Method for establishing narcissus protoplast separation and transformation system
Through a method including buffer and enzyme solution configuration, enzymatic digestion and other steps, the problems of low efficiency and complex operation of protoplasts in daffodils are solved, and efficient protoplast separation and transformation are achieved, providing a powerful tool for genetic improvement and biotechnology research of daffodils.
Patent Information
- Application Number
- CN202411843426.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-14
- Publication Date
- 2025-03-11
AI Technical Summary
The existing methods of protoplast isolation and transformation of daffodils have problems of inefficiency and complex operation, especially when the cell wall structure of daffodils is complex and the conditions for protoplast culture need to be optimized.
A method that includes configuring specific buffers and enzyme fluids, enzymatic digestion, filtration, washing, centrifugation and resuspension, efficient isolation and transformation of daffodil protoplasts is adopted. This method optimizes the separation and transformation process of protoplasts by using specific solution combinations and operating steps.
It has achieved efficient separation and transformation of daffodil protoplasts, improved the survival rate and regeneration ability of protoplasts, and provided a simple and efficient tool for genetic improvement and biotechnology research of daffodils.
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Figure CN119592495A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of narcissus protoplasts, and in particular to a method for establishing a narcissus protoplast separation and transformation system. Background Art
[0002] Narcissus is a flower with important ornamental value, with many varieties and rich colors, and is deeply loved by people. However, there are relatively few studies on the genetic improvement and biotechnology of narcissus.
[0003] Protoplasts refer to naked cells obtained after the cell wall is removed, and have many unique biological characteristics, such as easy genetic manipulation, fusion and culture. Therefore, the establishment of a daffodil protoplast isolation and transformation system is of great significance for in-depth research on the gene function, genetic improvement and biotechnology application of daffodils.
[0004] At present, there are some methods for the separation and transformation of plant protoplasts, but there are still some problems in the application of these methods in daffodils. For example, the cell wall structure of daffodils is relatively complex, and traditional cell wall degrading enzymes may not be able to effectively separate protoplasts; the culture conditions of daffodil protoplasts also need to be further optimized to improve the survival rate and regeneration ability of protoplasts.
[0005] Today, the Arabidopsis leaf protoplast isolation and transformation system has become relatively mature. Many other species have also established their leaf protoplast transient transformation systems based on this method, referring to the isolation and transformation methods of their leaf protoplasts, for functional research of related genes in the species. However, whether this method is also applicable to certain specific organs, such as flower organs and tuber organs, remains to be explored.
[0006] In addition, the existing protoplast transformation methods also have some limitations, such as low transformation efficiency and complex operation. Therefore, it is necessary to develop an efficient and simple daffodil protoplast isolation and transformation system to provide a powerful tool for the genetic improvement and biotechnology research of daffodils. Summary of the invention
[0007] The object of the present invention is to provide a method for isolating daffodil protoplasts and establishing a transformation system to solve the problems raised in the above-mentioned background technology.
[0008] In order to solve the above technical problems, the present invention provides a technical solution: a method for establishing a daffodil protoplast separation and transformation system, comprising the following steps:
[0009] (1) Prepare 10 mL buffer (containing 0.4 mol / L D-mannitol, 20 mmol / L KCl, 20 mmol / L MES, 10 mmol / L CaCl2, pH 5.7), tear off the perianth pieces of 4 Chinese narcissus flowers in the T5 period with transparent tape, cut off the edge of the transparent tape, and immerse the perianth pieces in the buffer;
[0010] (2) Vacuum the chamber in a dark place for 30 minutes;
[0011] (3) Take out the buffer and remove it with a pipette tip. Add 10% enzyme solution filtered with a 0.45 μm disposable sterile syringe filter.
