Method for improving success rate of instantaneous transformation system of wheat protoplast

Abstract

The invention provides a method for improving the success rate of a wheat protoplast instantaneous transformation system, which comprises the following steps: S1, fully rinsing a cell culture container for incubation by using a 10% bovine serum albumin solution, so that all structures in the container are infiltrated by the 10% bovine serum albumin solution, and discarding waste liquid after rinsing; and S2, resuspending the wheat protoplast into which the exogenous gene is transferred by using 900-1200mu L of a W5 solution, transferring the wheat protoplast into the cell culture container rinsed in the S1, wrapping the cell culture container by using tin foil paper, and incubating for 12-16 hours at room temperature. Compared with a traditional incubation process, the method has the advantages that the integrity rate and activity of the protoplast are remarkably improved, and the risks of protoplast rupture and death in the incubation process are effectively reduced. According to the system, the instantaneous conversion efficiency can stably reach more than 50%, and the reliability and operability of gene function verification and regulation mechanism research are greatly improved.

Description

Technical Field

[0001] This invention relates to the field of plant cell technology, and more specifically to a method for improving the success rate of a wheat protoplast transient transformation system. Background Technology

[0002] Wheat (Triticum aestivum L.) is one of the world's most important food crops, and its yield and quality are directly related to food security and national economic development. With the deepening of functional genomics research, there is a greater demand for rapid and efficient verification technologies for wheat gene function. Among these, the protoplast transient expression system plays a crucial role in studying the subcellular localization of wheat genes, the editing efficiency of target sites, and gene function due to its advantages such as short cycle, high efficiency, and no need for transgenic plants. Wheat protoplast transient transformation technology utilizes polyethylene glycol (PEG) to mediate the entry of DNA or RNA into cells, achieving transient gene expression. This system greatly facilitates wheat gene function research, especially in assessing transcription factor regulatory functions, verifying gene expression responses, and rapidly screening editing efficiency, where it plays an irreplaceable role.

[0003] However, a series of technical bottlenecks still exist in the transient expression system of wheat protoplasts, which restrict the success rate and stability of the transient transformation system. These bottlenecks mainly include: 1. The thick cell wall and complex cell structure of wheat limit the efficient release of cells during protoplast extraction; 2. Physical and osmotic stress during PEG-mediated transformation leads to high protoplast cell fragmentation and decreased physiological activity, affecting subsequent expression efficiency and the reliability of experimental results; 3. Some reports on improving the success rate of wheat transformation experiments use enzymatic hydrolysis conditions, centrifugation parameters, or incubation procedures that suffer from weak stability and poor reproducibility, making standardized operation difficult. Summary of the Invention

[0004] To address the issues of high cell disruption rate, poor result stability, and low success rate in wheat protoplast transient transformation systems, which affect gene expression and reduce gene editing efficiency, and the existing improved technologies for wheat protoplast transient expression systems suffer from technical bottlenecks, poor stability, poor reproducibility, and difficulty in achieving standardized operation, this invention provides a method to improve the success rate of wheat protoplast transient transformation systems, thus solving the aforementioned problems.

[0005] The technical solution of the present invention is as follows:

[0006] This invention provides a method for improving the success rate of a wheat protoplast transient transformation system, characterized by optimizing the incubation process, including the following steps:

[0007] S1: Thoroughly rinse the cell culture container used for incubation with 10% bovine serum albumin solution, ensuring that all internal structures of the container are wetted with the 10% bovine serum albumin solution. Discard the waste liquid after rinsing.

[0008] S2: Resuspend wheat protoplasts after introducing the exogenous gene in 900-1200 μL W5 solution, transfer them to the cell culture container rinsed in S1, wrap the cell culture container with aluminum foil, and incubate at room temperature for 12-16 h.

[0009] Preferably, the cell culture container used for incubation is a centrifuge tube or a cell culture plate.

[0010] Preferably, the cell culture plate parameters are: 6 wells, 3 ml, 9.60 cm. 2 PS stands for polystyrene, flat bottom, TC treatment (tissue culture treatment), sterilization, and free of DNase, RNase, and pyrogens.

[0011] Preferably, the volume of the 10% bovine serum albumin solution is 800-1200 μL; the volume of the W5 solution is 1 ml.

