Method for improving transformation efficiency of plant protoplast
By using sucrose enzymatic hydrolysis and centrifugation, highly active protoplasts were separated and enriched, solving the problem of protoplasts being easily broken during enzymatic hydrolysis and centrifugation, and achieving a significant improvement in protoplast conversion efficiency.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-22
- Publication Date
- 2026-04-10
AI Technical Summary
In existing technologies, plant protoplasts are prone to rupture during enzymatic hydrolysis, centrifugation, or transformation, resulting in low activity and thus affecting transformation efficiency.
Sucrose was used instead of mannitol as the enzymatic hydrolysate. The density difference between intact and broken protoplasts was used to separate and enrich highly active protoplasts by centrifugation. Competent protoplasts were then prepared and transformed using target plasmids.
It significantly improved the activity and transformation efficiency of protoplasts, and enhanced the stability and efficiency of downstream experiments.
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Figure CN121826044A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of plant cytology, and particularly relates to a method for improving the transformation efficiency of plant protoplasts. BACKGROUND
[0002] Plant protoplast refers to a plant cell with intact cell membrane and physiological activity after removing the cell wall by enzymatic or mechanical method. Due to the characteristic of no cell wall, plant protoplast has high application value in basic plant research and plant cell engineering, and is a good experimental system for researches such as distant hybrid breeding, polyploidy induction, organelle transfer, transient expression, gene editing, virus research, signal transduction, stress response and protein interaction.
[0003] The material for preparing plant protoplast is generally selected from tissues or organs with active metabolism, such as leaf, callus, shoot tip, young stem, cotyledon, hypocotyl and root tip, etc. However, the preparation efficiency is affected by many factors, including material source, enzymatic condition, osmotic pressure stabilizer and environmental factor. At present, the preparation method of Arabidopsis and tobacco leaf protoplast is relatively mature, and the preparation technology of gramineous plant protoplast has also made significant progress through optimization of enzymatic system and improvement of material selection in recent years.
[0004] At present, the most widely used experimental system in plant biology research is Arabidopsis leaf mesophyll protoplast, and the main preparation method of protoplast is to use enzymatic solution containing sucrose to enzymatically digest the leaf, and then to obtain protoplast through a series of washing processes. The method has the following advantages: ① Arabidopsis has short growth cycle, easy-to-obtain material and easy-to-digest genetic background; ② The experimental operation of protoplast is simple and does not require complex equipment, which can be carried out in ordinary laboratory. However, it also has some defects, such as: ① The protoplast after removing the cell wall is sensitive to osmotic pressure change and is easy to rupture during enzymatic digestion, centrifugation or transformation; ② The activity of protoplast is low due to the influence of plant growth state, enzymatic digestion time and centrifugation condition; and ③ The transformation efficiency of protoplast system is low due to the above reasons. Therefore, it is urgent to provide a new method for improving the transformation efficiency of plant protoplast. SUMMARY
[0005] The present application provides a method for improving the transformation efficiency of plant protoplast, which effectively improves the activity of plant protoplast and further improves the transformation efficiency of protoplast system, and better serves basic research and engineering application by using the method.
[0006] The technical scheme adopted by the present application is: The present application provides a method for improving the transformation efficiency of plant protoplast, and the steps are as follows: S1. Preparation of enzymatic hydrolysate: Each liter of enzymatic hydrolysate contains: 0.2mol~0.4mol sucrose, 20mmol MES at pH 5.7, 20mmol KCl, 5g~20g cellulase, 2g~6g dissociative enzyme, 10mmol CaCl2 and 1g BSA, and the remainder is made up to 1L with water. S2. Leaf enzymatic hydrolysis: Arabidopsis leaves that are 3 to 4 weeks old are used as enzymatic hydrolysis material; Arabidopsis leaves are chopped and placed in enzymatic hydrolysis solution, and enzymatically hydrolyzed at 25℃, 40 to 50 rpm in the dark for 2 to 4 hours. S3. Centrifugation: Collect the filtrate and centrifuge it at 4℃ for 1 min to 10 min at a rate of 50 g to 100 g. S4. Obtaining protoplasts: Collect the uppermost cells from S3, wash with 1 to 3 times the volume of W5 solution to obtain protoplasts; S5. Preparation of competent protoplasts: Add W5 solution to the protoplasts and place on ice for 30 min to obtain competent protoplasts; S6, MMG solution resuspension: MMG solution was added to competent protoplasts for resuspension to obtain protoplast resuspension; S7. Transform the protoplasmic resuspended solution using the target plasmid and incubate it.
