Pea cotyledon protoplast separation and instantaneous conversion method and application

Abstract

The invention discloses a pea cotyledon protoplast separation and instantaneous conversion method and application. The method discloses steps of pea cotyledon protoplast separation and instantaneous conversion and used reagents. The method comprises the steps of material acquisition, protoplast preparation, purification, activity detection, instantaneous conversion and the like. The method is easy to operate and short in digestion time, and a large amount of early-stage preparation work is saved. According to the method disclosed by the invention, the high-quality pea cotyledon protoplast suspension can be quickly and efficiently obtained, the activity of the protoplast can reach 90% or above, and a powerful technical support is provided for scientific researches such as subsequent gene function verification, gene editing and single cell transcriptome sequencing.

Description

Technical Field

[0001] The invention belongs to the field of plant biotechnology, and particularly relates to a method for separating and transiently transforming pea cotyledon protoplasts and applications thereof. Background Art

[0002] Plant protoplasts refer to the naked plant cells with vitality surrounded by plasma membranes after the cell walls are removed. A large number of relatively uniform protoplasts can be separated from the same plant tissue. Since there are no cell walls, protoplasts can easily absorb exogenous genetic materials. The transgenic plants obtained by protoplast gene transformation develop from a single cell, which effectively avoids the production of chimeras and provides good receptor materials for plant genetic improvement.

[0003] Plant protoplasts are also ideal materials for transient expression analysis and have been widely used in research work such as gene expression, protein interaction, single-cell sequencing, and gene editing vector screening, providing a convenient and effective research platform for plant research. However, the natural cell wall of plant cells makes important preliminary work full of difficulties and challenges. Therefore, the use of protoplasts for basic and applied research must be based on the establishment of an effective protoplast separation and transformation system.

[0004] pea( Pisum sativum L.) is the world's second largest edible legume crop. Peas can not only be used as food, but also have the ability to fix nitrogen. They are environmentally friendly crops with the functions of reducing weight and increasing efficiency, improving soil, etc., and play an important role in China's sustainable agricultural system. According to existing research, there are differences in the cell walls of the same part of different plants and different parts of the same plant, and the cell wall is a major difficulty in obtaining protoplasts. At present, plant protoplasts have been studied more on model plants and food crops, and there are few reports on the preparation of pea protoplasts. The establishment of pea cotyledon protoplast isolation and transient transformation technology will provide efficient and stable technical means for pea gene function verification, gene expression regulation, protein subcellular localization and gene editing. Summary of the invention

[0005] The purpose of the present invention is to provide a method for isolating and transiently transforming pea cotyledon protoplasts. The protoplast suspension prepared by the method of the present invention has high cell concentration, high quantity and activity of more than 90%, and can be used for pea gene function verification, subcellular localization and single-cell sequencing, etc., and has a wide range of applications.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The invention discloses a method for separating and transiently transforming pea cotyledon protoplasts, which comprises the following steps: (1) selecting pea seedlings of appropriate age, removing the top two cotyledons, cleaning them, and cutting the cotyledons into 0.5-1 mm filaments with a sharp double-sided blade; the pea seedlings of appropriate age are 3-5 days old seedlings; (2) After removing the surface water from the cut pea cotyledons, transfer them to an enzyme digestion solution and completely immerse the cotyledons in the enzyme digestion solution; sequentially perform light-protected digestion, rinsing, and centrifugation to obtain a pea cotyledon protoplast suspension; (3) Dilute the pea protoplast suspension with MMg buffer, add the plasmid to be transformed and the pea protoplast suspension and mix well; then PEG Ca 2+ The buffer solution was mixed by pipetting and incubated to complete the transient transformation of pea cotyledon protoplasts.

[0007] Furthermore, in step (2), the pea cotyledon protoplast suspension is obtained by light-protected digestion, rinsing, and centrifugation, specifically: (2.1) After the cotyledons are completely immersed in the enzyme digestion solution, digest in a sterile shaker at 28°C and 50 rpm / min in the dark for 4-5 h. After digestion, use a wide-mouth pipette to gently aspirate the protoplast suspension, rinse the cell sieve with W5 buffer, and pass the protoplast suspension through 70 μm and 40 μm cell sieves in turn to remove incompletely digested tissues; (2.2) The pea protoplast suspension with tissue blocks removed was collected by centrifugation to obtain a protoplast precipitate, and then W5 buffer was added and washed 2-3 times. After centrifugation, the supernatant was removed by aspiration; an appropriate amount of MMg buffer was added and gently pipetted to obtain a pea protoplast suspension; (2.3) The obtained pea protoplast suspension was observed under an optical microscope, and the protoplast concentration and activity were calculated using a hemocytometer and fluorescein diacetate staining.

