Separation and instantaneous conversion method of green Chinese onion leaf protoplast

Abstract

The invention discloses a separation and instantaneous conversion method of green Chinese onion leaf protoplast, and belongs to the technical field of protoplast preparation, the method comprises the following steps: 1) culturing green Chinese onion leaves; 2) separation and purification of green Chinese onion leaf protoplast; 3) determining the yield and vitality of the green Chinese onion leaf protoplast; 4) determining an optimal enzymolysis condition; and (5) PEG-mediated protoplast transformation: determining the optimal concentrations of cellulase, macerozyme and mannitol in the enzymolysis process, and determining the optimal enzymolysis time, so that the separated green Chinese onion leaf protoplast is higher in yield and activity, and the separated green Chinese onion leaf protoplast can not only be used for instantaneous transformation, but also can be used for enzymolysis of the green Chinese onion leaf protoplast. And a technical support is provided for research on molecular biology of the green Chinese onions.

Description

Technical Field

[0001] The invention relates to the technical field of protoplast preparation, and in particular to a method for separating and instantaneously transforming green onion leaf protoplasts. Background Art

[0002] As a widely cultivated plant, green onion is not only an indispensable condiment in daily diet, but also rich in a variety of compounds that are beneficial to health. With the continuous advancement of molecular breeding technology, genetic research on green onion has achieved many remarkable results, including the Af4CL gene related to flavonoid biosynthesis, the AfMAX1 gene that regulates tillering, and the AfCER2-LIKE1 gene related to wax synthesis. Although these genes have been cloned and preliminary functional analysis has been performed, the functional analysis of the green onion genome still faces challenges due to the lack of a stable genetic transformation system. In order to overcome this problem, it is crucial to establish an effective homologous expression system.

[0003] As an important experimental material, protoplasts play a key role in many biological fields. They have an application value that cannot be ignored in studying protein functions, cell signal transduction, gene expression regulation, cell biological processes, and comprehensive analysis of multi-omics. In particular, they play a key role in the evaluation of CRISPR / cas9-mediated genome editing efficiency. In some species, regenerated plants with desired traits can also be obtained, thereby accelerating plant genetic improvement and seed breeding. At present, there are still many species for which protoplast isolation methods have not been established. In addition, efficient protoplast systems remain challenging because different cell wall components require specific optimization. Enzymatic hydrolysis is often used for protoplast preparation, and the composition and conditions of the enzyme are the most critical factors affecting protoplast isolation and activity. Among many factors, the composition and pH value of the enzyme solution, the temperature, speed and duration of the enzymatic hydrolysis, different enrichment treatments, and the properties of the prefabricated solution will affect the yield and activity of the obtained protoplasts.

[0004] Protoplast transient transformation and expression systems play an important role in modern molecular biology analysis, such as subcellular localization, protein interaction and transcription factor activity detection. Among the commonly used methods for plant protoplast genetic transformation, PEG-mediated transfection is widely used because of its simple transfection operation, wide applicability and relatively stable efficiency. It is often used to introduce DNA, RNA or protein molecules into protoplasts.

[0005] There is no effective isolation and transient transformation protocol for the preparation of protoplasts of Welsh onion. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a method for separating and transiently transforming protoplasts from green onion leaves, which is achieved through the following technical solutions.

[0007] A method for separating and transiently transforming green onion leaf protoplasts, the method comprising the following steps:

[0008] 1) Cultivation of green onion leaves:

[0009] Seeds of the “Zhangqiu Dawutong” green onion variety were selected and planted in a culture medium made of nutrient soil and vermiculite in a mass ratio of 3:1, with a constant temperature of 23°C and a 12-hour alternating light and dark cycle. Seedlings grown for 20 days were selected and their new leaves were picked as protoplast extraction materials;

[0010] 2) Isolation and purification of onion leaf protoplasts:

[0011] Prepare enzymatic hydrolysis solutions with different concentrations of cellulase, cleavage enzyme, and mannitol; use different enzymatic hydrolysis solutions to enzymatically hydrolyze green onion leaves for different periods of time to separate protoplasts in the green onion leaves; and purify the protoplasts;

[0012] 3) Determine the yield and vitality of green onion leaf protoplasts:

[0013] Protoplast counts were performed under a fluorescence microscope using a double-chamber hemocytometer to calculate the yield and viability of protoplasts.