[0012] Preferably, the method for extracting wheat protoplasts is as follows:

[0013] A1: Prepare wheat seedlings aged 7-12 days, remove their roots and leaf tips, cut them into 0.5-1.0 mm sections along the vertical growth direction with a sharp blade, put them into a 50 mL beaker containing 20 mL of enzymatic hydrolysate, wrap the beaker with aluminum foil and leave a hole at the mouth of the beaker, and vacuum for 30 min under 15 Hg and 50 kPa conditions;

[0014] A2: Place the vacuum-sealed beaker on a shaker and react for 4-5 hours. Set the conditions to be protected from light, 28°C, and 45 rpm. During this period, gently invert and mix every hour to ensure that the wheat tissue attached to the tube wall is in full contact with the enzyme hydrolysate. After the reaction is complete, adjust the shaker speed to 80 rpm and shake for 5 minutes to promote the complete release of protoplasts from the mesophyll tissue.

[0015] A3: Take a new 50 mL centrifuge tube, place the cell sieve that has been rinsed with 1 mL W5 solution, filter the enzymatic hydrolysate in beaker A2, gently squeeze the remaining wheat seedling tissue to fully release the protoplasts until the green liquid drips down, then rinse the beaker used in A2 and the remaining wheat seedling tissue in it twice with 5 mL W5 solution, and collect the rinsing liquid into the 50 mL centrifuge tube.

[0016] A4: Centrifuge the liquid collected in A3 using a horizontal rotor. After removing the supernatant, the precipitate at the bottom of the centrifuge tube is the wheat protoplast. Purify the wheat protoplast precipitate using W5 solution, resuspend it, centrifuge, and then resuspend it using MMG solution, adjusting the wheat protoplast number to 2 × 10⁻⁶. 5 / mL.

[0017] Preferably, the centrifugation parameters in A4 are: 100g centrifugation at room temperature for 10 minutes, with an acceleration rate of 3 and a deceleration rate of 3.

[0018] Preferably, before centrifugation as described in step A4, the state of wheat protoplasts is observed and counted using a microscope and a hemocytometer, in order to adjust the wheat protoplast count to 2 × 10⁻⁶ later. 5 / mL for preparation; the calculation method for the amount of MMG solution used is: total volume (μL) = number of required reactions × 100 μL / reaction.

[0019] Preferably, the steps of the wheat protoplast transformation experiment are as follows:

[0020] B1: Add 3-20 µg of purified endotoxin-free plasmid to a sterilized round-bottom centrifuge tube, followed by adding 100 µL of protoplasts to adjust the number to 2 × 10⁻⁶. 5 / mL of wheat protoplasts, gently mix;

[0021] B2: Add 110µL of PEG conversion solution, mix gently to avoid generating bubbles, and react at room temperature for 10-15 minutes;

[0022] B3: Add 500 μL of W5 solution, mix gently, and terminate the conversion;

[0023] B4: Centrifuge for 10 min and remove the supernatant. Centrifugation parameters are set to 200g at room temperature, speed 3, and speed 3. Add 500μL of W5 solution, centrifuge again for 10 min and remove the supernatant. Centrifugation parameters are set to 100g at room temperature, speed 3, and speed 3. This completes the transformation of wheat protoplasts.

[0024] Preferably, the plasmid concentration in B1 is greater than 1000 ng / µL.

[0025] Preferably, the PEG conversion solution is completely dissolved in a 65°C water bath and then cooled to room temperature before use.

[0026] The beneficial effects of this invention are as follows:

[0027] This invention provides a method for improving the success rate of a wheat protoplast transient transformation system, wherein the method involves optimizing the incubation process of wheat protoplasts. The incubation process is a step in the wheat protoplast transient transformation system, aimed at ensuring sufficient expression of exogenous genes after transfer into wheat protoplasts. Factors such as incubation time, temperature, culture medium composition, whether light is blocked, and whether shaking or static incubation is used can significantly affect gene expression and editing efficiency: if the incubation time is too short, the expression of endonucleases such as Cas9 or sgRNA may be insufficient, potentially leading to a significant decrease in mutation rate; if the incubation environment is unsuitable (unstable temperature or poor culture conditions), the wheat protoplasts will rapidly inactivate or degrade, thus affecting subsequent experimental results. Therefore, the incubation process is crucial.

