A method for separating and purifying plant protoplasts
Through the combination of enzymatic solution and W5 solution, the problem of protoplast separation of woody plants was solved, and efficient and rapid protoplast purification was achieved. It is suitable for tea trees and other plants, meeting a variety of research needs.
Patent Information
- Application Number
- CN202310160107.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-02-24
- Publication Date
- 2024-12-27
- Estimated Expiration
- 2043-02-24
AI Technical Summary
It is difficult to effectively isolate and purify protoplasts of woody plants, especially leaf protoplasts of plants such as tea trees, resulting in research difficulties and limited application.
Using the combination of enzymatic lysate and W5 solution, high-quality protoplasts are obtained by cutting plant tissues, incubating and termination of digestion, filtration and centrifuging, especially adding snail enzymes and aluminum salts to improve cell release efficiency. and integrity.
The rapid isolation and purification of woody plant protoplasts has been achieved, cell viability and yield has been improved, and it is suitable for different tea tree varieties and other plants, meeting the research needs including protoplast culture, genetic transformation, gene editing, single-cell sequencing, etc.
Smart Images
Figure CN116144577B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a method for separating and purifying plant protoplasts, and more particularly to a method for separating and purifying protoplasts that can be used for different tissues of different plants. Background Art
[0002] Protoplasts are plant cells with cell walls removed, which can be used to construct transient expression systems and are widely used in research on protein subcellular localization, protein interaction, gene expression regulation, subcellular organelle preparation, and suspension cell culture; in addition, the single-cell sequencing technology widely used in the field of animal cells in recent years has also begun to be applied to the field of plants, and one of the technical bottlenecks of the application of this technology in the field of plants is the preparation of protoplasts. Limited by the protoplast preparation technology, only a small number of model plants can be used for single-cell sequencing. Due to reasons such as high cell wall lignin content, it is difficult to separate the protoplasts of woody plant leaves and the integrity is poor. In particular, some species with more secondary metabolites, such as tea trees (Camellia sinensis L.), are rich in a variety of unique natural compounds and lack a mature genetic transformation system, which makes it impossible to perform functional verification in plants for basic research such as molecular and cellular studies. Therefore, the separation and purification method of a plant protoplast provided by the present invention is of great significance to the basic research and production application of the preparation of protoplasts of non-model plants.
[0003] The existing reported methods for dissociating and purifying plant protoplasts are often limited by the materials, especially in woody plants, such as flowers, seedlings and special varieties. This makes it difficult to meet the experimental needs of researchers. Especially in tea, the buds, leaves and flowers of different tea tree varieties are important research objects. However, tea leaves are thick in wax and high in polyphenols, making it difficult to separate protoplasts. A protoplast preparation method that can break through material limitations has great application prospects. Summary of the invention
[0004] The invention aims to provide a rapid separation and purification method of protoplasts suitable for a wide range of dissociation objects, aiming at the problems of the existing plant protoplast separation being difficult, time-consuming, poor in integrity, and time-limited extraction of petals and seedling protoplasts. The method can be applied to different types of plants, and is particularly suitable for woody plants with high dissociation difficulty, especially for plants such as tea, Pittosporum tobira, Gardenia jasminoides, and Ophiopogon japonicus. The method can particularly obtain high-quality protoplasts from flowers, buds, and mature leaves of plants. BRIEF DESCRIPTION OF THE DRAWINGS
[0005] Figure 1The protoplasts of tea leaves prepared by the method of the present invention are shown under a microscope.
[0006] Figure 2 The present invention is a protoplast of tea tree flower prepared by the method of the present invention under a microscope.
[0007] Figure 3 The protoplasts of Ophiopogon japonicus leaves prepared by the method of the present invention are shown under a microscope.
[0008] Figure 4 The present invention is a protoplast of Pittosporum tobira leaves prepared by the method of the present invention under a microscope.
[0009] Figure 5 The protoplasts of gardenia leaves prepared by the method of the present invention are shown under a microscope. DETAILED DESCRIPTION
[0010] To achieve the above purpose, the technical solution of the present invention is:
[0011] A method for separating and purifying plant protoplasts, the method comprising the following steps:
[0012] Step 1: selection of dissociated material, wherein the dissociated material is plant tissue; Step 2: cutting the plant tissue into pieces, preferably into strips 2-10 mm wide, and quickly moving them into an enzymolysis solution; Step 3: incubating the enzymolysis solution, and shaking continuously or at intervals to help release cells; Step 4: after the incubation is completed, adding W5 solution to terminate digestion to obtain a mixed solution, filtering the mixed solution, collecting the filtrate, centrifuging the filtrate, removing the supernatant, and slowly adding W5 solution to resuspend the protoplasts to obtain high-quality protoplast cells. The enzymolysis solution components may include snail enzymes. The W5 solution components may include aluminum salts.