[0012] mL (containing 0.4 mol / L D-mannitol, 20 mmol / L KCl, 20 mmol / L MES, 10 mmol / L CaCl2, 1.50% cellulase RS, 0.40% macerate R10, 0.40% pectinase, pH 5.7);
[0013] (4) Vacuum the chamber in a dark place for 30 min;
[0014] (5) Place at room temperature of 20-22°C for enzymatic digestion for 2-6 hours, and at the same time, take out the K3 culture medium (containing 0.4 mol / L sucrose) and W5 solution from the -20°C refrigerator to thaw;
[0015] (6) Shake the enzyme solution containing narcissus perianth pieces on a microplate shaker at 200 g for 5 min to release protoplasts. After sufficient enzymatic hydrolysis, the perianth pieces become transparent, the protoplasts are released, and the enzyme solution becomes turbid; filter the released protoplasts through a clean 250-mesh nylon mesh and divide into two cell culture tubes;
[0016] (7) Gently add an equal volume of W5 solution along the wall edge of the culture dish originally filled with the enzymatic hydrolyzate of narcissus perianth pieces, shake at 200 g on a microplate shaker for 5 min to wash the protoplasts and terminate the enzymatic hydrolysis, and then filter and dispense the washing solution into the above-mentioned cell culture tube;
[0017] (8) Centrifuge in a horizontal rotor at a speed of 7:1 and a speed of 4:1 at 200 g, 4°C, for 3 min.
[0018] (9) Discard the supernatant, keep the precipitate, and add 2 mL of W5 to each tube to resuspend the protoplasts;
[0019] (10) Take two new cell culture tubes, add 1.3 mL of K3 culture medium (containing 0.4 mol / L sucrose) to each tube, and then slowly add the above protoplast resuspension along the tube wall. At this time, stratification can be seen;
[0020] (11) Gently centrifuge the sample in a swing-out rotor, at a speed of 7:0, at 200 g, at 4°C, for 3 min. This process may take about half an hour.
[0021] (12) After centrifugation, the liquid in the cell culture tube is divided into three layers: the top layer is W5, the middle layer is viable protoplasts, and the bottom layer is K3 culture medium and some dead protoplasts and cell debris. The thickness of the middle layer determines the amount of protoplasts separated;
[0022] (13) Use a sterile pipette with the tip cut off to transfer the protoplasts in the middle layer into a new 50-mL centrifuge tube, dilute to 25 mL with W5 solution, and gently shake to suspend the protoplasts;
[0023] (14) Use another centrifuge tube to balance and centrifuge, speed up 7 and down 4, 200g, 4°C, centrifuge for 3 min;
[0024] (15) Discard the supernatant, keep the precipitate, add 5 mL of W5 solution, and gently shake to suspend the protoplasts;
[0025] (16) Microscopic examination: Use a sterile pipette with the tip cut off to take 10 μL of the protoplast resuspension and spot it on a hemacytometer. Use a cell counter to calculate the protoplast concentration and total number.
[0026] (17) Add W5 solution to 25 mL to resuspend the protoplasts and gently place on ice for at least 1 h.
[0027] (18) Preparation for transformation of Narcissus chinensis protoplasts: prepare MMG solution, 40% PEG4000 solution, and W1 solution;
[0028] (19) The revived protoplast suspension was gently centrifuged in a horizontal rotor at a speed of 7:1 and a speed of 4:1 at 200 g, 4°C, for 3 min.
[0029] (20) Discard the supernatant and keep the precipitate. According to the results of microscopic examination and conversion on a blood cell counting plate, add MMG solution to make the final concentration of protoplasts reach the transformation requirement, that is, 1×10 6 Pieces / ml;
[0030] (21) Mixing plasmid and protoplasts: Add 15 μg of plasmid and 150 μL of protoplasts into a 1.5 mL EP tube and gently tap the tube wall to mix;
[0031] (22) Transformation: Add a volume of 40% PEG4000 solution equal to the total volume of plasmid + protoplasts, flick quickly to mix, do 1-2 tubes at a time, and incubate at room temperature at 20-22°C for 5-10 min;
[0032] (23) Termination of transformation: Add 4 times the volume of W5 solution (the total volume of plasmid + protoplasts), tap the tube wall to mix, and terminate the transformation;
[0033] (24) Centrifugation: 200 g, 4 °C for 5 min, remove as much supernatant as possible;
[0034] (25) Optional: Repeat steps (24) and (25), add 4 times the volume of W5, flick the tube to mix, centrifuge at 200g, 4°C for 5 min, and remove as much supernatant as possible;
[0035] (26) Add 150 μL of W1 solution, flick the bottom of the tube to mix, place horizontally in an aluminum box, and incubate at room temperature at 20-22°C in the dark.
[0036] After 9 to 12 hours, observe the subcellular localization of the transformed protein; if GFP fluorescent signal is observed, centrifuge the remaining sample and discard the supernatant, collect the protoplasts, and store them in a -80°C ultra-low temperature refrigerator for future use.