[0028] The method provided by this invention optimizes the incubation process. Compared to the conventional incubation process where wheat protoplasts with exogenous genes are directly transferred to the culture container without rinsing, this invention uses a 10% bovine serum albumin (BSA) solution to rinse the cell culture container. This not only forms a protein coating layer on the surface of the culture container, reducing non-specific adsorption and mechanical damage when wheat protoplasts with exogenous genes come into direct contact with the culture plate, thus avoiding cell membrane rupture or shrinkage due to water loss, but also reduces the adhesion and aggregation of wheat protoplasts with exogenous genes on the plate wall, improving their suspension distribution. This provides a more stable microenvironment for the subsequent survival and culture of wheat protoplasts with exogenous genes, greatly improving the integrity rate of wheat protoplasts with exogenous genes, thereby improving the overall success rate of the PEG-mediated transient transformation system of wheat protoplasts.

[0029] Furthermore, in the method provided by this invention, the cell culture container used in the incubation process is a cell culture plate. Compared with other cell culture containers used in conventional incubation processes, cell culture plates can provide a relatively closed and stable microenvironment, making the protoplasts more uniformly suspended and avoiding damage caused by aggregation or sedimentation. Simultaneously, the flat surface of the cell culture plate helps maintain constant osmotic pressure and temperature and humidity conditions, reducing the impact of external interference on protoplast survival and gene expression. Moreover, cell culture plates are easier to observe directly under a microscope, facilitating the evaluation of transformation efficiency and verification of subsequent functions. Therefore, using cell culture plates as containers to incubate wheat protoplasts after the introduction of exogenous genes not only improves the integrity rate of wheat protoplasts in the transient transformation system, thereby improving the overall expression efficiency of the PEG-mediated transient transformation system (i.e., increasing the success rate of the transient transformation system), but also helps improve cell viability and experimental reproducibility, enhancing the reproducibility and reliability of transient expression and gene function studies.

[0030] Existing methods for improving the success rate of wheat protoplast transient expression systems focus on protoplast isolation steps and transfection conditions, incubation media, and molecular strategies to enhance gene expression efficiency (such as vector design optimization). This invention, however, optimizes the incubation process, offering a simpler and more efficient approach compared to existing technologies. Other improvements to the wheat protoplast transient expression system involve vector construction and gene delivery technologies, which are costly, have low controllability, are influenced by numerous factors, have poor reproducibility, and are difficult to standardize. This invention optimizes the incubation process after the introduction of the exogenous gene, resulting in lower costs, stronger stability, and simpler operation compared to improvements in vector construction and gene delivery technologies, facilitating standardization. This not only optimizes the application value of the wheat protoplast transient transformation platform but also provides a more efficient and stable experimental system for wheat gene editing, functional genomics research, and molecular improvement of desirable traits. Attached Figure Description

[0031] Figure 1 Wheat protoplasts obtained by rinsing centrifuge tubes using the optimized incubation process provided by this invention (A, B, and C are the first, second, and third fields of view, respectively).

[0032] Figure 2 Wheat protoplasts obtained by rinsing cell culture plates using the optimized incubation process provided by this invention (A, B, and C are the first, second, and third fields of view, respectively).

[0033] Figure 3 : Observational images of wheat protoplasts obtained using the conventional incubation process (A, B, and C represent the first, second, and third fields of view, respectively).

[0034] Figure 4 Wheat protoplast cells obtained by rinsing centrifuge tubes using the optimized incubation process provided by this invention (A and B are wheat protoplasts observed in the first field of view in bright and dark fields, respectively; C and D are wheat protoplasts observed in the second field of view in bright and dark fields, respectively).

[0035] Figure 5 Wheat protoplast cells obtained by rinsing cell culture plates using the optimized incubation process provided by this invention (A and B are wheat protoplasts observed in the first field of view in bright and dark fields, respectively; C and D are wheat protoplasts observed in the second field of view in bright and dark fields, respectively).

[0036] Figure 6Cellular observation images of wheat protoplasts obtained using the conventional incubation procedure (A and B are wheat protoplasts observed in the first field of view in bright and dark fields, respectively; C and D are wheat protoplasts observed in the second field of view in bright and dark fields, respectively). Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the following detailed embodiments provide a method for improving the success rate of a wheat protoplast transient transformation system. The advantages and features of this invention will become clearer from the following description.

