Efficient isolation method of protoplast from durian callus

Abstract

The application discloses a high-efficiency method for separating protoplasts of durian callus. The method comprises the following steps: taking durian callus in good growth condition, placing the durian callus in an enzymolysis solution containing 0.4-0.8 mol / L mannitol, 0.5%-2.0% cellulase and 0.2%-0.6% macerozyme, and performing horizontal shaking enzymolysis at room temperature and under 30-120 rpm conditions for 2-8 hours in the dark, and centrifuging the product at 200-800 rpm for 5-7 min to obtain the protoplasts. The concentration of the protoplast suspension prepared by the method is 2.70x10 5 -2.91x10 5 / mL, and the activity is stably maintained at 72.27%-73.48%, the quantity is sufficient, and the physiological state is uniform, so that reliable experimental materials and technical support can be provided for durian somatic cell hybridization, germplasm innovation and genetic improvement.

Description

Technical Field

[0001] This invention belongs to the field of plant cell engineering and biotechnology, and specifically relates to a method for efficient separation of protoplasts adapted to durian callus tissue. Background Technology

[0002] Protoplasts are the collective term for all cellular structures enclosed by the plasma membrane after the cell wall of a plant cell has been removed. As a core experimental material in plant cell engineering and molecular biology research, they have multiple uses: they can be used to obtain mutants with superior traits such as stress resistance and high quality through somatic cell clonal variation; they are key materials for genetic transformation, providing efficient methods for gene editing; and they can also be used for the long-term stable preservation of superior fruit tree germplasm resources through cryopreservation and ultra-low temperature preservation. However, establishing a stable and efficient protoplast isolation system is the prerequisite and foundation for conducting the above research.

[0003] Durian (Durio zibethinus) is a perennial tropical fruit tree belonging to the genus Durio in the family Malvaceae of the order Malvaales. Its fruit is covered with hard thorns, has a strong aroma, and is rich in nutrients, earning it the title of "King of Southeast Asian Fruits." Due to its complex genetic background, self-incompatibility, long juvenile stage, and inconsistent flowering periods, traditional durian breeding methods have excessively long breeding cycles, slowing down the breeding process. There is an urgent need to utilize cell engineering technology to achieve germplasm innovation and genetic improvement. However, at present, research on durian protoplast isolation systems in domestic and international published literature and patent reports is still lacking. The absence of experimental materials restricts research in areas such as durian molecular breeding and in vitro germplasm resource preservation.

[0004] Currently, the commonly used raw materials for plant protoplast extraction in the industry are mostly leaves or suspended cells. However, durian leaves are thick and have a well-developed cuticle, and the tissue is rich in polysaccharides, polyphenols, mucilage, and secondary metabolites. When conventional leaf extraction methods are directly applied, the damage to the tissue cells will release a large amount of pectin-like polysaccharides and polyphenols into the enzymatic hydrolysis system. On the one hand, this causes the enzymatic hydrolysis system to become gel-like and its fluidity to decrease significantly, making it impossible to complete subsequent filtration and purification operations. On the other hand, polyphenol oxidation causes browning of the system, resulting in protoplast damage and decreased viability, making it difficult to obtain highly viable protoplasts.

[0005] Existing research on protoplasts from woody fruit trees largely focuses on temperate species such as apples, pears, and citrus, with very limited mature extraction systems for tropical high-polyphenol fruit trees. Because callus tissues from different species exhibit significant differences in cell wall composition, intracellular osmotic pressure, and the types and amounts of secondary metabolites, parameters from other species cannot be directly applied. Furthermore, to date, there are no publicly reported methods for isolating protoplasts from durian callus tissue, both domestically and internationally. This research direction represents a technological gap in the field. Key conditions such as the osmotic pressure of the enzymatic hydrolysate, enzyme concentration ratio, hydrolysis time, and purification parameters all require systematic screening based on the physiological characteristics of durian materials and cannot be derived through conventional limited experiments.