[0007] Preferably, the sucrose content in each liter of enzymatic hydrolysate is 0.4 mol.
[0008] Preferably, each liter of enzymatic hydrolysate contains 10g of cellulase and 4g of dissociative enzyme.
[0009] Preferably, the enzymatic hydrolysis time in S2 is 3 hours.
[0010] Preferably, the centrifugation conditions in S3 are 100g for 10min.
[0011] Preferably, the sieve used to collect the filtrate in S3 has a mesh size of 200.
[0012] Preferably, the amount of W5 solution added in S4 is 3 times that of the topmost cells.
[0013] Preferably, the ratio of Arabidopsis thaliana leaves to enzymatic hydrolysate is 1g:20mL.
[0014] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a method for improving the efficiency of plant protoplast transformation, comprising the following steps: S1. Preparation of enzymatic hydrolysate: Each liter of enzymatic hydrolysate contains: 0.2 mol–0.4 mol sucrose, 20 mmol MES (pH 5.7), 20 mmol KCl, 5 g–20 g cellulase, 2 g–6 g dissociative enzyme, 10 mmol CaCl2, and 1 g BSA. The remainder is added to a final volume of 1 L with water. S2. Leaf enzymatic hydrolysis: Use Arabidopsis thaliana leaves from 3–4 weeks old as the hydrolysis material. Chop the leaves and place them in the enzymatic hydrolysate. Incubate at 25°C, 40–50 rpm in the dark for 2–4 hours. S3. Centrifugation: Collect the filtrate and centrifuge at 4°C. Centrifuge at 50g-100g for 1-10 minutes; S4, Obtain protoplasts: Collect the uppermost layer of cells from S3, wash with 1-3 times the volume of W5 solution to obtain protoplasts; S5, Prepare competent protoplasts: Add W5 solution to the protoplasts and place on ice for 30 minutes to obtain competent protoplasts; S6, Resuspend in MMG solution: Resuspend the competent protoplasts in MMG solution to obtain a protoplast resuspension; S7, Transform the protoplast resuspension using the target plasmid and incubate.
[0015] In this invention, sucrose is used to replace mannitol in the enzymatic hydrolysate for Arabidopsis thaliana leaves. Utilizing the density difference between intact and fragmented protoplasts, centrifugation is used to separate and purify them. Intact, highly active protoplasts are enriched in the upper layer of the hydrolysate, while fragmented, less active protoplasts accumulate at the bottom of the tube. The method described in this invention yields high-quality protoplasts, which significantly improve transformation efficiency and stability in downstream experiments. In contrast, traditional methods typically use mannitol to prepare the hydrolysate for mesophyll cell hydrolysis. After hydrolysis, all protoplasts, including cell fragments, accumulate at the bottom of the centrifuge tube, making it impossible to distinguish between protoplast activity and integrity, often resulting in poor and unstable downstream experimental results. Attached Figure Description
[0016] Figure 1 This is a flowchart of the protoplast isolation, purification, and transient transformation process.
[0017] Figure 2 The effect of sucrose concentration in the enzymatic hydrolysate on protoplast yield. A: Representative image; B: Statistical results of image A.
[0018] Figure 3 The effect of cellulase content on protoplast yield. A: Representative image; B: Statistical results of image A. Figure 3 In the diagram, C represents cellulase and M represents dissociative enzyme.
[0019] Figure 4 The effect of dissociation enzyme content on protoplast yield. A: Representative image; B: Statistical results of image A. Figure 3In the diagram, C represents cellulase and M represents dissociative enzyme.
[0020] Figure 5 The effect of enzymatic hydrolysis time on protoplast yield. A: Representative image; B: Statistical results of image A.
[0021] Figure 6 These are representative images of protoplasts separated by different centrifugal forces during gradient centrifugation.
[0022] Figure 7 These are representative images of protoplasts separated at different centrifugation times during gradient centrifugation.
[0023] Figure 8 The effect of the amount of W5 solution added on the protoplast yield.
[0024] Figure 9 For protoplast activity assay. A: Example 1; B: Comparative Example 1; C: Statistical results of A and B.