[0038] The plasmid miniprep kit used in this embodiment is from Tiangen, and its specification is DP103.

[0039] Bovine serum albumin solution (BSA) was purchased from Sigma, CAS number 9048468.

[0040] Apart from the two mentioned above, all reagents used in the following examples were purchased from Thermo Fisher Scientific.

[0041] The preparation method for W5 solution is as follows: Add 2 mL of MES (0.2 M / L) to a sterile, enzyme-free glass bottle to achieve a concentration of 2 mM / L, 45.4 mL of NaCl (2 M / L) to achieve a concentration of 154 mM / L, 0.5 mL of KCl (2 M / L) to achieve a concentration of 5 mM / L, 0.18 g of glucose powder, and 25 mL of CaCl2 (1 M / L) to achieve a concentration of 125 mM / L. Make up the volume to 200 mL, mix well with a magnetic stirrer, and continuously add 1 M / L KOH solution to adjust the pH of the solution to 5.7. Autoclave the solution and store at 4°C.

[0042] Preparation of MMG solution: Add 1 mL of MES (0.2 M / L) to a sterile, enzyme-free glass bottle to achieve a concentration of 4 mM / L, 5.465 g of D-Mannitol, and 0.37 mL of MgCl2 (2 M / L) to achieve a concentration of 15 mM / L. Make up the volume to 50 mL, mix with a magnetic stirrer, and continuously add 1 M / L KOH solution to adjust the pH of the solution to 5.7. Autoclave the solution at high temperature and store at 4°C.

[0043] Preparation method of PEG conversion solution: Add 0.2186 g D-Mannitol, 0.8 g PEG4000 and 0.2 mL CaCl2 (1 M / L) to a sterile 2 mL centrifuge tube to achieve a concentration of 10 mM / L, make up to 2 mL, heat in a metal bath at 80℃, cool to room temperature before use, and filter for sterilization.

[0044] Example: Transient transformation system of wheat protoplasts

[0045] 1. Extraction of wheat protoplasts

[0046] Prepare 7-12 day old wheat seedlings. Take 5-10 wheat seedlings (sample size approximately 1g, can be increased appropriately), remove roots and leaf tips. Use a sharp blade to cut them perpendicular to the growth direction into 0.5-1.0mm segments. Place the cut segments into 50mL beakers containing 20mL of enzymatic hydrolysate, wrap with aluminum foil, leave a hole at the mouth of the beaker, and use a portable vacuum pump (Tianjin Keyilong Experimental Equipment Co., Ltd.) to evacuate for 30 minutes (15Hg, 50kPa). Incubate at 28℃ in the dark on a shaker at 45rpm for 4-5 hours (during which time gently invert and mix every hour to ensure sufficient contact between the wheat tissue attached to the tube wall and the enzymatic hydrolysate). Before collecting the protoplasts, shake at 80rpm for 5 minutes to completely release the protoplasts. Rinse the cell sieve with 1mL of W5 solution and discard the waste liquid. Filter the enzymatic hydrolysis product using a cell strainer. Gently squeeze the hydrolysate with tweezers or a sterile pipette tip to help fully release the protoplasts; at this point, a dark green liquid will be visible as it drips. Rinse the hydrolysis vessel and undigested leaves twice with 5 mL of W5 solution (using a blue pipette tip with a 2 mm tip cut off). Collect all the liquid into a 50 mL centrifuge tube.

[0047] Using a horizontal rotor, centrifuge at 100 g for 10 min at room temperature, with an acceleration of speed 3 and a deceleration of speed 3, then remove the supernatant. Add 5 mL of W5 solution using a sharpened blue pipette tip, gently resuspend the protoplasts at the bottom of the centrifuge tube, centrifuge at 100 g for 5 min, with an acceleration of speed 3 and a deceleration of speed 3, then remove the supernatant. Add 5 mL of W5 solution to resuspend the protoplasts and incubate on ice in the dark for 30 min. Before centrifugation, observe the protoplasts under a microscope and count them using a hemocytometer to adjust for appropriate cell concentration. Centrifuge at 100 g for 10 min, with an acceleration of speed 3 and a deceleration of speed 3, then remove the supernatant and resuspend the protoplasts with the appropriate volume of MMG solution to adjust the protoplast count to 2 × 10⁻⁶. 5 / mL (Add the corresponding MMG volume according to the number of cells and the number of reactions required, 100 μL / 1 reaction).

