterium-mediated transformation of tea embryos (Example 1); Figure 2Figure A shows the GUS staining results after Agrobacterium transformation, and Figure B shows that the tea embryos can germinate normally after Agrobacterium transformation.

[0020] Figure 3 The result of Agrobacterium-mediated pollen transformation (Example 2); Figure 3 In the image: A shows the GUS staining result after Agrobacterium transformation, and B shows the pollen viability staining (TTC staining) result after Agrobacterium transformation. Detailed Implementation

[0021] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto: Example 1: An apparatus for improving the permeation and transformation efficiency of Agrobacterium tumefaciens in tea trees, comprising an integrated air sterilization treatment apparatus consisting of an air sterilization apparatus and an air purification apparatus, and further comprising a vacuum contamination apparatus.

[0022] Air sterilization devices and air purification devices have similar structures, specifically: A sealed container filled with a suitable liquid, with an air inlet pipe and an air outlet pipe at the top of the container.

[0023] The air inlet pipe extends into the liquid inside the container, and an air stone is installed at the bottom of the inlet pipe to disperse the airflow. After the gas enters through the inlet pipe, it is dispersed into tiny bubbles by the air stone before entering the liquid, increasing the surface area and improving the treatment effect. The air treated by the liquid escapes from the liquid and returns to the upper space of the container above the liquid. The bottom of the exhaust pipe is located in the upper space of the container above the liquid (and near the top of the container), and the exhaust pipe is used to transport the air treated by the liquid to subsequent devices. The length of the inlet pipe inside the container is much greater than the length of the exhaust pipe inside the container.

[0024] The exhaust pipe of the air sterilization device is sealed to the air inlet pipe of the air purification device, the exhaust pipe of the air purification device is sealed to the air inlet valve of the vacuum contamination device, and the air extraction valve of the vacuum contamination device is connected to the vacuum pump, thus forming a sealed system consisting of the air sterilization device, the air purification device, and the vacuum contamination device.

[0025] When used as an air sterilization device, the liquid in the container is a water-soluble disinfectant, such as a high concentration of sodium hypochlorite, mercuric chloride, or an aqueous solution of a strong acid or strong alkali.

[0026] When used as an air purification device, the liquid in the container is sterile water, which washes away any bactericides that may be present in the air after sterilization, thus ensuring that the air entering the vacuum contamination device will not affect the growth of plant materials and Agrobacterium.

[0027] The actual usage process is as follows: 1. Connect the exhaust pipe of the air sterilizer and the inlet pipe of the air purifier with a latex hose. Then connect the exhaust pipe of the air purifier to the inlet valve of the vacuum contamination device. The exhaust valve of the vacuum contamination device is connected to the vacuum pump. Thus, the air sterilizer, air purifier and vacuum contamination device form a sealed system.

[0028] Plant materials and Agrobacterium are placed inside a vacuum inoculation device.

[0029] 2. During use, first turn on the vacuum pump and evacuate for several minutes. The original air in the system, after passing through the air sterilization device, air purification device, and vacuum contamination device, is discharged by the vacuum pump until the air in the entire closed system is sterilized and purified. Then, close the air inlet valve of the vacuum contamination device, evacuate to the set pressure, and close the vacuum pump and the exhaust valve to maintain the required negative pressure state in the vacuum contamination device. After the set contamination time is reached, slowly open the air inlet valve of the vacuum contamination device. Clean air, fully sterilized and purified by the air sterilization and air purification devices, enters the vacuum contamination device for repressurization (restoring to normal pressure). At this time, the Agrobacterium solution also enters the interior of the treated material (plant material) during the repressurization process, restoring the internal and external pressure balance.

[0030] Example 1: Agrobacterium tumefaciens LBA4404-mediated genetic transformation of tea embryos

[0031] 1. Experimental materials Plant material: After peeling the pericarp from tea tree seeds, rinse them under running water, soak them in 75% ethanol for 60 seconds, soak them in 10% sodium hypochlorite for 20 minutes, rinse them three times with sterile water, and blot them dry with filter paper. Peel off the seed coat, leaving half of the cotyledon with the embryo, cut it into pieces about 4mm in size, and gently make 1-2 longitudinal cuts on the embryo with a scalpel to create infection wounds.

[0032] Strains: Agrobacterium tumefaciens LBA4404, carrying the pCambia2301 binary vector plasmid.