[0037] The above-mentioned W5 solution refers to a mixed solution containing 154mmol / L NaCl, 125mmol / L CaC12, 5mmol / L KCI, 2mmol / L MES, and pH5.7; MMG solution refers to a mixed solution containing 0.4mol / L D-mannitol, 15mmol / L MgCl2, 4mmol / L MES, and pH5.7; 40% PEG4000 solution refers to a mixed solution containing 0.2mol / L D-mannitol, 100mmol / L CaCl2, and 400g / L polyethylene glycol 4000; W1 solution refers to a mixed solution containing 0.5mol / L D-mannitol, 20mmol / L KCl, 4mmol / L MES, and pH5.7.
[0038] Preferably, the volume of W5 added in step (13) is at least 4 times the volume of the protoplasts.
[0039] Preferably, in step (16), the resuspended liquid is spotted on the blood cell counting plate and covered with a coverslip to allow the plate to enter the plate by siphoning.
[0040] Preferably, if the number of protoplast cells in step (16) is particularly large and inconvenient to count, further dilution with 5 to 10 mL of W is required and then re-examination under the microscope is performed.
[0041] Preferably, in step (21), the plasmid concentration is adjusted to 1 μg / ul.
[0042] The invention has the advantages of reasonable step design, efficient and simple establishment of a narcissus protoplast separation and transformation system, providing a powerful tool for genetic improvement and biotechnology research of narcissus, and having good market prospects, biological value and economic value. BRIEF DESCRIPTION OF THE DRAWINGS
[0043] Figure 1 It is a line chart of Table 1. DETAILED DESCRIPTION
[0044] For the purpose of the following detailed description, it should be understood that the present invention may adopt various alternative changes and step sequences, unless expressly specified to the contrary. In addition, except in any operating examples, or otherwise indicated, all numbers representing the amount of ingredients used in the specification and claims should be understood to be modified by the term "about" in all cases. Therefore, unless otherwise indicated, the numerical parameters set forth in the following specification and the attached claims are approximate values that vary according to the desired performance to be obtained by the present invention. At least it is not intended to limit the application of the doctrine of equivalents to the scope of the claims, and each numerical parameter should at least be interpreted according to the number of reported significant figures and by applying ordinary rounding techniques.
[0045] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
[0046] In addition, it should be understood that any numerical range described herein is intended to include all subranges subsumed therein. For example, a range of "1 to 10" is intended to include all subranges between (and including) the stated minimum value of 1 and the stated maximum value of 10, i.e., having a minimum value equal to or greater than 1 and a maximum value equal to or less than 10.
[0047] Embodiment 1:
[0048] The invention discloses a method for establishing a narcissus protoplast separation and transformation system, which specifically comprises the following steps: preparing 10 mL buffer (containing 0.4 mol / L D-mannitol, 20 mmol / L KCl, 20 mmol / L MES, 10 mmol / L CaCl2, pH 5.7), tearing off tepals of 4 Chinese narcissus flowers in the T5 period with transparent adhesive tape, cutting off the edge of the transparent adhesive tape, and immersing the tepals in the buffer.
[0049] Vacuum the container for 30 min in the dark.
[0050] Take it out, remove the buffer with a pipette tip, and add 10 mL of enzyme solution filtered with a 0.45 μm disposable sterile syringe filter (containing 0.4 mol / L D-mannitol, 20 mmol / L KCl, 20 mmol / L MES, 10 mmol / L CaCl2, 1.50% cellulase RS, 0.40% chromatinase R10, 0.40% pectinase, pH 5.7).
[0051] Vacuum the container for 30 min in the dark.
[0052] The culture medium was placed at 20°C for 4 h for enzymatic digestion. Meanwhile, the K3 culture medium (containing 0.4 mol / L sucrose) and W5 solution were taken out from the -20°C refrigerator and thawed.
[0053] The enzyme solution containing narcissus perianth pieces was shaken at 200 g on a microplate shaker for 5 minutes to release the protoplasts. After sufficient enzymatic hydrolysis, the perianth pieces became transparent, the protoplasts were released, and the enzyme solution became turbid. The released protoplasts were filtered through a clean 250-mesh nylon mesh and divided into two cell culture tubes.