[0048] 2. Transformation of wheat protoplasts

[0049] Plasmids were extracted using a plasmid miniprep kit, with a plasmid concentration ≥1000 ng / µL. 3–20 µg of purified, high-quality, endotoxin-free plasmid was added to a 2 mL sterile round-bottom centrifuge tube, followed by 100 µL of the adjusted concentration (2 × 10⁻⁶). 5 Mix the protoplasts ( / mL) with a gentle tap to ensure thorough mixing (if the plasmid concentration is too low, the expression efficiency will usually decrease significantly, especially for long-fragment plasmids, such as CRISPR / Cas9 expression vectors).

[0050] Add 110 µL of PEG conversion solution to a centrifuge tube, gently tap to mix or gently pipette with a blue pipette tip with a 2 mm tip cut off, avoiding the formation of air bubbles during the process. Incubate at room temperature for 10-15 minutes (no more than 15 minutes for routine experiments). Note: PEG conversion solution is difficult to dissolve; it can be placed in a 65°C water bath for about 10 minutes to dissolve completely, then removed and cooled to room temperature before use.

[0051] Add 500 μL of W5 solution to the centrifuge tube, gently invert to mix, and terminate the transformation. Centrifuge at 200g for 10 min at room temperature, with an acceleration of speed 3 and a deceleration of speed 3, removing as much supernatant as possible without losing protoplasts. Resuspend the protoplasts in 500 μL of W5 solution, centrifuge at 100g for 10 min at room temperature, with an acceleration of speed 3 and a deceleration of speed 3, and discard the supernatant.

[0052] 3. Incubation of wheat protoplasts

[0053] Rinse the cell culture vessel with 1 mL of 10% bovine serum albumin (BSA) to ensure that all internal structures are wetted with the 10% BSA solution. Discard the waste liquid after rinsing.

[0054] The cell culture containers include commonly used cell culture containers such as cell culture plates, centrifuge tubes, and culture dishes.

[0055] In some embodiments of the present invention, the cell culture container is a centrifuge tube with parameters of 2 mL, free of enzymes and pyrogens, and purchased from Shanghai Kejin Biotechnology Co., Ltd.

[0056] In some embodiments of the present invention, the cell culture container is a cell culture plate, and the cell culture plate parameters are 6 wells, 3 ml, and 9.60 cm. 2 PS, flat bottom, TC treated, sterilized, free of DNase, RNase and pyrogens, purchased from Sangon Biotech Co., Ltd.

[0057] Add 1 mL of W5 solution to resuspend the cells, then transfer the resuspended wheat protoplasts to a rinsed cell culture plate, wrap the cell culture plate with aluminum foil, and incubate at room temperature for 12-16 h.

[0058] Experiment Example 1: Effects of different incubation procedures on cell membrane integrity in wheat transient transformation experiment

[0059] Cells after being transferred with the foreign gene were divided into a control group and an experimental group, and treated with different incubation procedures.

[0060] (1) Control group: The conventional incubation procedure was used. 500 μL of W5 solution was added to a 2 mL centrifuge tube to resuspend the protoplasts after the foreign gene was introduced, and the tubes were kept in the dark at room temperature for 12-16 h.

[0061] (2) Experimental Group 1: The optimized incubation process described in the examples was adopted. 2 ml centrifuge tubes were rinsed with 1 mL of 10% bovine serum albumin solution to ensure thorough rinsing (sterilized, free of DNase, RNase, and pyrogens, Shanghai Kejin Biotechnology Co., Ltd.). The waste liquid was discarded after rinsing. 1 mL of W5 solution was added to the rinsed centrifuge tubes to resuspend the cells. The resuspended wheat protoplasts were then transferred to the rinsed centrifuge tubes, wrapped with aluminum foil, and incubated at room temperature for 12-16 h.