[0008] Furthermore, in the step (2.1), the protoplast suspension is gently sucked and beaten using a wide-mouthed pipette tip, and the cell sieve is rinsed with W5 buffer. Specifically, after the digestion is completed, the protoplast suspension is gently sucked and beaten using a wide-mouthed pipette tip for 5-8 times, the cell sieve is rinsed with W5 buffer, and the protoplast suspension is passed through a 70 μm and a 40 μm cell sieve in sequence to remove incompletely digested tissue; wherein the composition of the W5 buffer is: 9 g / L NaCl, 13.9 / L CaCl2·2H2O, 3.7 g / L KCl, 0.4 g / L MES (2-(N-morpholino)ethanesulfonic acid), 91.1 g / L D-mannitol and sterile water.

[0009] Furthermore, the concentration of the protoplast suspension in step (2.2) is 5-10×10 5 / mL; the composition of the MMg buffer is: 1.4 g / L MgCl2·6H2O, 0.9 g / L MES, 91.1 g / L D-mannitol and sterile water, and its pH is 5.7-5.8. After the solution is prepared, it is filtered and sterilized using a sterile filter membrane.

[0010] Specifically, the centrifugation conditions in step (2.2) are 4°C, 400 rpm / min, and 3 min.

[0011] Furthermore, the step (3) specifically includes the following sub-steps: (3.1) Dilute the pea protoplast suspension to 5-10×10 5 Pieces / mL; (3.2) Add the plasmid to be transformed and the pea protoplast suspension to a 2 mL round-bottom centrifuge tube, gently pipette to mix, and then add an equal volume of PEG Ca 2+ Mix the buffer by pipetting and incubate at 26-28℃ in the dark for 20 min. (3.3) After incubation, add 880 μL of W5 buffer to terminate the transformation reaction, remove the supernatant after centrifugation, add 400 μL of W5 buffer, and incubate at 26-28°C in the dark for 12-16 h; (3.4) After the culture is completed, the protoplast mixture that has undergone transformation culture is briefly separated, 2 / 3 of the supernatant is removed, and the remaining 1 / 3 of the supernatant is mixed with the protoplast precipitate by pipetting and then the transformation status of the pea protoplasts is observed under a confocal microscope.

[0012] Specifically, in step (3.2), PEG Ca 2+ The composition of the buffer solution is: 200 g / L PEG4000, 22.2 g / L CaCl2·2H2O, 36.4 g / L D-mannitol and sterile water, and its pH is 5.7-5.8. After the solution is prepared, it is filtered and sterilized using a sterile filter membrane.

[0013] Specifically, the composition of the enzyme digestion solution in step (2) is: 20 g / L cellulase, 10 g / L macerate, 4.2 g / L MES, 2.2 g / L CaCl2·2H2O, 0.7 g / L KCl, 91.1 g / L D-mannitol and sterile water, with a pH of 5.7-5.8; and after the solution is prepared, it is filtered and sterilized using a sterile filter membrane.

[0014] The present invention also discloses a method for separating and transiently transforming pea cotyledon protoplasts and its application in gene function verification, gene expression regulation, protein subcellular localization, signal transduction, RNA interference and / or gene editing.

[0015] Beneficial effects of the invention: The invention establishes for the first time a fast and efficient method for isolating and transiently transforming pea cotyledon protoplasts by exploring enzyme concentration combinations, osmotic pressure and other enzymatic conditions. The protoplast activity can reach more than 90%, providing strong technical support for subsequent scientific research such as cell fusion culture, transient expression, and single-cell transcriptome sequencing, and providing efficient and stable technical means for pea gene function verification, gene expression regulation, protein subcellular localization, and gene editing. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The figure is a sampling position diagram of pea seedlings of the present invention; Figure 2 This is a schematic diagram of the separation and purification process of pea cotyledon protoplasts of the present invention; Figure 3 This is a diagram showing the effects of different digestion times on pea cotyledon protoplasts of the present invention; Figure 4 The figure is a diagram showing the effects of different purification methods of the present invention on pea cotyledon protoplasts; Figure 5 This is a diagram for detecting the activity of the purified protoplasts of the present invention; Figure 6 This is a schematic diagram of the transformation of pea cotyledon protoplasts by pCAMBIA1300 of the present invention; Figure 7 The figure is a schematic diagram of subcellular localization of pea cotyledon protoplasts of the present invention. DETAILED DESCRIPTION