[0014] 4) Determine the optimal enzymatic hydrolysis conditions:

[0015] According to the data in step 3), determining the optimal concentrations of cellulase, cleavage enzyme and mannitol in the enzymolysis solution, and determining the optimal enzymolysis time;

[0016] 5) PEG-mediated protoplast transformation:

[0017] The plasmid DNA of the plant expression vector pAL902-GFP containing the GFP gene is transformed into the green onion leaf protoplasts separated under the optimal enzymatic hydrolysis conditions by PEG-mediated method; the integration of GFP in the transfected protoplasts is detected under a fluorescence microscope, wherein the GFP is green fluorescent protein and the PEG is polyethylene glycol.

[0018] Preferably, in the step 2):

[0019] The enzymatic hydrolysate comprises the following components: 20mmol / L KCl, 20mmol / L MES, 10mmol / L CaCl2, 1.0g / L BSA, cellulase, macerate and mannitol, wherein the MES is 4-morpholineethanesulfonic acid with a pH value of 5.8, and the BSA is a bovine serum albumin solution;

[0020] The green onion leaves were rinsed with ddH2O, wiped with absorbent paper and placed in a petri dish to dry; the dried green onion leaves were cut into pieces with a width of 0.5 to 1.0 mm; 0.5 g of the pieces were weighed and mixed with 10 mL of enzymatic hydrolysate; the mixture of the enzymatic hydrolysate and the pieces was placed in a dark environment at 26°C, and after a vacuum treatment for 0.5 h, the mixture was shaken at 50 r / min for 4 to 10 h to release the protoplasts in the green onion leaves;

[0021] After the enzymatic hydrolysis, the mixed solution was filtered through a cell strainer with a pore size of 70 μm, and the filtered liquid was collected in a 50 mL round-bottom centrifuge tube. An equal volume of pre-cooled W5 solution was added to the filtered protoplast suspension, and the mixture was centrifuged at 100 rcf for 3 min. The supernatant was discarded, and 5 mL of W5 solution was added to the obtained precipitate to resuspend the protoplasts. The mixture was placed in ice and away from light for 30 min, the supernatant was discarded, and the precipitate was resuspended in 1 mL of MMG solution.

[0022] Preferably, the W5 solution comprises 4mmol / L MES, 154mmol / L NaCl, 125mmol / L CaCl2 and 5mmol / L KCl, and the pH value of the W5 solution is 5.7; the MMG solution comprises 0.4mol / L mannitol, 15mmol / L MgCl2 and 4mmol / L MES.

[0023] Preferably, in step 3), every 100 μL of protoplast resuspension is stained with 1 μL of 0.02% FDA solution, incubated in the dark for 5 min, 20 μL of the mixture is taken, and the protoplasts are counted using a hemocytometer, and the protoplasts emitting green fluorescence are counted as viable.

[0024] Preferably, in the step 3), the calculation formulas for the yield and vitality of the protoplasts are as follows:

[0025] Protoplast yield (g -1 ) = number of protoplasts in the large square of the counting plate × 10 4 / Mass of enzymatic hydrolysis material (g);

[0026] Protoplast viability (%) = (number of protoplasts emitting green fluorescence / total number of protoplasts) × 100%.

[0027] Preferably, the method for determining the optimal cellulose and cleavage enzyme concentrations in the enzymatic hydrolyzate is as follows:

[0028] In step 2), an orthogonal test group was set, and all test groups had the same mannitol concentration and enzymolysis time. The two influencing factors of the orthogonal test group were the concentrations of cellulase and macerate. The test concentrations of cellulase were 5 g / L, 10 g / L and 15 g / L, and the test concentrations of macerate were 3 g / L, 5 g / L and 7 g / L, thereby setting 9 control tests;

[0029] The yield and activity of protoplasts in each control test were statistically analyzed to determine the optimal concentrations of cellulase and macerate.