[0062] (3) Experimental Group 2: The optimized incubation procedure described in the examples was used. The bottom and walls of the cell culture plates were rinsed with 1 mL of 10% bovine serum albumin solution to ensure thorough rinsing (cell culture plates with parameters of 6 wells, 3 ml, and 9.60 cm⁻¹ are recommended). 2 The cells were prepared using PS, flat-bottomed, TC-treated, sterilized, and free of DNase, RNase, and pyrogens (Sangon Biotech Co., Ltd.). The waste liquid was discarded after rinsing. 1 mL of W5 solution was added to the rinsed cell culture plate to resuspend the cells. The resuspended wheat protoplasts were then transferred to the rinsed cell culture plate, wrapped with aluminum foil, and incubated at room temperature for 12-16 hours.

[0063] Wheat protoplasts obtained using the optimized incubation process in experimental group 1 were observed. Random sampling was performed, and three fields of view were randomly selected for statistical observation. Results are as follows: Figure 1 As shown, in the first field of view, wheat protoplasts contained 127 intact cells and 59 broken cells, with a cell integrity rate of 68.28% (A); in the second field of view, wheat protoplasts contained 117 intact cells and 75 broken cells, with a cell integrity rate of 60.94% (B); in the third field of view, wheat protoplasts contained 58 intact cells and 69 broken cells, with a cell integrity rate of 45.67% (C). The average cell integrity rate reached 58.3%.

[0064] Wheat protoplasts obtained using the optimized incubation process in experimental group 2 were observed, and three fields of view were randomly selected for statistical observation. The results are as follows: Figure 2As shown, the first field of view contained 239 wheat protoplasts, of which 227 were intact cells and 12 were broken cells, with a cell integrity rate of 94.98% (A); the second field of view contained 166 wheat protoplasts, of which 152 were intact cells and 14 were broken cells, with a statistical cell integrity rate of 91.56% (B); the third field of view contained 208 wheat protoplasts, of which 196 were intact cells and 12 were broken cells, with a statistical cell integrity rate of 94.23% (C), and the average cell integrity rate reached 93.80%.

[0065] Wheat protoplasts obtained from the control group using the conventional incubation process were observed, and three fields of view were randomly selected for statistical analysis. Results are as follows: Figure 3 As shown, the first field of view contains 112 protoplasts, of which 39 are intact cells and 73 are broken cells, with a cell integrity rate of 34.82% (A); the second field of view contains 110 cells, of which 32 are intact cells and 78 are broken cells, with a cell integrity rate of 29.09% (B); the third field of view contains 115 cells, of which 38 are intact cells and 77 are broken cells, with a cell integrity rate of 33.04% (C), and an average cell membrane integrity rate of 32.34%.

[0066] The wheat protoplasm obtained using the optimized incubation process provided by this invention has an average cell integrity rate increased by 25.69~61.46%.

[0067] Experiment Example 2: Effects of different incubation processes on transient expression rate in wheat protoplast transient transformation system

[0068] Wheat protoplasts were transformed using a PEG-mediated transformation of an eGFP-carrying green fluorescent signaling vector. The plasmid PDH51 carrying the eGFP gene was extracted using a plasmid miniprep kit, with a plasmid concentration ≥1000 ng / µL. 3–20 µg of purified, high-quality, endotoxin-free plasmid was added to a 2 mL sterile round-bottom centrifuge tube, followed by 100 µL of the adjusted concentration (2 × 10⁻⁶). 5 Mix the protoplasts ( / mL) thoroughly with a light tap.

[0069] Add 110 µL of PEG conversion solution to a centrifuge tube, gently tap to mix or gently aspirate with a pointed blue pipette tip, avoiding the formation of air bubbles during the mixing process. Incubate at room temperature for 10-15 minutes (no more than 15 minutes for routine experiments). Note: PEG conversion solution is difficult to dissolve; it can be placed in a 65°C water bath for about 10 minutes to fully dissolve it, then removed and cooled to room temperature before use.

[0070] Add 500 μL of W5 solution to the centrifuge tube, gently invert to mix, and terminate the transformation. Centrifuge at 200 g for 10 min at room temperature, with an acceleration of speed 3 and a deceleration of speed 3, removing the supernatant as much as possible without losing protoplasts. Resuspend the protoplasts in 500 μL of W5 solution, centrifuge at 100 g for 10 min at room temperature, with an acceleration of speed 3 and a deceleration of speed 3, and discard the supernatant.