[0033] 2. Implementation Steps Step 1: Activation and Cultivation of Bacterial Solution Pick a loopful of Agrobacterium from the plate and inoculate it into 5 mL of liquid LB medium containing the corresponding antibiotics (100 mg / L rifampin and 50 mg / L kanamycin). Incubate at 28°C and 200 rpm with shaking for 3 days. Then, inoculate the same amount into liquid LB medium at a 1:100 (v / v) ratio and continue incubating at 28°C and 200 rpm with shaking until OD (dose retardation) is reached. 600 The value reaches approximately 0.8. Centrifuge at 5000 rpm for 10 minutes, discard the supernatant, and suspend the precipitate in permeate (until the OD600 is approximately 0.8).

[0034] The permeate consisted of: MES (10 mM, pH 5.5), MgCl2 (10 mM), acetosyringone (100 μM), and Silwet L-77 (0.03%).

[0035] Step 2: Explant Infection and Vacuum Infiltration Treatment As described in Example 1 above: Connect the air sterilization device (containing a 20% sodium hypochlorite solution), the air purification device (containing sterilized distilled water, sterilized using conventional methods: 121°C, 0.1 MPa, constant temperature for 20-30 minutes), the vacuum contamination device, and the vacuum pump sequentially using latex tubing. Under aseptic conditions, pour the cultured bacterial solution into a sterile beaker containing cotyledons, ensuring complete immersion of the cotyledons. Wrap the mouth of the beaker with sterile aluminum foil. Place the beaker containing the bacterial solution and stem segments into the vacuum contamination device. Start the vacuum pump and pre-evacuate for several minutes (until all air in the closed system is sterilized and purified). Close the inlet valve of the vacuum contamination device and continue evacuating until the pressure inside the device reaches approximately 0.08 MPa. Then close the evacuation valves of both the vacuum pump and the vacuum contamination device.

[0036] The vacuum treatment time is set to 10 minutes. Therefore, after 10 minutes, the air inlet valve of the vacuum contamination device is slowly opened to release the vacuum. Ambient air enters the vacuum contamination device through the air sterilization device and air purification device of the present invention, and returns to normal pressure after about 5 to 10 minutes.

[0037] Note: In this invention, the residence time of air in the sterilization solution and sterilized distilled water is approximately 2 to 3 seconds.

[0038] Step 3: Co-cultivation Under aseptic conditions: After opening the sterile aluminum foil, use sterile forceps to remove the cotyledons with embryos that have undergone vacuum permeation treatment from the bacterial solution, and blot off any excess bacterial solution on the surface with sterile filter paper. Spread these stem segments evenly on hormone-free solid MS medium in a petri dish. Seal the petri dish with sealing film and incubate in the dark at 28°C for 2 days.

[0039] Step 4: Debacterial treatment and induction culture The following steps must be performed under aseptic conditions. After co-culture, wash the cotyledons with embryos 3-5 times with sterile water, then transfer them to a sterile aqueous solution of 500 mg / L cefotaxime sodium and soak for 30 minutes to completely kill any remaining Agrobacterium. Blot the solution off the surface of the material with sterile filter paper and transfer it to induction medium (MS solid medium supplemented with 500 mg / L cefotaxime sodium, used as the induction medium). Seal the culture dish with sealing film and continue dark culture at 25°C for 15 days. Transfer the explants to a fresh medium every 15 days.

[0040] Step 5: Results Statistics and Validation Six treatments were randomly selected and GUS stained; three materials showed a color reaction. Figure 2 (A) The embryo that has undergone cutting can continue to grow roots and shoots ( Figure 2 (B in the middle).

[0041] Example 2: Agrobacterium tumefaciens LBA4404-mediated genetic transformation of tea pollen 1. Experimental Materials Plant material: Unopened tea tree flower buds, rinsed under running water, soaked in 75% ethanol for 60 seconds, soaked in 10% sodium hypochlorite for 20 minutes, rinsed three times with sterile water, and blotted dry with filter paper. The flower stalks were removed, bracts and petals were removed, and the filaments were cut at the base to form single filaments with anthers.

[0042] Strains: Agrobacterium tumefaciens LBA4404, carrying the pCambia2301 binary vector plasmid.