[0054] Gently add an equal volume of W5 solution along the wall edge of the culture dish originally filled with the enzymatic hydrolyzate of narcissus perianth pieces, shake at 200g on a microplate shaker for 5 minutes to wash the protoplasts and terminate the enzymatic hydrolysis, and then filter the washing solution and dispense it into the above-mentioned cell culture tubes.
[0055] Centrifuge in a horizontal rotor, speed up 7 and down 4, 200g, 4℃, centrifuge for 3 minutes.
[0056] Discard the supernatant, keep the precipitate, and add 2 mL of W5 to each tube to resuspend the protoplasts.
[0057] Take two new cell culture tubes, add 1.3mL K3 culture medium (containing 0.4mol / L sucrose) to each tube, and then slowly add the above protoplast resuspension along the tube wall. At this time, stratification can be seen.
[0058] The sample was centrifuged gently in a horizontal rotor, with the speed increased to 7 and the speed decreased to 0, at 200 g, 4°C, and centrifuged for 3 min. This process takes about half an hour.
[0059] At the end of centrifugation, the liquid in the cell culture tube is divided into three layers: the top layer is W5, the middle layer is viable protoplasts, and the bottom layer is K3 culture medium and some dead protoplasts and cell fragments. The thickness of the middle layer determines the amount of protoplasts separated.
[0060] Use a sterile pipette with a cut tip to transfer the protoplasts in the middle layer to a new 50 mL centrifuge tube, add W5 solution to dilute to 25 mL (the volume of W5 added is 4 times the volume of the protoplasts), and gently shake to suspend the protoplasts.
[0061] Use another centrifuge tube to balance and centrifuge, increase speed 7 and decrease speed 4, 200g, 4℃, centrifuge for 3 minutes.
[0062] Discard the supernatant, keep the precipitate, add 5 mL of W5 solution, and gently shake to suspend the protoplasts.
[0063] Microscopic examination: Use a sterile pipette with the tip cut off to take 10 μL of the protoplast resuspension and spot it on the hemacytometer. Cover it with a coverslip and use the siphon phenomenon to enter. Use a cell counter to calculate the protoplast concentration and total number. If the number of protoplast cells is particularly large and it is inconvenient to count, further dilute and add W5 to 10 mL before re-examination under the microscope.
[0064] Add W5 solution to 25 mL to resuspend the protoplasts and gently place on ice for at least 1 hour.
[0065] Transformation preparation of Chinese narcissus protoplasts: prepare MMG solution, 40% PEG4000 solution and W1 solution.
[0066] The revived protoplast suspension was gently centrifuged in a horizontal rotor, with a speed of 7 and a speed of 4, at 200g, 4°C, and centrifuged for 3 minutes.
[0067] Discard the supernatant and keep the precipitate. According to the results of microscopic examination and conversion of the blood cell counting plate, add MMG solution to make the final concentration of protoplasts reach the transformation requirement, that is, 1×10 6 Pieces / ml.
[0068] Mix plasmid and protoplasts: Add 15 μg of plasmid (adjust the plasmid concentration to 1 μg / ul) and 150 μL of protoplasts into a 1.5 mL EP tube and gently tap the tube wall to mix.
[0069] Transformation: Add 40% PEG4000 solution in a volume equal to the total volume of plasmid + protoplasts, flick quickly to mix, do one tube at a time, and incubate at room temperature at 20℃ for 8 minutes.
[0070] Terminate transformation: Add 4 times the volume of W5 solution (the total volume of plasmid + protoplasts), flick the tube wall to mix, and terminate transformation.
[0071] Centrifugation: 200g, 4℃ for 5min, remove as much supernatant as possible.
[0072] Optional: Repeat the above steps, add 4 times the volume of W5, flick the tube to mix, centrifuge at 200g, 4℃ for 5min, and remove as much supernatant as possible.
[0073] Add 150 μL of W1 solution, flick the bottom of the tube to mix, place horizontally in an aluminum box, incubate at room temperature at 20°C in the dark, and observe the subcellular localization of the transformed protein after 9 hours; if GFP fluorescence signal is observed, centrifuge the remaining sample and discard the supernatant, collect the protoplasts, and store them in a -80°C ultra-low temperature refrigerator for later use.
[0074] Embodiment 2:
[0075] Prepare the buffer and process the perianth pieces as described above, perform vacuuming and other operations, and then add the enzyme solution.