[0071] Protoplasts after being transferred with exogenous genes were divided into control and experimental groups and treated with different incubation procedures.

[0072] (1) Control group: The conventional incubation procedure was used. 500 μL of W5 solution was added to a 2 mL centrifuge tube to resuspend the protoplasts after the foreign gene was introduced, and the tubes were kept in the dark at room temperature for 12-16 h.

[0073] (2) Experimental Group 1: The optimized incubation process described in the examples was adopted. 2 mL centrifuge tubes were rinsed with 1 mL of 10% bovine serum albumin solution (enzyme-free, pyrogen-free, Shanghai Kejin Biotechnology Co., Ltd.) to ensure thorough rinsing, and the waste liquid was discarded after rinsing. 1 mL of W5 solution was added to the rinsed centrifuge tubes to resuspend the cells, and the resuspended wheat protoplasts were then transferred to the rinsed centrifuge tubes. The centrifuge tubes were wrapped with aluminum foil and incubated at room temperature for 12-16 h.

[0074] (2) Experimental Group 2: The optimized incubation procedure described in the examples was adopted. The bottom and walls of the cell culture plates were rinsed with 1 mL of 10% bovine serum albumin solution to ensure thorough rinsing (cell culture plates with parameters of 6 wells, 3 ml, and 9.60 cm⁻¹ are recommended). 2 The cells were prepared using PS, flat-bottomed, TC-treated, sterilized, and free of DNase, RNase, and pyrogens (Sangon Biotech Co., Ltd.). The waste liquid was discarded after rinsing. 1 mL of W5 solution was added to the rinsed cell culture plate to resuspend the cells. The resuspended wheat protoplasts were then transferred to the rinsed cell culture plate, wrapped with aluminum foil, and incubated at room temperature for 12-16 hours.

[0075] Wheat protoplasts obtained using the optimized incubation process in experimental group 1 were observed in both light and dark fields. The experimental results are as follows: Figure 4 As shown, the conversion efficiency reached 46.15% in the first field of view (A, B), and 45% in the second field of view (C, D).

[0076] Wheat protoplasts obtained using the optimized incubation process in experimental group 2 were observed in both light and dark fields. The experimental results are as follows: Figure 5 As shown, the transient expression rate reached 53.84% in the first field of view (A, B), and the conversion efficiency reached 50% in the second field of view (C, D).

[0077] Bright-dark field observations were performed on wheat protoplasts obtained using the conventional incubation procedure in the control group. The results are as follows: Figure 6 As shown, the transient expression rate reached 4% in the first field of view (A, B), and the transient expression rate reached 8.33% in the second field of view (C, D).

[0078] The above transient expression rate is calculated as follows: the ratio of protoplasts carrying green fluorescent signals to intact protoplasts in the field of view is calculated.

[0079] The experimental results show that this study innovatively established a novel wheat protoplast incubation process, which significantly improved cell integrity and viability compared to traditional incubation methods, effectively reducing the risk of protoplast rupture and death during incubation. Simultaneously, this method can stably achieve a transient transformation efficiency of over 50%, greatly improving the reliability and operability of gene function verification and regulatory mechanism research. This method not only optimizes the application value of the wheat protoplast transient transformation system but also provides a more efficient and robust experimental system for verifying the editing efficiency of wheat target genes, studying gene function, and improving the molecular traits of superior traits.

Claims

1. A method for improving the success rate of a wheat protoplast transient transformation system, characterized in that, The incubation process is optimized, including the following steps: S1: using 10% bovine serum albumin solution to fully rinse the cell culture container for incubation, so that all the structures inside the container are infiltrated with 10% bovine serum albumin solution, and the waste liquid is discarded after rinsing; S2: using 900-1200 μL W5 solution to resuspend the wheat protoplast after introducing the exogenous gene, transferring to the cell culture container rinsed in S1, wrapping the cell culture container with tin foil, and incubating at room temperature for 12-16 h.

2. The method for improving the success rate of a wheat protoplast transient transformation system according to claim 1, characterized in that, The cell culture container for incubation is a centrifuge tube or a cell culture plate.