[0025] Figure 10 This is a graph showing the instantaneous gene conversion efficiency. A: Example 1; B: Comparative Example 1; C: Statistical results of A and B. Detailed Implementation
[0026] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.
[0027] The inventive concept of this invention is as follows: As a model plant in plant biology research, Arabidopsis thaliana has a relatively mature method for preparing protoplasts from its mesophyll cells. However, the protoplasts after cell wall removal are sensitive to changes in osmotic pressure and are prone to rupture during enzymatic hydrolysis, centrifugation, or transformation. Furthermore, the protoplasts are affected by factors such as their agglomerated growth state, enzymatic hydrolysis time, and centrifugation conditions, resulting in low protoplast activity. These factors contribute to the low transformation efficiency of the protoplast system. Therefore, this invention provides a method to improve the transformation efficiency of plant protoplasts. Compared with existing technologies, the improvements of this invention are as follows:
[0028] 1) Replace mannitol in the enzymatic hydrolysate of traditional methods with sucrose; 2) In this invention, after the enzymatic hydrolysis mixture is centrifuged, intact and highly active protoplasts are suspended in the upper layer of the enzymatic hydrolysate, while broken or abnormal protoplasts are aggregated in the lower layer of the enzymatic hydrolysate; whereas in the traditional method, both intact and broken, low-activity protoplasts exist at the bottom of the enzymatic hydrolysate and cannot be distinguished.
[0029] Based on the above two improvements, this invention provides a complete protoplast transformation system, the steps of which are as follows: S1. Preparation of enzymatic hydrolysate: Each liter of enzymatic hydrolysate contains: 0.2mol~0.4mol sucrose, 20mmol MES at pH 5.7, 20mmol KCl, 5g~20g cellulase, 2g~6g dissociative enzyme, 10mmol CaCl2 and 1g BSA, and the remainder is made up to 1L with water. S2. Leaf enzymatic hydrolysis: Arabidopsis leaves that are 3 to 4 weeks old are used as enzymatic hydrolysis material; Arabidopsis leaves are chopped and placed in enzymatic hydrolysis solution, and enzymatically hydrolyzed at 25℃, 40 to 50 rpm in the dark for 2 to 4 hours. S3. Centrifugation: Collect the filtrate and centrifuge it at 4℃ for 1 min to 10 min at a rate of 50 g to 100 g. S4. Obtaining protoplasts: Collect the uppermost cells from S3, wash with 1 to 3 times the volume of W5 solution to obtain protoplasts; S5. Preparation of competent protoplasts: Add W5 solution to the protoplasts and place on ice for 30 min to obtain competent protoplasts; S6, MMG solution resuspension: MMG solution was added to competent protoplasts for resuspension to obtain protoplast resuspension; S7. Transform the protoplasmic resuspended solution using the target plasmid and incubate it.
[0030] The method flow described in this invention is as follows: Figure 1 The protoplasts obtained by the method described in this invention are of high quality and are significantly more efficient than traditional methods for downstream experiments such as transient gene transformation.
[0031] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.
[0032] The list of abbreviations for this invention is shown in Table 1.
[0033] Table 1. List of abbreviations for this invention The basic information of the cellulase and ionizing enzyme used in this invention is as follows: Cellulase and ionizing enzyme: both purchased from Yakult, with activity >10000U / g.
[0034] Example 1 A method for improving the efficiency of plant protoplast transformation is as follows: S1. Prepare the enzyme hydrolysate.
[0035] 1) Prepare the enzyme stock solution using the following formula: Each liter of enzyme stock solution contains: 0.4 mol sucrose, 20 mmol MES at pH 5.7, 20 mmol KCl, and the remainder is made up to 1 L with water.
[0036] 2) Preparation of enzymatic hydrolysate: Continue to add cellulase and cleavage enzyme to the enzyme stock solution, so that the final concentrations of cellulase and cleavage enzyme are 0.01 g / mL and 0.004 g / mL, respectively. Then heat in a 55°C water bath for 10 min and cool to room temperature; then add CaCl2 and BSA, so that the final concentrations of CaCl2 and BSA are 10 mM and 0.001 g / mL, respectively, to obtain the enzymatic hydrolysate.
[0037] S2, leaf enzymatic hydrolysis.