[0006] Therefore, establishing a stable, efficient, and applicable method for protoplast extraction from durian callus, screening suitable starting materials, and systematically optimizing key technical parameters are crucial to overcoming defects such as protoplast fragmentation and easy browning. This method will break through the technical bottlenecks of browning and polysaccharide interference, gently degrade cell walls, and obtain protoplasts with sufficient yield and stable viability, filling the gap in durian protoplast extraction technology research. This is a key technical problem that urgently needs to be solved in current durian molecular breeding and functional genomics research, and has important theoretical significance and application value. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the purpose of this invention is to provide a highly efficient method for isolating protoplasts adapted to durian callus tissue. This invention provides a method for isolating protoplasts with stable isolation efficiency and a gentle release process, yielding a sufficient quantity of durian callus tissue protoplasts with relatively intact cell membrane structures and uniform physiological states. It maintains the membrane stability and basic vitality of the protoplasts, reduces interference with cell surface structures during enzymatic digestion, and provides suitable donor and recipient materials for subsequent protoplast fusion. Furthermore, it enhances the feasibility of fusion operations, providing reliable in vitro experimental materials for durian somatic cell hybridization and cytoplasmic gene introgression studies, and offering technical support for durian germplasm innovation and genetic improvement.

[0008] The technical solution adopted by this invention to solve its technical problem is:

[0009] In a first aspect, the present invention provides a method for efficient separation of protoplasts adapted to durian callus tissue. The method includes the following steps: taking durian callus tissue in good growth condition, placing it in an enzymatic hydrolysate containing 0.4~0.8 mol / L mannitol, 0.5%~2.0% cellulase, and 0.2%~0.6% (preferably 0.2%~0.4%) dissociative enzyme, and performing enzymatic hydrolysis at room temperature and 30~120 rpm under light-protected conditions for 2~8 h (preferably 2~6 h). After the enzymatic hydrolysis is completed, adding W5 solution, centrifuging at 200~800 rpm for 5~7 min, and collecting the protoplasts.

[0010] In a specific implementation scheme, the concentration of cellulase is 1.0% to 2.0%, preferably 1.0% to 1.5% or 1.5% to 2.0%.

[0011] In a specific implementation scheme, the enzymatic hydrolysate also contains 20 mmol / L 2-(N-morpholino)ethanesulfonic acid (MES), 10 mmol / L potassium chloride (KCl) solution, 10 mmol / L calcium chloride (CaCl2) solution and 0.1% bovine serum albumin (BSA), with a pH of 5.7 ± 0.1.

[0012] In a specific implementation plan, the W5 solution is prepared as follows: 80 mL of sterile water is added to the bottle, followed by glucose, MES, potassium chloride, calcium chloride, and sodium chloride, to make the final concentrations 5 mmol / L glucose, 2 mmol / L MES, 5 mmol / L potassium chloride, 125 mmol / L calcium chloride, and 154 mmol / L sodium chloride, respectively. The solution is then diluted to 100 mL with sterile water.

[0013] In a specific implementation plan, the durian callus tissue is first pretreated in a 0.4~0.8 mol / L mannitol solution for a pretreatment time greater than 0 h and less than or equal to 0.5 h.

[0014] In a specific implementation scheme, the mannitol concentration is 0.6~0.8 mol / L.

[0015] In the specific implementation plan, room temperature refers to 20~25℃.

[0016] In the specific implementation plan, the oscillation rate is 30~90 rpm.

[0017] In a specific implementation, the centrifugation rate is 200~600 rpm, more preferably 400~600 rpm.

[0018] In the specific implementation plan, the durian variety is either 'Golden Pillow' or 'Musang King'.

[0019] In a more specific embodiment, the present invention provides a method for isolating protoplasts from 'Golden Pillow' agaric: Protoplasts are extracted from well-grown callus tissue. The protoplasts are pretreated in a 0.6 mol / L mannitol solution for 0.5 h, then transferred to an enzymatic hydrolysate containing 0.6 mol / L mannitol, 2.0% cellulase, and 0.2% cleavage enzyme. The hydrolysate is then incubated at 22°C and 60 rpm under light-protected conditions with horizontal shaking for 6 h. After hydrolysis, the protoplasts are centrifuged at 600 rpm for 7 min, yielding a yield of 2.70 × 10⁻⁶. 5 per mL.

[0020] In a more specific embodiment, this invention provides a method for isolating protoplasts from 'Musang King' grapes: Protoplasts are extracted from vigorously growing callus tissue and placed in an enzymatic hydrolysate containing 0.8 mol / L mannitol, 2.0% cellulase + 0.4% ionizing enzyme. The mixture is then kept in the dark at 22°C with horizontal shaking at 60 rpm for 6 hours. The protoplasts are purified by centrifugation at 600 rpm for 7 minutes, yielding 2.91 × 10⁻⁶ protoplasts. 5 per mL.

[0021] The present invention has at least the following beneficial effects:

[0022] (1) This invention establishes a stable isolation system for durian protoplasts for the first time. Based on the tissue characteristics of durian, this invention screens and optimizes the enzymatic hydrolysis materials and key technical parameters, which can stably obtain durian protoplasts with intact morphology and biological activity.