[0030] Preferably, the method for determining the optimal mannitol concentration and optimal enzymolysis time in the enzymolysis solution is as follows:

[0031] In step 2), a control experiment group was set, under the optimum concentrations of cellulase and macerate, the enzymolysis time was controlled to be unchanged, the concentration of mannitol was set to 0.3, 0.4, 0.5, 0.6 mol / L, respectively, the yield and viability of the protoplasts in each control experiment were counted, and the optimum concentration of mannitol was determined;

[0032] In step 2), a control experimental group is set, and the enzymolysis time is set to 4, 6, 8, and 10 h at the optimal concentrations of cellulase, macerate and mannitol, and the yield and activity of protoplasts in each control experiment are statistically analyzed to determine the optimal enzymolysis time.

[0033] Preferably, in the step 5), the plant expression vector pAL902-GFP is amplified, the plasmid DNA is purified, 20 μg of the plasmid DNA is mixed with 200 ul of the prepared protoplast resuspension in a 2 mL round-bottom centrifuge tube; then 200 ul of PEG solution is added, and the round-bottom centrifuge tube is inverted to prepare a suspension; the suspension is allowed to stand at room temperature for 20 min, 1 mL of pre-cooled W5 solution is added for washing, the suspension is centrifuged at 100 rcf for 2 min, and the supernatant is discarded; 1 mL of W5 solution is added again for resuspending, and the suspension is then placed in a 35 mm cell culture dish for culture, incubated in the dark at 24° C. for 20 h, and the integration of GFP in the transfected protoplasts is detected under a fluorescence microscope.

[0034] Preferably, the plant expression vector pAL902-GFP is amplified using the Escherichia coli DB3.1 strain, and then the plasmid DNA is extracted and purified, and the purity and concentration of the plasmid DNA are determined using electrophoresis technology to ensure that its OD260 / OD280 ratio is between 1.8 and 2.0, the concentration reaches 1 μg / μL, and it is stored at -20°C.

[0035] The beneficial effects of the present invention are that a method for separating green onion leaf protoplasts is determined, the optimal concentrations of cellulase, maceration enzyme and mannitol in the enzymatic hydrolysis process are determined, and the optimal enzymatic hydrolysis time is determined, so that the yield and vitality of the separated green onion leaf protoplasts are higher, and the separated green onion leaf protoplasts can not only be used for instantaneous transformation, but also provide technical support for the research of green onion in molecular biology. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solution of the present invention, the drawings required for use in the description of the specific implementation methods will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1 : Schematic diagram of the process of extraction and transformation of green onion protoplasts in the present invention;

[0038] Figure 2 :Image of protoplasts isolated from green onion leaves under optimal enzymatic hydrolysis conditions;

[0039] Figure 3 :Effects of mannitol and enzymatic hydrolysis time on the yield and vitality of protoplasts from green onion leaves;

[0040] Figure 4 :Observation images of transient expression of GFP gene in protoplasts of green onion leaves under fluorescence microscope. DETAILED DESCRIPTION

[0041] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] The specific implementation of the present invention is as follows: a method for separating and transiently transforming green onion leaf protoplasts, the method comprising the following steps:

[0043] 1) Cultivation of green onion leaves:

[0044] Seeds of the "Zhangqiu Da Wutong" green onion variety were selected and planted in a culture medium made of a mixture of nutrient soil and vermiculite in a mass ratio of 3:1, provided with a constant temperature of 23°C and a 12-hour alternating light and dark cycle. Seedlings that had grown for 20 days were selected and their new leaves were picked as protoplast material.

[0045] 2) Isolation and purification of onion leaf protoplasts:

[0046] Enzyme hydrolysis solutions with different concentrations of cellulase, cleavage enzyme and mannitol are prepared; different enzyme hydrolysis solutions are used to enzymatically hydrolyze green onion leaves for different time periods to separate protoplasts in the green onion leaves; and the protoplasts are purified.