[0013] The enzymolysis solution comprises mannitol, calcium chloride, aluminum chloride, 2-(N-morpholino)ethane sulfonic acid (pH 6.0), potassium chloride, snail enzyme, cellulase R-10, macerate R-10, bovine serum albumin, and β-mercaptoethanol, and the W5 solution comprises one or more of calcium chloride, aluminum chloride, 2-(N-morpholino)ethane sulfonic acid, potassium chloride, and sucrose. The mannitol can also be replaced by other balancing agents, and the macerate R-10 can be replaced by pectinase.
[0014] The step 4 may further comprise rinsing the tissue block using W5 solution.
[0015] The step 4 may further comprise passing the resuspended protoplasts through a cell sieve, then centrifuging, removing the supernatant, and adding an appropriate amount of W5 for resuspending. Preferably, the ratio of the components of the enzymatic hydrolysis solution is 0.1-1.6M mannitol, 1-20mM calcium chloride, 20-160μM aluminum chloride, 10-40mM 2-(N-morpholino)ethane sulfonic acid, 1-40mM potassium chloride, 0.1-2.0% (w / v) snail enzyme, 0.5-3% (w / v) cellulase R-10, 0.1-0.8% (w / v) cleavage enzyme R-10, 0.01-0.2% (w / v) bovine serum albumin, 1-1000mM β-mercaptoethanol, and the concentration ratio of the W5 solution is 60-250mM calcium chloride, 20-160μM aluminum chloride, 0.1‰-0.6‰ (w / v) 2-(N-morpholino)ethane sulfonic acid, 2-10mM potassium chloride, and 2-10mM sucrose.
[0016] More preferably, the ratio of the components of the enzymatic hydrolysis solution is 0.4-0.8M mannitol, 2-10mM calcium chloride, 40-80μM aluminum chloride, 15-30mM 2-(N-morpholino)ethane sulfonic acid, 15-30mM potassium chloride, 0.5-1.0% (w / v) snail enzyme, 1-1.5% (w / v) cellulase R-10, 0.2-0.4% (w / v) chromatase R-10, 0.05-0.15% (w / v) bovine serum albumin, 5-500mM β-mercaptoethanol, and the ratio of the W5 solution is 60-125mM calcium chloride, 40-80μM aluminum chloride, 0.2‰-0.4‰ (w / v) 2-(N-morpholino)ethane sulfonic acid, 4-6mM potassium chloride, and 4-6mM sucrose.
[0017] The plant tissue may be a flower, a bud, a root or a leaf. Preferably, the leaf or flower of the plant; the leaf may be all the buds and leaves, and particularly preferably the buds and leaves or petals of the new branches of the plant.
[0018] The enzymatic hydrolysate and the W5 solution are filtered and sterilized with a nylon filter membrane to reduce contamination and increase the yield of protoplasts.
[0019] The above method can be used to prepare a kit for separating and purifying plant protoplasts.
[0020] Example 1
[0021] Step 1: Selection of dissociation materials: Select the woody plant tea tree as the target plant. Select the flowers or leaves of the tea tree as the dissociation material. The tea tree can be put into the flowering period under the greenhouse conditions in a short-day mode to meet the supply of flowers in the non-flowering period; the leaves can be selected from all the buds and leaves of the new branches in the tea garden. If cultivated in a greenhouse, appropriate pruning can promote the continuous growth of new branches of 3-5-year-old tea seedlings to meet the year-round acquisition of experimental materials.
[0022] Step 2: Cut the leaves or petals into pieces, preferably into strips 2-3 mm wide, and quickly transfer them to the enzymatic solution. Preferably, about 0.5-1.0 g of fresh tissue is dissociated per 15 mL of enzymatic solution.
[0023] Step 3: Incubate the enzymatic hydrolysate from step 2 in a dark incubator at 23-25°C for 4-6 hours, shaking gently every hour to help release the cells.