[0076] The enzymatic digestion conditions were 22°C for 3 h.
[0077] The subsequent protoplast isolation steps are the same as those in Example 1, including filtering, washing, centrifugation, resuspension and other operations.
[0078] After microscopic examination, the revived protoplasts were resuspended.
[0079] After the transformation preparation is completed, the protoplasts and plasmids are mixed. 15 μg of plasmid (concentration 1 μg / ul) and 150 μL of protoplasts are added to a 1.5 mL EP tube and mixed.
[0080] During transformation, an equal volume of 40% PEG4000 solution was added and incubated at room temperature of 22°C for 5 min.
[0081] The termination of transformation and subsequent centrifugation and treatment steps are the same as those in Example 1.
[0082] After adding 150 μL of W1 solution, incubate at 22°C in the dark for 10 h to observe the transformation. If there is a GFP fluorescent signal, process and store the protoplasts as required.
[0083] Embodiment three:
[0084] After preparing the buffer to treat the perianth segments and performing related operations, enzymatic digestion was performed at 21°C for 6 hours.
[0085] The protoplast separation procedure remains unchanged. When aspirating the middle layer protoplasts, strictly follow the requirement that the added W5 volume is at least 4 times the protoplast volume.
[0086] During the microscopic examination step, if the number of cells is large, appropriate dilution should be performed and the cells should be re-examined. The protoplast concentration should be determined and then resuspended and revived.
[0087] For protoplast transformation, mix plasmid and protoplasts, the plasmid concentration is 1 μg / ul, and add 15 μg of plasmid and 150 μL of protoplasts.
[0088] The transformation incubation was carried out at 21°C for 10 min.
[0089] Termination of transformation and subsequent centrifugation and treatment are the same as in Example 1.
[0090] Example 1 After adding W1 solution, incubate at 21°C in dark for 12 hours, observe the transformation results, and process and store the protoplasts when there is GFP fluorescence signal. The number of protoplasts under microscopic examination corresponding to different enzymolysis times is shown in Table 1 below:
[0091] Table 1
[0092] Enzymatic digestion time / h 2 3 4 5 Number of protoplasts / ml <![CDATA[2.5×10 6 ]]> <![CDATA[1.0×10 7 ]]> <![CDATA[1.025×10 8 ]]> <![CDATA[1.25×10 7 ]]>
[0093] It can be seen from the above table that the optimal time for enzymatic hydrolysis is 4h.
[0094] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.
Claims
1. A method for establishing a daffodil protoplast separation and transformation system, characterized in that: The specific steps include: (1) Prepare 10 mL of buffer (containing 0.4 mol / L D-mannitol, 20 mmol / L KCl, 20 mmol / L MES, 10 mmol / L CaCl2, pH 5.7), tear off the perianth pieces of 4 Chinese narcissus flowers in the T5 period with transparent tape, cut off the edge of the transparent tape, and immerse the perianth pieces in the buffer; (2) Vacuum the chamber in a dark place for 30 minutes; (3) Take out the buffer and remove it with a pipette tip. Add 10% enzyme solution filtered with a 0.45 μm disposable sterile syringe filter. mL (containing 0.4 mol / L D-mannitol, 20 mmol / L KCl, 20 mmol / L MES, 10 mmol / L CaCl2, 1.50% cellulase RS, 0.40% macerate R10, 0.40% pectinase, pH 5.7); (4) Vacuum the chamber in a dark place for 30 min; (5) Place at room temperature of 20-22°C for enzymatic digestion for 2-6 hours, and at the same time, take out the K3 culture medium (containing 0.4 mol / L sucrose) and W5 solution from the -20°C refrigerator to thaw; (6) Shake the enzyme solution containing narcissus perianth pieces on a microplate shaker at 200 g for 5 min to release protoplasts. After sufficient enzymatic hydrolysis, the perianth pieces become transparent, the protoplasts are released, and the enzyme solution becomes turbid; filter the released protoplasts through a clean 250-mesh nylon mesh and divide into two cell culture tubes; (7) Gently add an equal volume of W5 solution along the wall edge of the culture dish originally filled with the enzymatic hydrolyzate of narcissus perianth pieces, shake at 200 g on a microplate shaker for 5 min to wash the protoplasts and terminate the enzymatic hydrolysis, and then filter and dispense the washing solution into the above-mentioned cell culture tube; (8) Centrifuge in a horizontal rotor at a speed of 