[0017] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0018] Example 1: Effects of different factors on pea cotyledon protoplasts (1) Effects of enzyme digestion combinations on pea cotyledon protoplasts Divide the cut cotyledons into 6 equal parts (such as Figure 1 As shown in FIG. 1 , it is a diagram showing the source and sampling location of pea seedlings of the present invention), as shown in FIG. Figure 2As shown, the cotyledons were placed in 3 mL of enzyme digestion solution with different enzyme combinations. The mannitol concentration was 0.4M, and the enzymatic digestion was performed for 5 hours. Each group was repeated 3 times. The experimental results are shown in Table 1. The results show that the protoplast yields of the enzymatic digestion solutions with different concentration combinations are very different. When the concentration of cellulase R10 is 2%, the concentration of cleavage enzyme R10 is 1%, and the enzymatic digestion is performed for 5 hours, the enzymatic digestion effect is the best, and the protoplast yield can reach 3.76×10 6 pcs / g FW.

[0019] Table 1 Cellulase R-10 (%) Mesase R-10 (%) Protoplast yield (pcs / g·FW) Protoplast activity (%) 1 1.0 1.0 <![CDATA[1.36×10 6 ]]> 81.77 2 1.0 2.0 <![CDATA[0.93×10 6 ]]> 72.13 3 2.0 1.0 <![CDATA[3.76×10 6 ]]> 85.72 4 2.0 2.0 <![CDATA[2.40×10 6 ]]> 81.72 5 3.0 1.0 <![CDATA[1.40×10 6 ]]> 80.10 6 3.0 2.0 <![CDATA[1.73×10 6 ]]> 76.52 (2) Effect of osmotic pressure on pea cotyledon protoplasts The cut cotyledons were evenly divided into 6 parts and placed in 3 mL of enzyme digestion solution with different osmotic pressures. The enzyme digestion solution was 2.0% cellulase and 1.0% mashase, and the enzymatic digestion was performed for 5 hours. Each group was repeated 3 times. The experimental results are shown in Table 2. The results show that the protoplast activity is very different at different osmotic pressure concentrations. When the osmotic pressure concentration is 0.5M, the enzymatic hydrolysis effect is the best, the activity is higher, and the protoplast yield can reach 5.76×10 6 / g FW, and the activity reached 85%.

[0020] Table 2 Mannitol concentration (M) Protoplast yield (pcs / g·FW) Protoplast activity (%) 1 0.3 <![CDATA[3.61×10 6 ]]> 72.63 2 0.4 <![CDATA[4.31×10 6 ]]> 84.23 3 0.5 <![CDATA[6.70×10 6 ]]> 86.20 4 0.6 <![CDATA[4.91×10 6 ]]> 80.43 5 0.7 <![CDATA[3.40×10 6 ]]> 74.32 6 0.8 <![CDATA[2.15×10 6 ]]> 71.19 (3) Effect of digestion time on pea cotyledon protoplasts Divide the cut cotyledons into three equal parts and place each into 3 mL of enzyme digestion solution. The enzyme digestion solution contains 2.0% cellulase and 1.0% macerate, with an osmotic pressure of 0.5M. Figure 3 As shown in the figure, within the designed time gradient, observation was performed once every hour. The experimental results are shown in Table 3. The results show that the yield and vitality of protoplasts are different at different digestion times, such as Figure 3 When the digestion time reaches 3h, the enzymatic hydrolysis effect is the best, the activity is higher, and the yield of protoplasts can reach 12.48×10 6 / g FW, and the activity reached 90%.

[0021] Table 3 Digestion time (h) Protoplast yield (pcs / g·FW) Protoplast activity (%) 1 1 <![CDATA[5.65×10 6 ]]> 85.18 2 2 <![CDATA[11.41×10 6 ]]> 87.94 3 3 <![CDATA[12.48×10 6 ]]> 91.08 4 4 <![CDATA[9.95×10 6 ]]> 85.76 5 5 <![CDATA[6.40×10 6 ]]> 82.70 6 6 <![CDATA[6.08×10 6 ]]> 75.44 7 7 <![CDATA[5.13×10 6 ]]> 65.93 8 8 <![CDATA[4.09×10 6 ]]> 50.67 (4) Effects of purification methods on pea cotyledon protoplasts After the digestion is completed, the protoplast suspension is filtered and collected. Figure 4 As shown in Figure 1, three different centrifugation methods (floatation, interface and sedimentation) were used to purify protoplasts. Figure 4 The results show that the protoplasts obtained by the precipitation method have a clean background and a large number.