[0024] Step 4: Microscope examination of the number of released cells, round and full cells, diverse cell types, and fewer cell fragments are preferred. At the end of incubation, add an equal volume of W5 solution to the enzymatic solution to terminate digestion, filter the solution through a cell sieve (select the appropriate pore size according to the target cell size), collect the filtrate in a centrifuge tube, rinse the tissue block with W5 solution, and use it to elute the cell protoplasts on the tissue block. Centrifuge the filtrate with 50-200g for 1min, remove the supernatant, slowly add W5 solution to the wall to resuspend the protoplasts, and pass through a cell sieve with a pore size of 40μm. Then centrifuge at 50g for 1min, remove the supernatant, and ensure that the enzymatic solution is removed. Resuspend with an appropriate amount of W5 to obtain high-quality protoplast cells. For protoplast transformation, it can be stored in a 4°C refrigerator for one week and is still active.
[0025] The concentration ratio of the enzymatic solution is 0.1-1.6M mannitol, 1-20mM calcium chloride, 20-160μM aluminum chloride, 10-40mM 2-(N-morpholino)ethanesulfonic acid, 1-40mM potassium chloride, 0.1-2.0% (w / v) snail enzyme, 0.5-3% (w / v) cellulase R-10, 0.1-0.8% (w / v) chromatase R-10, 0.01-0.2% (w / v) bovine serum albumin, and 1-1000mM β-mercaptoethanol. Preferably, 0.4-0.8 M mannitol, 10 mM calcium chloride, 40-80 μM aluminum chloride, 20 mM 2-(N-morpholino)ethanesulfonic acid (pH 6.0), 20 mM potassium chloride, 0.5-1.0% (w / v) snail enzyme, 1-1.5% (w / v) cellulase R-10, 0.2-0.4% (w / v) mesenzyme R-10, 0.1% (w / v) bovine serum albumin, 5-500 mM β-mercaptoethanol, and sterilized by filtration with a 0.45 μm nylon filter membrane.
[0026] The concentration ratio of W5 solution is 60-250mM calcium chloride, 20-160μM aluminum chloride, 0.1‰-0.6‰ (w / v) 2-(N-morpholino)ethane sulfonic acid, 2-10mM potassium chloride, 2-10mM sucrose. Preferably, 125mM calcium chloride, 40-80μM aluminum chloride, 0.3‰ (w / v) 2-(N-morpholino)ethane sulfonic acid (pH 6.0), 5mM potassium chloride, 5mM sucrose, and sterilized by filtration with a 0.45μm nylon filter membrane.
[0027] The addition of snail enzyme accelerates the dissociation, shortening the digestion time of the woody plant tea tree to 2-4h. It can also be applied to leaves of different tea varieties, tea petals and other plant leaves. The addition of aluminum salt improves the integrity and cell viability of tea leaf cells, making the protoplast yield between 0.3-2.4×10 7 The cell suspension has a viability of 70-95%, rich cell types, and few impurities and cell fragments. It can be filtered to obtain high-quality cell suspension. It can meet the needs of various subsequent studies including protoplast culture, somatic cell hybridization, genetic transformation, gene editing, single-cell sequencing, etc.
[0028] According to the characteristics of tea tissue cells, a kit for separating and purifying plant protoplasts from tea is manufactured.
[0029] Example 2
[0030] Step 1: Selection of dissociation materials: Select the herb Ophiopogon japonicus as the target plant. Select the flowers or leaves of Ophiopogon japonicus as the dissociation materials. The leaves can be selected from the entire leaves of Ophiopogon japonicus, and the flowers can be selected from the petals.
[0031] Step 2: Cut the leaves or petals into pieces, preferably into strips 2-3 mm wide, and quickly transfer them to the enzymatic solution. Preferably, about 0.5-1.0 g of fresh tissue is dissociated per 15 mL of enzymatic solution.
[0032] Step 3: Incubate the enzymatic hydrolysate from step 2 in a dark incubator at 23-25°C for 4-6 hours, shaking gently every hour to help release the cells.