[0038] Take healthy, non-bolting Arabidopsis leaves that have grown for 4 weeks and place them in a container filled with double-distilled water. Take 1g of leaves and cut them into 1mm wide strips with a scalpel. Place the strips in the enzymatic hydrolysate at a ratio of 1g:20mL. Use a water bath shaker in the dark at 25℃ and 40rpm for 3 hours, until no obvious mesophyll tissue remains. At this point, the hydrolysate will turn dark green.
[0039] S3, centrifugation.
[0040] Filter the solution using a 200-mesh sieve and discard the leaves. Collect the filtrate and transfer it to a 50 mL centrifuge tube. Centrifuge the tube at 4°C and 100 g for 10 minutes until the filtrate is clearly separated into layers.
[0041] S4. Obtain protoplasts.
[0042] Carefully aspirate the top layer of dark green solution containing intact protoplasts using a 1 mL pipette, place it in a new 50 mL centrifuge tube, and gently wash with 3 times the volume of ice-cold W5 solution. Centrifuge at 4°C and 100 g for 1 min, and the protoplasts will precipitate at the bottom of the solution.
[0043] The W5 solution formulation is: 154mM NaCl, 125mM CaCl2, 5mM KCl, 2mM MES at pH 5.7, and water.
[0044] S5. Preparation of competent protoplasts.
[0045] Discard the supernatant from the previous step, resuspend the protoplast precipitate in 4 mL of ice-cold W5 solution, and place on ice for 30 min to obtain competent protoplasts.
[0046] Subsequently, competent protoplasts were taken and stained with the cell membrane dye FDA for observation.
[0047] Resuspended in S6 and MMG solutions.
[0048] The unstained competent protoplasts obtained above were centrifuged at 25°C and 100g for 1 min. The supernatant was discarded, and the precipitated cells were resuspended in 0.5 mL of MMG solution to obtain a protoplast resuspension. This provides an ideal cation and osmotic pressure environment for the binding of protoplasts to the DNA-PEG complex, thereby maximizing the transformation efficiency.
[0049] The MMG solution formulation is as follows: 0.4M mannitol, 4mM MES at pH 5.7, 15mM MgCl2, and water.
[0050] S7, Transformation.
[0051] Take 20 μg of PBI221-GFP plasmid (1 g / L) into a 1.5 mL sterile centrifuge tube, add 100 μL of freshly prepared protoplasmic resuspension, and then add 110 μL of PEG / Ca. 2+ For the conversion solution, use a 200μL pipette tip with the tip cut off to repeatedly pipette and mix, avoiding vigorous handling that could cause protoplast rupture. Let stand at room temperature for 20-30 minutes.
[0052] PEG / Ca 2+ The formula for the conversion solution is as follows: PEG / Ca per liter 2+ The conversion solution contains: 400g PEG4000, 0.2mol mannitol, 0.1mol CaCl2, and the remainder is made up with water.
[0053] S8. Continue to add 440 μL of W5 solution, mix thoroughly, and centrifuge at 100 g for 1 min at 25 °C.
[0054] S9. Carefully aspirate the supernatant, add 1 mL of W5 solution to the precipitate, and gently resuspend the protoplasts.
[0055] S10. The transformed protoplast solution was incubated at 25°C for 16 hours in the dark, and the fluorescence signal was observed using a laser confocal microscope.
[0056] Example 2 A method for improving the efficiency of plant protoplast transformation is as follows: Each liter of enzyme stock solution contains: 0.2 mol sucrose, 20 mmol MES (pH 5.7), 20 mmol KCl, and the remainder is made up to 1 L with water. Everything else is exactly the same as in Example 1.
[0057] Example 3 A method for improving the efficiency of plant protoplast transformation is as follows: Each liter of enzyme stock solution contains: 0.6 mol sucrose, 20 mmol MES (pH 5.7), 20 mmol KCl, and the remainder is made up to 1 L with water. Everything else is exactly the same as in Example 1.
[0058] As shown in Examples 1-3, the protoplast yield was highest when the sucrose concentration in the enzymatic hydrolysate was 0.4 M. (See the results below.) Figure 2 .
[0059] Example 4 A method for improving the efficiency of plant protoplast transformation is as follows: The final concentration of cellulase in the enzymatic hydrolysate was 0.005 g / mL, and the rest was exactly the same as in Example 1.