[0043] 2. Implementation Steps Equivalent to steps one through three of Example 1; Step 4: GUS staining and pollen viability detection: GUS staining was performed on the co-cultured anthers; approximately 50% of the anthers showed a color reaction. Figure 3 (A in the middle).

[0044] Several anthers were also tested for viability using triphenyltetrazolium chloride (TTC) staining. Approximately 50% of the pollen turned red. These viable pollen samples can be used for subsequent experiments. Figure 3 (B in the middle).

[0045] Comparative Experiment 1, in relation to Example 2: 1. If the air sterilization treatment device of the present invention is not used, and the outside air is directly introduced into the vacuum contamination device for repressurization (restoring to normal pressure), during the subsequent cultivation process, it was observed that about 20% to 50% of the leaf materials became contaminated with fungi after 10 and 20 days of observation. The longer the time, the more contamination there was, which affected the smooth progress of subsequent experiments.

[0046] This indicates that air is the main source of pollution, and the microorganisms carried in the air are the main factor leading to a large amount of pollution.

[0047] 2. After employing the air sterilization treatment integrated device of the present invention, i.e., after being thoroughly sterilized and purified by the air sterilization and purification devices, the clean air was introduced into the vacuum contamination device for repressurization (restoring to normal pressure), and then placed on a culture medium for cultivation. A survey after 30 days revealed that fungal contamination of the leaves was less than 5%; the fungal contamination rate of the experimental materials was significantly different from the aforementioned control. This indicates that the air sterilization treatment integrated device in the entire closed system played its due role in the experiment, effectively killing airborne microorganisms. Because the air in the entire closed system was sterilized, microbial contamination of the experimental materials was prevented. Therefore, the cases of the present invention using different experimental materials all successfully obtained the final results according to the expected experimental plan.

[0048] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A device for improving the permeation transformation efficiency of Agrobacterium tumefaciens in tea, comprising a vacuum infection device, characterized in that: It also includes an integrated air sterilization treatment device; The air sterilization treatment integrated device includes a connected air sterilization device and an air purification device.

2. The device for improving the permeation transformation efficiency of Agrobacterium tumefaciens in tea according to claim 1, characterized in that: The air sterilization device and the air purification device have similar structures, both including a sealed container filled with liquid, with an air inlet pipe and an air outlet pipe respectively located at the top of the container; the air inlet pipe extends into the liquid inside the container, and an air stone is installed at the bottom of the air inlet pipe; after the gas enters through the air inlet pipe, it is dispersed into tiny bubbles under the action of the air stone and then enters the liquid; the air treated by the liquid escapes from the liquid and returns to the upper space of the container above the liquid; the bottom of the air outlet pipe is located in the upper space of the container above the liquid. The exhaust pipe of the air sterilization device is sealed to the air inlet pipe of the air purification device, the exhaust pipe of the air purification device is sealed to the air inlet valve of the vacuum contamination device, and the air extraction valve of the vacuum contamination device is connected to the vacuum pump, thereby assembling the air sterilization device, the air purification device, and the vacuum contamination device into a sealed system.

3. The device for improving the permeation conversion efficiency of Agrobacterium tumefaciens in tea according to claim 2, characterized in that: The liquid in the air sterilization device is a water-soluble disinfectant; The liquid in the air purification device is sterile water.

4. The device for improving the permeation conversion efficiency of Agrobacterium tumefaciens in tea according to claim 3, characterized in that: The bactericide is any one of the following: sodium hypochlorite, mercuric chloride, strong acid, or strong alkali.

5. A method for improving the permeation transformation efficiency of Agrobacterium tumefaciens in tea using the apparatus as described in any one of claims 1 to 4, characterized in that... Includes the following steps: 1) Place the container containing both plant material and Agrobacterium in a vacuum inoculation device; 2) First, turn on the vacuum pump to evacuate the system. The original gas in the system is discharged by the vacuum pump until the gas in the entire closed system is sterilized and purified air. Then, close the air inlet valve of the vacuum contamination device, evacuate to the set air pressure, and close the vacuum pump and the air extraction valve to maintain the vacuum contamination device in the required negative pressure state. After the set infiltration time is reached, the air inlet valve of the vacuum infiltration device is opened. Clean air, which has been fully sterilized and purified by the air sterilization and air purification devices, enters the vacuum infiltration device for repressurization. The Agrobacterium solution also enters the interior of the plant material during the repressurization process.

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