[0033] Step 4: Microscope examination of the number of released cells, round and full cells, diverse cell types, and fewer cell fragments are preferred. At the end of incubation, add an equal volume of W5 solution to the enzymatic solution to terminate digestion, filter the solution through a single layer of magic cloth, collect the filtrate in a centrifuge tube, rinse the tissue block with W5 solution, and use it to elute the cell protoplasts on the tissue block. Centrifuge the filtrate with 50g for 1min, remove the supernatant, adhere to the wall and slowly add W5 solution to resuspend the protoplasts, and pass through a cell sieve with a pore size of 40μm. Then centrifuge at 50g for 1min, remove the supernatant, and ensure that the enzymatic solution is removed. Resuspend with an appropriate amount of W5 to obtain high-quality protoplast cells. For protoplast transformation, it can be stored in a 4℃ refrigerator for one week and still be active.
[0034] The concentration ratio of the enzymatic solution is 0.1-1.6M mannitol, 1-20mM calcium chloride, 20-160μM aluminum chloride, 10-40mM 2-(N-morpholino)ethanesulfonic acid, 1-40mM potassium chloride, 0.1-2.0% (w / v) snail enzyme, 0.5-3% (w / v) cellulase R-10, 0.1-0.8% (w / v) chromatase R-10, 0.01-0.2% (w / v) bovine serum albumin, and 1-1000mM β-mercaptoethanol. Preferably, 0.4-0.8 M mannitol, 10 mM calcium chloride, 40-80 μM aluminum chloride, 20 mM 2-(N-morpholino)ethanesulfonic acid (pH 6.0), 20 mM potassium chloride, 0.5-1.0% (w / v) snail enzyme, 1-1.5% (w / v) cellulase R-10, 0.2-0.4% (w / v) mesenzyme R-10, 0.1% (w / v) bovine serum albumin, 5-500 mM β-mercaptoethanol, and sterilized by filtration with a 0.45 μm nylon filter membrane.
[0035] The concentration ratio of W5 solution is 60-250mM calcium chloride, 20-160μM aluminum chloride, 0.1‰-0.6‰ (w / v) 2-(N-morpholino)ethane sulfonic acid, 2-10mM potassium chloride, 2-10mM sucrose. Preferably, 125mM calcium chloride, 40-80μM aluminum chloride, 0.3‰ (w / v) 2-(N-morpholino)ethane sulfonic acid (pH 6.0), 5mM potassium chloride, 5mM sucrose, and sterilized by filtration with a 0.45μm nylon filter membrane.
[0036] The addition of snail enzyme accelerated dissociation and shortened digestion time. The addition of aluminum salt improved the integrity and cell viability of Ophiopogon japonicus cells, resulting in a protoplast yield of 1-6.5×10 6 The cell suspension contains more than 100 cells / gram fresh weight, with a viability of 70-95%, rich cell types, and few impurities and cell fragments. High-quality cell suspension can be obtained by simple filtration.
[0037] According to the characteristics of Ophiopogon japonicus tissue cells, a kit for isolating and purifying plant protoplasts of Ophiopogon japonicus is manufactured.
[0038] Example 3
[0039] Step 1: Selection of dissociation materials: Select the woody plant Pittosporum tobira as the target plant. Select the flowers or leaves of Pittosporum tobira as the dissociation materials. The leaves can be selected from all the buds and leaves of the new branches of Pittosporum tobira.
[0040] Step 2: Cut the leaves or petals into 2-10 mm pieces, preferably into 2-3 mm wide strips, and quickly transfer them to the enzymatic solution. Preferably, about 0.5-1.0 g of fresh tissue is dissociated per 15 mL of enzymatic solution.
[0041] Step 3: Incubate the enzymatic hydrolysate from step 2 in a dark incubator at 23-25°C for 4-6 hours, shaking gently every hour to help release the cells.
[0042] Step 4: Microscope examination of the number of released cells, round and full cells, diverse cell types, and fewer cell fragments are preferred. At the end of incubation, add an equal volume of W5 solution to the enzymatic solution to terminate digestion, filter the solution through a single layer of magic cloth, collect the filtrate in a centrifuge tube, rinse the tissue block with W5 solution, and use it to elute the cell protoplasts on the tissue block. Centrifuge the filtrate with 50g for 1min, remove the supernatant, adhere to the wall and slowly add W5 solution to resuspend the protoplasts, and pass through a cell sieve with a pore size of 40μm. Then centrifuge at 50g for 1min, remove the supernatant, and ensure that the enzymatic solution is removed. Resuspend with an appropriate amount of W5 to obtain high-quality protoplast cells. For protoplast transformation, it can be stored in a 4℃ refrigerator for one week and still be active.