[0060] Example 5 A method for improving the efficiency of plant protoplast transformation is as follows: The final concentration of cellulase in the enzymatic hydrolysate was 0.015 g / mL, and the rest was exactly the same as in Example 1.
[0061] Example 6 A method for improving the efficiency of plant protoplast transformation is as follows: The final concentration of cellulase in the enzymatic hydrolysate was 0.02 g / mL, and the rest was exactly the same as in Example 1.
[0062] As shown in Examples 1, 4-6, the highest protoplast yield was achieved when the final concentration of cellulase in the enzymatic hydrolysate was 0.01 g / mL. See the results below. Figure 3 .
[0063] Example 7 A method for improving the efficiency of plant protoplast transformation is as follows: The final concentration of the isolated enzyme in the enzymatic hydrolysate was 0.002 g / mL, and the rest was exactly the same as in Example 1.
[0064] Example 8 A method for improving the efficiency of plant protoplast transformation is as follows: The final concentration of the isolated enzyme in the enzymatic hydrolysate was 0.006 g / mL, and the rest was exactly the same as in Example 1.
[0065] As shown in Examples 1, 7, and 8, the highest protoplast yield was achieved when the amount of cellulase added was 0.004 g / mL. See the results below. Figure 4 .
[0066] Example 9 A method for improving the efficiency of plant protoplast transformation is as follows: The enzymatic hydrolysis time for leaves in S2 was 2 hours, and the rest was exactly the same as in Example 1.
[0067] Example 10 A method for improving the efficiency of plant protoplast transformation is as follows: The enzymatic hydrolysis time for leaves in S2 was 4 hours, and the rest was exactly the same as in Example 1.
[0068] As shown in Examples 1, 9, and 10, the highest protoplast yield was obtained when the enzymatic hydrolysis time was 3 hours. The results are shown below. Figure 5 .
[0069] Example 11 A method for improving the efficiency of plant protoplast transformation is as follows: During centrifugation in S3, the rotation speed was 50g, and the rest was exactly the same as in Example 1.
[0070] Example 12 A method for improving the efficiency of plant protoplast transformation is as follows: During centrifugation in S3, the rotation speed was 200g, and the rest was exactly the same as in Example 1.
[0071] As shown in Examples 1, 11, and 12, the protoplast separation effect was best when the rotation speed was 100g. The results are shown below. Figure 6 .
[0072] Example 13 A method for improving the efficiency of plant protoplast transformation is as follows: During centrifugation in S3, the centrifugation time was 1 minute, and the rest was exactly the same as in Example 1.
[0073] Example 14 A method for improving the efficiency of plant protoplast transformation is as follows: During centrifugation in S3, the centrifugation time was 4 minutes, and the rest was exactly the same as in Example 1.
[0074] Example 15 A method for improving the efficiency of plant protoplast transformation is as follows: During centrifugation in S3, the centrifugation time was 7 minutes, and the rest was exactly the same as in Example 1.
[0075] As shown in Examples 1, 13-15, the protoplast separation effect is best when the centrifugation time is 10 min. The results are shown below. Figure 7 .
[0076] Example 16 A method for improving the efficiency of plant protoplast transformation is as follows: In S4, when obtaining complete protoplasts, the amount of W5 solution added is 1 volume of the protoplasts, and the rest is exactly the same as in Example 1.
[0077] Example 17 A method for improving the efficiency of plant protoplast transformation is as follows: In S4, when obtaining complete protoplasts, the amount of W5 solution added is twice the volume of the protoplasts, and the rest is exactly the same as in Example 1.
[0078] As shown in Examples 1, 16, and 17, the highest protoplast yield was achieved when the amount of W5 solution added was three times the volume of the protoplasts. See the results below. Figure 8 .
[0079] Comparative Example 1 A method for improving the efficiency of plant protoplast transformation is as follows: S1. Prepare the enzyme hydrolysate.
[0080] 1) Prepare the enzyme stock solution using the following formula: Each liter of enzyme stock solution contains: 0.4 mol M mannitol, 20 mmol MES at pH 5.7, 20 mmol KCl, and the remainder is made up to 1 L with water.
[0081] 2) Preparation of enzyme hydrolysate: Same as in Example 1.
[0082] S2, leaf enzymatic hydrolysis.