7:1 and a speed of 4:1 at 200 g, 4°C, for 3 min. (9) Discard the supernatant, keep the precipitate, and add 2 mL of W5 to each tube to resuspend the protoplasts; (10) Take two new cell culture tubes, add 1.3 mL of K3 culture medium (containing 0.4 mol / L sucrose) to each tube, and then slowly add the above protoplast resuspension along the tube wall. At this time, stratification can be seen; (11) Gently centrifuge the sample in a swing-out rotor, at a speed of 7:0, at 200 g, at 4°C, for 3 min. This process may take about half an hour. (12) After centrifugation, the liquid in the cell culture tube is divided into three layers: the top layer is W5, the middle layer is viable protoplasts, and the bottom layer is K3 culture medium and some dead protoplasts and cell debris. The thickness of the middle layer determines the amount of protoplasts separated; (13) Use a sterile pipette with the tip cut off to transfer the protoplasts in the middle layer into a new 50-mL centrifuge tube, dilute to 25 mL with W5 solution, and gently shake to suspend the protoplasts; (14) Use another centrifuge tube to balance and centrifuge, speed up 7 and down 4, 200g, 4°C, centrifuge for 3 min; (15) Discard the supernatant, keep the precipitate, add 5 mL of W5 solution, and gently shake to suspend the protoplasts; (16) Microscopic examination: Use a sterile pipette with the tip cut off to take 10 μL of the protoplast resuspension and spot it on a hemacytometer. Use a cell counter to calculate the protoplast concentration and total number. (17) Add W5 solution to 25 mL to resuspend the protoplasts and gently place on ice for at least 1 h. (18) Preparation for transformation of Narcissus chinensis protoplasts: prepare MMG solution, 40% PEG4000 solution, and W1 solution; (19) The revived protoplast suspension was gently centrifuged in a horizontal rotor at a speed of 7:1 and a speed of 4:1 at 200 g, 4°C, for 3 min. (20) Discard the supernatant and keep the precipitate. According to the results of microscopic examination and conversion on a blood cell counting plate, add MMG solution to make the final concentration of protoplasts reach the transformation requirement, i.e., 1 × 106 cells / ml; (21) Mixing plasmid and protoplasts: Add 15 μg of plasmid and 150 μL of protoplasts into a 1.5 mL EP tube and gently tap the tube wall to mix; (22) Transformation: Add a volume of 40% PEG4000 solution equal to the total volume of plasmid + protoplasts, flick quickly to mix, do 1-2 tubes at a time, and incubate at room temperature at 20-22°C for 5-10 min; (23) Termination of transformation: Add 4 times the volume of W5 solution (the total volume of plasmid + protoplasts), tap the tube wall to mix, and terminate the transformation; (24) Centrifugation: 200 g, 4 °C for 5 min, remove as much supernatant as possible; (25) Optional: Repeat steps (24) and (25), add 4 times the volume of W5, flick the tube to mix, centrifuge at 200g, 4°C for 5 min, and remove as much supernatant as possible; (26) Add 150 μL of W1 solution, gently tap the bottom of the tube to mix, place horizontally in an aluminum box, and incubate at room temperature at 20-22°C in the dark. After 9-12 h, observe the subcellular localization of the transformed protein. If GFP fluorescence signal is observed, centrifuge the remaining sample and discard the supernatant. Collect the protoplasts and store them in a -80°C ultra-low temperature refrigerator for later use.
2. A method for establishing a daffodil protoplast separation and transformation system according to claim 1, characterized in that: The volume of W5 added in step (13) is at least 4 times the volume of the protoplasts.
3. A method for establishing a daffodil protoplast separation and transformation system according to claim 1, characterized in that: In the step (16), the resuspended liquid is placed on the cell counting plate and covered with a coverslip to allow the cell counting plate to enter the plate by siphoning.
4. A method for establishing a daffodil protoplast separation and transformation system according to claim 1, characterized in that: If the number of protoplast cells in step (16) is particularly large and inconvenient to count, it is necessary to further dilute and add 5 to 10 mL of W and then re-examine under the microscope.
5. A method for establishing a narcissus protoplast separation and transformation system according to claim 1, characterized in that: In the step (22), the plasmid concentration was adjusted to 1 μg / ul.