[0047] The enzymatic hydrolysate includes the following components: 20mmol / L KCl, 20mmol / L MES, 10mmol / L CaCl2, 1.0g / L BSA, cellulase, macerate and mannitol, MES is 4-morpholineethanesulfonic acid, its pH value is 5.8, BSA is bovine serum albumin solution;

[0048] The green onion leaves were rinsed with ddH2O, wiped with absorbent paper and placed in a petri dish to dry; the dried green onion leaves were cut into pieces with a width of 0.5 to 1.0 mm; 0.5 g of the pieces were weighed and mixed with 10 mL of enzymatic hydrolysate; the mixture of the enzymatic hydrolysate and the pieces was placed in a dark environment at 26°C, and after a vacuum treatment for 0.5 h, the mixture was shaken at 50 r / min for 4 to 10 h to release the protoplasts in the green onion leaves;

[0049] After the enzymatic hydrolysis, the mixed solution was filtered through a cell strainer with a pore size of 70 μm, and the filtered liquid was collected in a 50 mL round-bottom centrifuge tube. An equal volume of pre-cooled W5 solution was added to the filtered protoplast suspension, and the mixture was centrifuged at 100 rcf for 3 min. The supernatant was discarded, and 5 mL of W5 solution was added to the obtained precipitate to resuspend the protoplasts. The mixture was placed in ice and away from light for 30 min, the supernatant was discarded, and the precipitate was resuspended in 1 mL of MMG solution.

[0050] W5 solution includes 4mmol / L MES, 154mmol / L NaCl, 125mmol / L CaCl2 and 5mmol / L KCl, and the pH value of W5 solution is 5.7; MMG solution includes 0.4mol / L mannitol, 15mmol / L MgCl2 and 4mmol / L MES.

[0051] 3) Determine the yield and vitality of green onion leaf protoplasts:

[0052] The protoplast counts were performed using a double-chamber hemacytometer under a fluorescence microscope to calculate the protoplast yield and viability.

[0053] Every 100 μL of protoplast resuspension was stained with 1 μL of 0.02% FDA solution and incubated in the dark for 5 min. 20 μL of the mixture was taken and the protoplasts were counted using a hemocytometer. Protoplasts emitting green fluorescence were counted as viable.

[0054] The calculation formula for protoplast yield and vitality is as follows:

[0055] Protoplast yield (g -1 ) = number of protoplasts in the large square of the counting plate × 10 4 / Mass of enzymatic hydrolysis material (g);

[0056] Protoplast viability (%) = (number of protoplasts emitting green fluorescence / total number of protoplasts) × 100%.

[0057] 4) Determine the optimal enzymatic hydrolysis conditions:

[0058] Determine the optimal cellulase, cleavage enzyme and mannitol concentrations in the enzymatic hydrolysis solution, and determine the optimal enzymatic hydrolysis time.

[0059] The method for determining the optimal cellulose and cleavage enzyme concentrations in the enzymatic hydrolysate is as follows:

[0060] Step 2) in, orthogonal test group is set, all test groups have identical mannitol concentration and enzymolysis time, two influencing factors of orthogonal test group are the concentration of cellulase and analyte, the test concentration of cellulase is 5g / L, 10g / L and 15g / L, the test concentration of analyte is 3g / L, 5g / L and 7g / L, 9 control experiments are set with this. The output and the vigor of protoplasts in each control experiment are counted.

[0061] The results of each control test are shown in Table 1:

[0062] Table 1. The orthogonal test results of the protoplast isolation conditions of green onion leaves

[0063]

[0064] As shown in Table 1, 10 g / L cellulase and 7 g / L macerate (Control Experiment 6) showed the best separation effect among all treatments. Under the conditions of Control Experiment 6, the yield of protoplasts reached 1.84×106 g-1 and the activity was 89.67%.

[0065] The data in Table 1 were analyzed by range analysis to obtain Table 2.

[0066] Table 2 Results of orthogonal test range analysis of onion leaf protoplast isolation conditions

[0067]

[0068] The results of the range analysis in Table II show that the effect of macerate on protoplast yield is greater than that of cellulase, which indicates that macerate plays a more critical role in the separation process and helps to release protoplasts more effectively.