3. The method for improving the success rate of a wheat protoplast transient transformation system according to claim 2, characterized in that, The cell culture plate parameters are: 6 well, 3 ml, 9.60 cm 2 PS, i.e. polystyrene, flat bottom, TC treated, i.e. tissue culture treated, sterilized, free of DNase, RNase and pyrogen.

4. The method for improving the success rate of a wheat protoplast transient transformation system according to claim 3, characterized in that, The amount of 10% bovine serum albumin solution is 800-1200 μL; the amount of W5 solution is 1 ml.

5. The method for improving the success rate of a wheat protoplast transient transformation system according to claim 4, characterized in that, The extraction method of the wheat protoplast is: A1: preparing 7-12 d seedling age wheat seedlings, removing their roots and leaf tips, cutting into 0.5-1.0 mm sections along the vertical growth direction with a sharp knife, and placing them in a 50 mL volume small beaker containing 20 mL enzyme solution, wrapping the small beaker with tin foil and leaving a hole at the beaker opening, and vacuuming at 15 Hg, 50 kpa for 30 min; A2: placing the vacuumed small beaker on a shaker for 4-5 h, setting the conditions to be light-proof, 28°C, 45 rpm, and gently inverting and mixing every 1 hour to ensure that the wheat tissue attached to the wall fully contacts with the enzyme solution; after the reaction is completed, the shaker speed is adjusted to 80 rpm for 5 min to completely release the protoplasts from the mesophyll tissue; A3: taking a new 50 mL centrifuge tube, placing the cell screen rinsed with 1 mL W5 solution, filtering the enzyme solution in the A2 beaker, gently squeezing the residual wheat seedling tissue to fully release the protoplasts, until green liquid drops, and then washing the beaker used in A2 and the residual wheat seedling tissue in it with 5 mL of W5 solution twice, and collecting the liquid obtained by washing into the 50 mL centrifuge tube; A4: The liquid collected in A3 is centrifuged using a horizontal rotor, and the precipitate at the bottom of the centrifuge tube is the wheat protoplast after removing the supernatant. The wheat protoplast is purified using W5 solution, resuspended and centrifuged, and then resuspended using MMG solution, and the number of wheat protoplasts is adjusted to 2 x 10 5 / mL.

6. The method for improving the success rate of a wheat protoplast transient transformation system according to claim 5, characterized in that, The centrifugation parameters in A4 are 100g at room temperature for 10 min, 3 for the increase speed, and 3 for the decrease speed.

7. The method for improving the success rate of a wheat protoplast transient transformation system according to claim 6, characterized in that, Before centrifugation in A4, the state of the wheat protoplasts was observed and counted using a microscope and a hemocytometer, in preparation for adjusting the number of wheat protoplasts to 2 x 10 5 The amount of MMG solution used was calculated as follows: total volume (μL) = number of reactions required x 100 μL / reaction.

8. The method for improving the success rate of a wheat protoplast transient transformation system according to claim 7, characterized in that, The steps of the wheat protoplast transformation experiment are: B1 : Add 3-20 µg purified endotoxin-free plasmid to a sterile round bottom centrifuge tube, followed by 100 µL of protoplasts adjusted to 2 x 10 5 / mL of wheat protoplasts, mix gently; B2: adding 110 µL of PEG transformation solution, gently mixing to avoid air bubbles, and reacting at room temperature for 10-15 min; B3: adding 500 μL of W5 solution, gently mixing, and terminating the transformation; B4: centrifuging for 10 min to remove the supernatant, setting the centrifugation parameters to 200g at room temperature, 3 for the increase speed, and 3 for the decrease speed; then adding 500 μL of W5 solution, centrifuging for 10 min to remove the supernatant, setting the centrifugation parameters to 100g at room temperature, 3 for the increase speed, and 3 for the decrease speed, which completes the transformation of the wheat protoplast.

9. The method for improving the success rate of a wheat protoplast transient transformation system according to claim 8, characterized in that, The plasmid concentration in B1 is greater than 1000 ng / µL.

10. The method for improving the success rate of a wheat protoplast transient transformation system according to claim 9, characterized in that, The PEG transformation solution is completely dissolved in a 65°C water bath, and then cooled to room temperature before use.

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