[0022] Example 2: Method for isolating pea cotyledon protoplasts (1) Preparation of test materials Select mature pea seeds with full grains, soak them in running water for 1-2 days, sow them in moist sand and culture them at room temperature. After the cotyledons of the pea plants unfold, cut the healthy and disease-free cotyledons as experimental materials, such as Figure 1 shown.

[0023] (2) Isolation of pea cotyledon protoplasts like Figure 2 As shown, the newly grown pea cotyledons were removed and quickly cut into 0.5-1 mm filaments on a sterile culture dish with a sharp double-sided blade, and the cut filaments were placed in sterile water; all the cut pea cotyledon filaments were rinsed with sterile water for 3 times, then the surface moisture was wiped dry with absorbent paper and transferred into enzyme digestion solution, and the material was immersed below the liquid surface of the enzyme digestion solution, and digested in a sterile shaker at 28°C and 50 rpm / min in the dark for 3 h; the pea protoplast suspension was gently sucked and beaten with a wide-mouthed gun tip for 5-8 times, and then the cell sieve was rinsed with buffer, and the pea protoplast suspension was passed through 70 μm and 40 μm cell sieves in turn; the pea protoplast suspension without tissue blocks was centrifuged and collected to obtain protoplast precipitates, and then W5 buffer was added, washed 2-3 times, and the supernatant was sucked off after centrifugation at 500 rpm and 4 min. Add MMg buffer and gently pipette to obtain pea protoplast suspension; take 20 μL of protoplast suspension and use a hemocytometer and diacetate fluorescein staining to observe the protoplast state under an optical microscope. Protoplast viability test Figure 5 .

[0024] Example 3: Effects of different factors on transient transformation of pea cotyledon protoplasts (1) Effect of plasmid dosage on transient transformation of pea cotyledon protoplasts Different amounts of plasmid were added to 2 mL round-bottom centrifuge tubes, followed by 100 μL of the protoplast suspension prepared in Example 2, and then 110 μL of 40% PEG Ca 2+ Buffer, gently pipette and mix, and then place incubate at 26-28℃ in dark for 10 min. After the incubation, add 880 μL of W5 buffer and mix by inversion to terminate the transformation reaction; centrifuge at 500 rpm for 4 min, discard the supernatant; then add 400 μL of W5 buffer to resuspend the transformed pea cotyledon protoplasts, and culture at 25℃ in dark for 12-16 h to obtain successfully transformed protoplasts. The experimental results are shown in Table 4, which show that the higher the amount of plasmid used, the higher the transformation efficiency of the protoplasts.

[0025] Table 4 Plasmid dosage (μg) Conversion efficiency (%) 1 5 17.24 2 10 30.75 3 15 23.71 4 20 14.44 (2) Effect of PEG concentration on transient transformation of pea cotyledon protoplasts 10 μg and 100 μL of plasmid and protoplast suspension were pipetted into 2 mL round-bottom centrifuge tubes and mixed, followed by adding 110 μL of PEG Ca 2+ Buffer, gently pipette and mix, and then place incubate at 26-28℃ in dark for 10 min. After the incubation, add 880 μL of W5 buffer and mix inverted to stop the transformation reaction; centrifuge at 500rpm for 4 min, discard the supernatant; add 400 μL of W5 buffer to resuspend the transformed pea cotyledon protoplasts, and culture at 25℃ in dark for 12-16 h to obtain successfully transformed protoplasts. The experimental results are shown in Table 5, which show that the transformation efficiency is best when the PEG concentration is 40%, which can reach 40%.