[0069] ANOVA and multiple comparisons were performed to obtain Table III.

[0070] Table 3 Results of orthogonal test variance analysis of onion leaf protoplast isolation conditions

[0071]

[0072] Table 3 further confirms that cellulase and macerate have a very significant effect on the experimental results. These findings emphasize the importance of the two enzymes in optimizing the conditions for protoplast isolation. Taking into account the experimental results, the best enzymatic solution formula was determined for the isolation of green onion leaf protoplasts: 10g / L cellulase and 7g / L macerate.

[0073] The method for determining the optimal mannitol concentration in the enzymatic hydrolysis solution and the optimal enzymatic hydrolysis time is as follows:

[0074] In step 2), a control experiment group was set. Under the optimal concentrations of cellulase and macerate (10 g / L cellulase and 7 g / L macerate), the enzymolysis time was controlled to be constant, and the concentration of mannitol was set to 0.3, 0.4, 0.5, and 0.6 mol / L, respectively. The yield and vigor of protoplasts in each control experiment were counted to determine the optimal concentration of mannitol.

[0075] like Figure 3 As shown in Figure 2, with the gradient change of mannitol concentration, the yield of protoplasts first increased. When 0.4 mol / L mannitol was added, the yield of protoplasts reached a peak of 3.3×10 6 g -1 , the activity was 90%. Further increase in mannitol concentration resulted in decreased yield and activity, and it was determined that the optimal concentration of mannitol was 0.4 mol / L.

[0076] In step 2), a control experiment group was set, and the enzymolysis time was set to 4, 6, 8, and 10 h at the optimal concentrations of cellulase, macerate and mannitol (10 g / L cellulase, 7 g / L macerate, and 0.4 mol / L mannitol), and the yield and vigor of the protoplasts in each control experiment were counted to determine the optimal enzymolysis time.

[0077] like Figure 3 As shown in the figure, the experiment was carried out in a solution containing 0.4 mol / L mannitol, 10 g / L cellulase and 7 g / L macerate. The yield and activity of protoplasts were counted at 4, 6, 8, and 10 h of digestion time. The enzymatic hydrolysis time had no significant effect on the activity of protoplasts, but the yield had a significant difference. As the time went on, the yield first increased (3.3 × 10 6 g -1 ), and then decreased to 1.3×10 6 g -1 The optimal processing time is 6h.

[0078] In summary, 10 g / L cellulase, 7 g / L macerate and 0.4 mol / L mannitol, enzymatic hydrolysis for 6 h, is the best choice for isolating protoplasts from green onion leaves.

[0079] like Figure 2 As shown, Figure 2 In the middle, a is the protoplast after separation and purification. Figure 2 (b) shows protoplasts detected using a fluorescence microscope, and the scale bar = 200 μm.

[0080] from Figure 2 It can be seen that under the optimal enzymatic hydrolysis conditions, the yield and quality of the obtained protoplasts reached a high level and can be used for subsequent transformation experiments.

[0081] 5) PEG-mediated protoplast transformation:

[0082] The plasmid DNA of the plant expression vector pAL902-GFP containing the GFP gene was transformed into the green onion leaf protoplasts separated under the optimal enzymatic hydrolysis conditions by PEG-mediated method; the integration of GFP in the transfected protoplasts was detected under a fluorescence microscope, GFP is green fluorescent protein, and PEG is polyethylene glycol.

[0083] Amplify the plant expression vector pAL902-GFP, purify the plasmid DNA, mix 20 μg of plasmid DNA with 200 ul of the prepared protoplast resuspension in a 2 mL round-bottom centrifuge tube; then add 200 ul of PEG solution, invert the round-bottom centrifuge tube to make a suspension; let stand at room temperature for 20 min, add 1 mL of pre-cooled W5 solution to wash, centrifuge at 100 rcf for 2 min and discard the supernatant; add 1 mL of W5 solution again to resuspend, then place it in a 35 mm cell culture dish, incubate it in the dark at 24 ° C for 20 h, and detect the integration of GFP in the transfected protoplasts under a fluorescence microscope.