[0043] The concentration ratio of the enzymatic solution is 0.1-1.6M mannitol, 1-20mM calcium chloride, 20-160μM aluminum chloride, 10-40mM 2-(N-morpholino)ethanesulfonic acid, 1-40mM potassium chloride, 0.1-2.0% (w / v) snail enzyme, 0.5-3% (w / v) cellulase R-10, 0.1-0.8% (w / v) chromatase R-10, 0.01-0.2% (w / v) bovine serum albumin, and 1-1000mM β-mercaptoethanol. Preferably, 0.4-0.8 M mannitol, 10 mM calcium chloride, 40-80 μM aluminum chloride, 20 mM 2-(N-morpholino)ethanesulfonic acid (pH 6.0), 20 mM potassium chloride, 0.5-1.0% (w / v) snail enzyme, 1-1.5% (w / v) cellulase R-10, 0.2-0.4% (w / v) mesenzyme R-10, 0.1% (w / v) bovine serum albumin, 5-500 mM β-mercaptoethanol, and sterilized by filtration with a 0.45 μm nylon filter membrane.
[0044] The concentration ratio of W5 solution is 60-250mM calcium chloride, 20-160μM aluminum chloride, 0.1‰-0.6‰ (w / v) 2-(N-morpholino)ethane sulfonic acid, 2-10mM potassium chloride, 2-10mM sucrose. Preferably, 125mM calcium chloride, 40-80μM aluminum chloride, 0.3‰ (w / v) 2-(N-morpholino)ethane sulfonic acid (pH 6.0), 5mM potassium chloride, 5mM sucrose, and sterilized by filtration with a 0.45μm nylon filter membrane.
[0045] The addition of snail enzyme accelerated the dissociation. The addition of aluminum salt improved the integrity and cell viability of Pittosporum leaf cells, making the protoplast yield between 1 and 6.5 × 10 6The cell suspension is high in quality and contains 70-95% of fresh weight, 70-95% of vitality, rich cell types, few impurities and cell fragments, and can be obtained by simple filtration. This allows the experimental materials to meet the needs of various subsequent studies, including protoplast culture, somatic cell hybridization, genetic transformation, gene editing, single-cell sequencing, etc.
[0046] According to the characteristics of Pittosporum tobira tissue cells, a kit for isolating and purifying plant protoplasts from Ophiopogon japonicus was manufactured.
[0047] Example 4
[0048] Step 1: Selection of dissociation materials: Select the woody plant Gardenia as the target plant. Select the flowers or leaves of Gardenia as the dissociation materials. The leaves are all the buds and leaves of the new branches of Gardenia.
[0049] Step 2: Cut the leaves or petals into pieces, preferably into strips 2-3 mm wide, and quickly transfer them to the enzymatic solution. Preferably, about 0.5-1.0 g of fresh tissue is dissociated per 15 mL of enzymatic solution.
[0050] Step 3: Incubate the enzymatic hydrolysate from step 2 in a dark incubator at 23-25°C for 4-6 hours, shaking gently every hour to help release the cells.
[0051] Step 4: Microscope examination of the number of released cells, round and full cells, diverse cell types, and fewer cell fragments are preferred. At the end of incubation, add an equal volume of W5 solution to the enzymatic solution to terminate digestion, filter the solution through a single layer of magic cloth, collect the filtrate in a centrifuge tube, rinse the tissue block with W5 solution, and use it to elute the cell protoplasts on the tissue block. Centrifuge the filtrate with 50g for 1min, remove the supernatant, adhere to the wall and slowly add W5 solution to resuspend the protoplasts, and pass through a cell sieve with a pore size of 40μm. Then centrifuge at 50g for 1min, remove the supernatant, and ensure that the enzymatic solution is removed. Resuspend with an appropriate amount of W5 to obtain high-quality protoplast cells. For protoplast transformation, it can be stored in a 4℃ refrigerator for one week and still be active.