[0083] Take healthy, non-bolting Arabidopsis leaves that have been growing for 4 weeks and place them in a container filled with double-distilled water. Cut the leaves into 1mm wide strips with a scalpel and place them in the enzymatic hydrolysate at a ratio of 1g:20mL. Enzymatically hydrolyze the leaves in a water bath at 25℃ and 40rpm for 3 hours in the dark, until no obvious mesophyll tissue remains and the hydrolysate turns dark green.
[0084] S3, centrifugation.
[0085] Filter the solution using a 200-mesh sieve and discard the leaves. Collect the filtrate and transfer it to a 50 mL centrifuge tube. Centrifuge the tube at 4°C and 100 g for 10 min.
[0086] S4. Obtain the complete protoplast.
[0087] Discard the supernatant and gently wash the precipitate with ice-cold W5 solution. Then centrifuge at 4°C and 100g for 1 min.
[0088] S5~S10 are exactly the same as in Example 1.
[0089] The activity and transformation efficiency of the protoplasts prepared in Example 1 and Comparative Example 1 were verified as follows: 1. Protoplast activity.
[0090] Protoplast production statistical analysis: Protoplast production and viability were statistically analyzed using a hemocytometer.
[0091] .
[0092] In the formula, A Protoplast yield, in units per fresh weight; B Number of protoplasts per milliliter, expressed as protoplasts / ml; C Total volume of protoplast hydrolysate, in ml; D Total mass of leaves, in fresh weight.
[0093] See results Figure 9 The protoplasts prepared in Example 1 of this invention have good integrity and high activity, and their activity can reach more than 80% as detected by FDA fluorescent staining; while the protoplasts prepared in Comparative Example 1 have an activity of less than 60%.
[0094] 2. Conversion efficiency.
[0095] See results Figure 10 The transformed protoplasts were observed using a confocal microscope. Statistical analysis showed that the protoplasts extracted in Example 1 of this invention achieved a transformation efficiency of over 30%, while those extracted using traditional methods achieved less than 10%.
[0096] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0097] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.
Claims
1. A method for improving the efficiency of plant protoplast transformation, characterized in that, The steps are as follows: S1. Preparation of enzymatic hydrolysate: Each liter of enzymatic hydrolysate contains: 0.2mol~0.4mol sucrose, 20mmol MES at pH 5.7, 20mmol KCl, 5g~20g cellulase, 2g~6g dissociative enzyme, 10mmol CaCl2 and 1g BSA, and the remainder is made up to 1L with water. S2. Leaf enzymatic hydrolysis: Arabidopsis leaves that are 3 to 4 weeks old are used as enzymatic hydrolysis material; Arabidopsis leaves are chopped and placed in enzymatic hydrolysis solution, and enzymatically hydrolyzed at 25℃, 40 to 50 rpm in the dark for 2 to 4 hours. S3. Centrifugation: Collect the filtrate and centrifuge it at 4℃ for 1 min to 10 min at a rate of 50 g to 100 g. S4. Obtaining protoplasts: Collect the uppermost cells from S3, wash with 1 to 3 times the volume of W5 solution to obtain protoplasts; S5. Preparation of competent protoplasts: Add W5 solution to the protoplasts and place on ice for 30 min to obtain competent protoplasts; S6, MMG solution resuspension: MMG solution was added to competent protoplasts for resuspension to obtain protoplast resuspension; S7. Transform the protoplasmic resuspended solution using the target plasmid and incubate it.
2. The method as described in claim 1, characterized in that, The sucrose content in each liter of enzymatic hydrolysate is 0.4 mol.
3. The method as described in claim 1, characterized in that, Each liter of enzymatic hydrolysate contains 10g of cellulase; Each liter of enzymatic hydrolysate contains 4g of ionizing enzyme.
4. The method as described in claim 1, characterized in that, The enzymatic hydrolysis time in S2 is 3 hours.
5. The method as described in claim 1, characterized in that, The centrifugation conditions in S3 are 100g for 10 minutes.
6. The method as described in claim 1, characterized in that, The sieve used to collect the filtrate in S3 has a mesh size of 200.
7. The method as described in claim 1, characterized in that, The amount of W5 solution added in S4 is three times that of the topmost cells.
8. The method as described in claim 1, characterized in that, The ratio of Arabidopsis thaliana leaves to enzymatic hydrolysate was 1g:20mL.