[0084] The plant expression vector pAL902-GFP was amplified using the Escherichia coli DB3.1 strain, and then the plasmid DNA was extracted and purified. The purity and concentration of the plasmid DNA were determined using electrophoresis technology to ensure that its OD260 / OD280 ratio was between 1.8 and 2.0, the concentration reached 1 μg / μL, and it was stored at -20°C.

[0085] like Figure 4 As shown, Figure 4 Ac represent the bright field, GFP, and fused field images, respectively.

[0086] from Figure 4 A clear and bright green fluorescence signal can be observed in c, indicating that the GFP gene has been successfully expressed with an efficiency of (10.47±2.63)%.

[0087] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to only specific implementation methods. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for separating and transiently transforming green onion leaf protoplasts, characterized in that: The method comprises the following steps: 1) Cultivation of green onion leaves: Seeds of the "Zhangqiu Dawutong" scallion variety were selected and planted in a culture medium made of a mixture of nutrient soil and vermiculite in a mass ratio of 3:1, with a constant temperature of 23°C and a 12-hour alternating light and dark cycle. Seedlings grown for 20 days were selected and their new leaves were picked as protoplast extraction materials; 2) Isolation and purification of onion leaf protoplasts: Prepare enzymatic hydrolysis solutions with different concentrations of cellulase, cleavage enzyme, and mannitol; use different enzymatic hydrolysis solutions to enzymatically hydrolyze green onion leaves for different periods of time to separate protoplasts in the green onion leaves; and purify the protoplasts; 3) Determine the yield and vitality of green onion leaf protoplasts: Protoplast counts were performed under a fluorescence microscope using a double-chamber hemocytometer to calculate the yield and viability of protoplasts. 4) Determine the optimal enzymatic hydrolysis conditions: According to the data in step 3), determining the optimal concentrations of cellulase, cleavage enzyme and mannitol in the enzymolysis solution, and determining the optimal enzymolysis time; 5) PEG-mediated protoplast transformation: The plasmid DNA of the plant expression vector pAL902-GFP containing the GFP gene is transformed into the green onion leaf protoplasts separated under the optimal enzymatic hydrolysis conditions determined in step 4) by PEG-mediated method; the integration of GFP in the transfected protoplasts is detected under a fluorescence microscope, wherein the GFP is green fluorescent protein and the PEG is polyethylene glycol.

2. The method for separating and transiently transforming green onion leaf protoplasts according to claim 1, characterized in that: In the step 2): The enzymatic hydrolysate comprises the following components: 20mmol / L KCl, 20mmol / L MES, 10mmol / L CaCl2, 1.0g / L BSA, cellulase, macerate and mannitol, wherein the MES is 4-morpholineethanesulfonic acid, and its pH value is 5.8, and the BSA is bovine serum albumin solution; The green onion leaves were rinsed with ddH2O, wiped with absorbent paper and placed in a petri dish to dry; the dried green onion leaves were cut into pieces with a width of 0.5 to 1.0 mm; 0.5 g of the pieces were weighed and mixed with 10 mL of enzymatic hydrolysate; the mixture of the enzymatic hydrolysate and the pieces was placed in a dark environment at 26°C, and after a vacuum treatment for 0.5 h, the mixture was shaken at 50 r / min for 4 to 10 h to release the protoplasts in the green onion leaves; After the enzymatic hydrolysis, the mixed solution was filtered through a cell strainer with a pore size of 70 μm, and the filtered liquid was collected in a 50 mL round-bottom centrifuge tube. An equal volume of pre-cooled W5 solution was added to the filtered protoplast suspension, and the mixture was centrifuged at 100 rcf for 3 min. The supernatant was discarded, and 5 mL of W5 solution was added to the obtained precipitate to resuspend the protoplasts. The mixture was placed in ice and away from light for 30 min, the supernatant was discarded, and the precipitate was resuspended in 1 mL of MMG solution.