[0052] The concentration ratio of the enzymatic solution is 0.1-1.6M mannitol, 1-20mM calcium chloride, 20-160μM aluminum chloride, 10-40mM 2-(N-morpholino)ethanesulfonic acid, 1-40mM potassium chloride, 0.1-2.0% (w / v) snail enzyme, 0.5-3% (w / v) cellulase R-10, 0.1-0.8% (w / v) chromatase R-10, 0.01-0.2% (w / v) bovine serum albumin, and 1-1000mM β-mercaptoethanol. Preferably, 0.4-0.8 M mannitol, 10 mM calcium chloride, 40-80 μM aluminum chloride, 20 mM 2-(N-morpholino)ethanesulfonic acid (pH 6.0), 20 mM potassium chloride, 0.5-1.0% (w / v) snail enzyme, 1-1.5% (w / v) cellulase R-10, 0.2-0.4% (w / v) mesenzyme R-10, 0.1% (w / v) bovine serum albumin, 5-500 mM β-mercaptoethanol, and sterilized by filtration with a 0.45 μm nylon filter membrane.
[0053] The concentration ratio of W5 solution is 60-250mM calcium chloride, 20-160μM aluminum chloride, 0.1‰-0.6‰ (w / v) 2-(N-morpholino)ethane sulfonic acid, 2-10mM potassium chloride, 2-10mM sucrose. Preferably, 125mM calcium chloride, 40-80μM aluminum chloride, 0.3‰ (w / v) 2-(N-morpholino)ethane sulfonic acid (pH 6.0), 5mM potassium chloride, 5mM sucrose, and sterilized by filtration with a 0.45μm nylon filter membrane.
[0054] The addition of snail enzyme accelerates dissociation and shortens digestion time. It can also be applied to different plant tissues, such as leaves, petals, and other plant leaves. The addition of aluminum salt improves the integrity and cell viability of leaf cells, making the protoplast yield between 1-6.5×10 6 The cell suspension has a viability of 70-95%, rich cell types, and few impurities and cell fragments. High-quality cell suspension can be obtained by simple filtration, which can meet the needs of various subsequent research including protoplast culture, somatic cell hybridization, genetic transformation, gene editing, single-cell sequencing, etc.
[0055] According to the characteristics of gardenia tissue cells, a kit for separating and purifying plant protoplasts from gardenia is manufactured.
[0056] Exemplary embodiments of the present invention have been described above. It will be appreciated by those skilled in the art that modifications may be made without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered as descriptive rather than restrictive. The scope of the present invention is defined by the claims, rather than by the above description, and it should be understood that all differences within their equivalents are included in the present invention.
Claims
1. A method for separating and purifying plant protoplasts, the method comprising the following steps: Step 1: selection of dissociation material, wherein the dissociation material is plant tissue; Step 2: cutting the plant tissue into pieces and quickly transferring them into an enzymatic solution; Step 3: Incubate the enzymatic solution, and shake continuously or intermittently to help release the cells; Step 4: After the incubation is completed, W5 solution is added to terminate the digestion to obtain a mixed solution, the mixed solution is filtered, the filtrate is collected, the filtrate is centrifuged, the supernatant is removed, and the W5 solution is slowly added to resuspend the protoplasts to obtain high-quality protoplast cells. The enzymatic hydrolysate comprises snail enzyme, the plant tissue is woody plant tissue, the W5 solution comprises aluminum salt, and the ratio of the enzymatic hydrolysate components is 0.4-0.8M mannitol, 2-10mM calcium chloride, 40-80μM aluminum chloride, 15-30mM 2-(N-morpholino)ethane sulfonic acid, 15-30mM potassium chloride, 0.5-1.0% w / v snail enzyme, and 1-1.5% w / v cellulase R-10 , 0.2-0.4% w / v cleavage enzyme R-10, 0.05-0.15% w / v bovine serum albumin, 5-500mM β-mercaptoethanol, the plant tissue is the flower, bud, root or leaf of the tea plant, the W5 solution is composed of 60-125mM calcium chloride, 40-80μM aluminum chloride, 0.2‰-0.4‰ w / v 2-(N-morpholino)ethane sulfonic acid, 4-6mM potassium chloride, and 4-6mM sucrose.
2. A method for separating and purifying plant protoplasts according to claim 1, characterized in that: The step 4 further comprises rinsing the tissue block using W5 solution.
3. A method for separating and purifying plant protoplasts according to claim 1, characterized in that: The step 4 further comprises passing the resuspended protoplasts through a cell sieve, then centrifuging, removing the supernatant, and adding an appropriate amount of W5 for resuspending.
4. A method for separating and purifying plant protoplasts according to claim 1, characterized in that: The enzymatic hydrolysate and the W5 solution were sterilized by filtration using a nylon filter membrane.