[0026] Table 5 PEG concentration (%) Conversion efficiency (%) 1 10 28.32 2 20 43.31 3 30 26.92 4 40 19.69 (3) Effect of transformation time on transient transformation of pea cotyledon protoplasts 10 μg and 100 μL of plasmid and protoplast suspension were pipetted into 2 mL round-bottom centrifuge tubes and mixed, followed by addition of 110 μL of 20% PEG Ca 2+ Buffer, gently pipette and mix, then place incubate in dark at 26-28℃. The incubation time gradient design is shown in Table 6. After the incubation, add 880 μL of W5 buffer and mix inverted to terminate the transformation reaction; centrifuge at 500rpm for 4min, discard the supernatant; then add 400 mL of W5 buffer to resuspend the transformed pea cotyledon protoplasts, and culture at 25℃ and shade for 12-16h to obtain successfully transformed protoplasts. The experimental results are shown in Table 6, which show that the transformation efficiency is best when the transformation time is 20min, which can reach 60%.

[0027] Table 6 Conversion time (min) Conversion efficiency (%) 1 10 43.72 2 20 62.41 3 30 45.98 4 40 31.70 Example 4: Transient transformation of pea cotyledon protoplasts (1) Preparation of pea cotyledon protoplast transient transformation materials Add MMg buffer to adjust the protoplast concentration to 5-10×10 5 / mL, and the pCAMBIA1300 plasmid with a concentration of 1000 ng / μL was obtained using a plasmid reagent extraction kit.

[0028] (2) Transient transformation of pea cotyledon protoplasts 10 μg and 100 μL of the prepared plasmid pCAMBIA1300 and protoplast suspension were pipetted into a 2 mL round-bottom centrifuge tube and mixed, followed by the addition of 110 μL of 20% PEG Ca 2+ Buffer, gently pipette to mix, and then place incubate at 26-28℃ in dark for 20 min. After the incubation, add 880 μL of W5 buffer and mix by inversion to terminate the transformation reaction; centrifuge at 500rpm for 4min, discard the supernatant; then add 400 μL of W5 buffer to resuspend the transformed pea cotyledon protoplasts, and culture at 25℃ in dark for 12-16h to obtain successfully transformed protoplasts.

[0029] (3) Observation and statistics of transient transformation efficiency of pea cotyledon protoplasts After the culture was completed, the protoplast mixture after transformation culture was briefly separated, 2 / 3 of the supernatant was removed, and the remaining 1 / 3 of the supernatant was mixed with the protoplast precipitate by pipetting and beating, and then placed under a confocal laser microscope with a fluorescence excitation wavelength of 488 nm to observe the transient expression of green fluorescent protein (GFP) in pea cotyledon protoplasts. Pea cotyledon protoplasts with GFP fluorescent signals were 35S-GFP protein successfully expressed in pea cotyledon protoplasts. The transformation efficiency was calculated based on the pea cotyledon protoplasts with GFP fluorescent signals and pea cotyledon protoplasts without GFP fluorescent signals in the fusion image of green fluorescent protein and bright field. The transformation rate was above 60%. The protoplast transformation efficiency was shown in Figure 2. Figure 6 .

[0030] Example 5: Subcellular localization of pea cotyledon protoplasts The operation steps are consistent with those in Example 4, except that the protein localized in the nucleus is selected and transferred into the protoplasts. The chloroplast autofluorescence in the pea cotyledon protoplasts is observed under a confocal laser microscope at a wavelength of 680 nm for fluorescence excitation. This is used as a reference. The subcellular localization is shown in Figure 7 .

[0031] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A method for isolating and transiently transforming pea cotyledon protoplasts, characterized in that: The method comprises the following steps: (1) Select pea seedlings, remove the top two cotyledons, clean them, and cut the cotyledons into 0.5-1 mm filaments with a sharp double-sided blade; (2) After removing the surface water from the cut pea cotyledons, transfer them to an enzyme digestion solution and completely immerse the cotyledons in the enzyme digestion solution; sequentially perform light-protected digestion, rinsing, and centrifugation to obtain a pea cotyledon protoplast suspension; (3) Dilute the pea protoplast suspension with MMg buffer, add the plasmid to be transformed and the pea protoplast suspension and mix well; then PEG Ca 2+ The buffer solution was mixed by pipetting and incubated to complete the transient transformation of pea cotyledon protoplasts.