3. A method for separation and transient transformation of green onion leaf protoplasts according to claim 2, characterized in that: The W5 solution includes 4mmol / L MES, 154mmol / L NaCl, 125mmol / L CaCl2 and 5mmol / L KCl, and the pH value of the W5 solution is 5.7; the MMG solution includes 0.4mol / L mannitol, 15mmol / L MgCl2 and 4mmol / L MES.

4. A method for separation and transient transformation of green onion leaf protoplasts according to claim 3, characterized in that: In the step 3), every 100 μL of protoplast resuspension is stained with 1 μL of 0.02% FDA solution, incubated in the dark for 5 minutes, 20 μL of the mixture is taken, and the protoplasts are counted using a hemocytometer, and the protoplasts emitting green fluorescence are counted as viable.

5. A method for separation and transient transformation of green onion leaf protoplasts according to claim 4, characterized in that: In the step 3), the calculation formulas for the yield and vitality of the protoplasts are as follows: Protoplast yield (g -1 ) = number of protoplasts in the large square of the counting plate × 10 4 / Mass of enzymatic hydrolysis material (g); Protoplast viability (%) = (number of protoplasts emitting green fluorescence / total number of protoplasts) × 100%.

6. The method for separation and transient transformation of green onion leaf protoplasts according to claim 5, characterized in that: The method for determining the optimal cellulose and cleavage enzyme concentrations in the enzymatic hydrolysate is as follows: In step 2), an orthogonal test group was set, and all test groups had the same mannitol concentration and enzymolysis time. The two influencing factors of the orthogonal test group were the concentrations of cellulase and macerate. The test concentrations of cellulase were 5 g / L, 10 g / L and 15 g / L, and the test concentrations of macerate were 3 g / L, 5 g / L and 7 g / L, thereby setting 9 control tests; The yield and activity of protoplasts in each control test were statistically analyzed to determine the optimal concentrations of cellulase and macerate.

7. The method for separation and transient transformation of green onion leaf protoplasts according to claim 6, characterized in that: The method for determining the optimal mannitol concentration in the enzymatic hydrolysis solution and the optimal enzymatic hydrolysis time is as follows: In step 2), a control experiment group was set, under the optimum concentrations of cellulase and macerate, the enzymolysis time was controlled to be unchanged, the concentration of mannitol was set to 0.3, 0.4, 0.5, 0.6 mol / L, respectively, the yield and viability of the protoplasts in each control experiment were counted, and the optimum concentration of mannitol was determined; In step 2), a control experimental group is set, and the enzymolysis time is set to 4, 6, 8, and 10 h at the optimal concentrations of cellulase, macerate and mannitol, and the yield and activity of protoplasts in each control experiment are statistically analyzed to determine the optimal enzymolysis time.

8. The method for separation and transient transformation of green onion leaf protoplasts according to claim 7, characterized in that: In the step 5), the plant expression vector pAL902-GFP is amplified, the plasmid DNA is purified, and 20 μg of the plasmid DNA is mixed with 200 ul of the prepared protoplast resuspension in a 2 mL round-bottom centrifuge tube; then 200 ul of PEG solution is added, and the round-bottom centrifuge tube is inverted to prepare a suspension; the suspension is allowed to stand at room temperature for 20 min, 1 mL of pre-cooled W5 solution is added for washing, and the suspension is centrifuged at 100 rcf for 2 min and the supernatant is discarded; 1 mL of W5 solution is added again for resuspending, and the suspension is then placed in a 35 mm cell culture dish for culture, incubated in the dark at 24° C. for 20 h, and the integration of GFP in the transfected protoplasts is detected under a fluorescence microscope.

9. The method for separation and transient transformation of green onion leaf protoplasts according to claim 8, characterized in that: The plant expression vector pAL902-GFP was amplified using the Escherichia coli DB3.1 strain, and then the plasmid DNA was extracted and purified. The purity and concentration of the plasmid DNA were determined using electrophoresis technology to ensure that its OD260 / OD280 ratio was between 1.8 and 2.0, the concentration reached 1 μg / μL, and it was stored at -20°C.

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