2. The method for separation and transient transformation of pea cotyledon protoplasts according to claim 1, characterized in that: In the step (2), the pea cotyledon protoplast suspension is obtained by light-protected digestion, rinsing, and centrifugation, specifically: (2.1) When the cotyledons are completely immersed in the enzyme digestion solution, digest in a sterile shaker at 28°C and 50 rpm / min in the dark for 4-5 hours. After digestion, use a wide-mouth pipette to gently aspirate the protoplast suspension, rinse the cell sieve with W5 buffer, and pass the protoplast suspension through 70 μm and 40 μm cell sieves in turn to remove incompletely digested tissues; (2.2) The pea protoplast suspension with tissue blocks removed was collected by centrifugation to obtain a protoplast precipitate, and then W5 buffer was added and washed 2-3 times. After centrifugation, the supernatant was removed by aspiration; an appropriate amount of MMg buffer was added and gently pipetted to obtain a pea protoplast suspension; (2.3) The obtained pea protoplast suspension was observed under an optical microscope, and the protoplast concentration and activity were calculated using a hemocytometer and fluorescein diacetate staining.

3. The method for separation and transient transformation of pea cotyledon protoplasts according to claim 2, characterized in that: In the step (2.1), the protoplast suspension is gently sucked and beaten with a wide-mouthed pipette tip, and the cell sieve is rinsed with W5 buffer. Specifically, after the digestion is completed, the protoplast suspension is gently sucked and beaten with a wide-mouthed pipette tip for 5-8 times, and the cell sieve is rinsed with W5 buffer. The protoplast suspension is passed through a 70 μm and a 40 μm cell sieve in sequence to remove incompletely digested tissues; wherein the composition of the W5 buffer is: 9 g / L NaCl, 13.9 / L CaCl2·2H2O, 3.7 g / L KCl, 0.4 g / L MES, 91.1 g / L D-mannitol and sterile water; the MES is 2-(N-morpholino)ethanesulfonic acid.

4. The method for separation and transient transformation of pea cotyledon protoplasts according to claim 2, characterized in that: The concentration of the protoplast suspension in step (2.2) is 5-10×10 5 / mL; the composition of the MMg buffer is: 1.4 g / LMgCl2·6H2O, 0.9 g / L MES, 91.1 g / L D-mannitol and sterile water, and its pH is 5.7-5.

8. After the solution is prepared, it is filtered and sterilized using a sterile filter membrane.

5. The method for separation and transient transformation of pea cotyledon protoplasts according to claim 2, characterized in that: The centrifugation conditions in step (2.2) are 4°C, 400 rpm / min, and 3 min.

6. The method for isolating and transiently transforming pea cotyledon protoplasts according to claim 1, characterized in that: The step (3) specifically includes the following sub-steps: (3.1) Dilute the pea protoplast suspension to 5-10×10 5 Pieces / mL; (3.2) Add the plasmid to be transformed and the pea protoplast suspension to a 2 mL round-bottom centrifuge tube, gently pipette to mix, and then add an equal volume of PEG Ca 2+ Mix the buffer by pipetting and incubate at 26-28°C in the dark for 20 min. (3.3) After the incubation, add 880 μL of W5 buffer to terminate the transformation reaction, remove the supernatant after centrifugation, add 400 μL of W5 buffer, and incubate at 26-28°C in the dark for 12-16 h; (3.4) After the culture is completed, the protoplast mixture that has undergone transformation culture is briefly separated, 2 / 3 of the supernatant is removed, and the remaining 1 / 3 of the supernatant is mixed with the protoplast precipitate by pipetting and then the transformation status of the pea protoplasts is observed under a confocal microscope.

7. The method for isolating and transiently transforming pea cotyledon protoplasts according to claim 6, characterized in that: In the step (3.2), PEG Ca 2+ The composition of the buffer solution is: 200 g / L PEG4000, 22.2 g / L CaCl2·2H2O, 36.4 g / L D-mannitol and sterile water, and its pH is 5.7-5.

8. After the solution is prepared, it is filtered and sterilized using a sterile filter membrane.

8. The method for isolating and transiently transforming pea cotyledon protoplasts according to claim 1, characterized in that: The composition of the enzyme digestion solution in step (2) is: 20 g / L cellulase, 10 g / L macerate, 4.2 g / L MES, 2.2 g / L CaCl2·2H2O, 0.7 g / L KCl, 91.1 g / L D-mannitol and sterile water, with a pH of 5.7-5.8; and after the solution is prepared, it is filtered and sterilized using a sterile filter membrane.

9. Use of the method for isolating and transiently transforming pea cotyledon protoplasts according to any one of claims 1 to 8 in gene function verification, gene expression regulation, protein subcellular localization, signal transduction, RNA interference and / or gene editing.

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