[0023] (2) Regarding the selection of enzymatic hydrolysis materials, this invention uses callus tissue instead of conventionally used leaves. Compared with leaf materials, the durian callus tissue suspension used in this invention is clear and transparent after enzymatic hydrolysis, without the production of viscous gel-like substances. It does not require the addition of antioxidants, polysaccharide removal reagents, or complex purification operations, nor does it require dilution or repeated filtration. The sieving and centrifugation purification processes are smooth, which can effectively avoid browning and polysaccharide adhesion problems, greatly simplifying the operation process and reducing reagent costs.

[0024] (3) The protoplasts prepared using the method of this invention have stable and reliable extraction results, which can meet the needs of downstream experiments. The yield of protoplasts from 'Golden Pillow' durian callus tissue can reach 2.70±0.13×10⁻⁶. 5 The activity was 73.48±1.63%; the yield of 'Musang King' reached 2.91±0.22×10⁶ cells / mL. 5 The yield / mL was 72.27±2.02%. Both varieties met the dual requirements of somatic cell hybridization and subsequent genetic manipulation for both quantity and quality of materials.

[0025] (4) The separation process of the present invention is mild, the membrane structure is highly intact, and mechanical damage and excessive enzymatic degradation are reduced, providing donor and acceptor materials with relatively intact membrane structures for protoplast fusion. Attached Figure Description

[0026] Figure 1 Different enzymatic hydrolysis materials; among them, Figure 1 Image A: Young leaves of seedlings in the field; Figure 1 Figure B in the diagram shows the shredded leaf blades in the enzymatic hydrolysate. Figure 1 Figure C in the diagram: Callus tissue; Figure 1 Figure D in the diagram: Callus fragments in the enzymatic hydrolysate;

[0027] Figure 2 Enzymatic hydrolysis results of different enzymatic hydrolysis materials; among them, Figure 2 Figure A in the diagram shows the results of enzymatic hydrolysis of callus tissue. Figure 2 Figure B in the figure shows the results of enzymatic hydrolysis of tender leaves from seedlings in the field.

[0028] Figure 3 The effect of mannitol concentration on protoplast yield and viability of 'Golden Pillow' genus; among which... Figure 3 Figure A in the figure shows the changes in protoplast production in 'Golden Pillow' genus. Figure 3 Figure B in the figure shows the changes in protoplast activity in 'Golden Pillow' genus.

[0029] Figure 4 The effect of mannitol concentration on protoplast yield and viability of 'Golden Pillow' genus; among which... Figure 4 Figure A in the diagram: 0.2 mol / L mannitol; Figure 4 Figure B in the diagram: 0.4 mol / L mannitol; Figure 4 Figure C in the diagram: 0.6 mol / L mannitol; Figure 4 Figure D in the diagram: 0.8 mol / L mannitol; Figure 4 Figure E in the diagram: 1.0 mol / L mannitol;

[0030] Figure 5 The effect of mannitol concentration on protoplast yield and viability in 'Musang King' varietal plants; among which, Figure 5 Figure A in the figure shows the changes in protoplast production in 'Musang King'. Figure 5 Figure B in the image shows changes in protoplast activity in 'Musang King' dinosaurs.

[0031] Figure 6 The effect of mannitol concentration on protoplast yield and viability in 'Musang King' varietal plants; among which, Figure 6 Figure A in the diagram: 0.2 mol / L mannitol; Figure 6 Figure B in the diagram: 0.4 mol / L mannitol; Figure 6 Figure C in the diagram: 0.6 mol / L mannitol; Figure 6 Figure D in the diagram: 0.8 mol / L mannitol; Figure 6 Figure E in the diagram: 1.0 mol / L mannitol;

[0032] Figure 7 The effect of enzymatic hydrolysis time on the yield and viability of 'Golden Pillow' protoplasts; among which... Figure 7 Figure A in the figure shows the changes in protoplast production in 'Golden Pillow' genus. Figure 7 Figure B in the figure shows the changes in protoplast activity in 'Golden Pillow' genus.

[0033] Figure 8 The effect of enzymatic hydrolysis time on the yield and viability of 'Golden Pillow' protoplasts; among which... Figure 8 Figure A in the diagram: 2h; Figure 8Figure B in the diagram: 4h; Figure 8 Figure C in the diagram: 6h; Figure 8 D diagram in the image: 8h; Figure 8 E diagram in the image: 10h;

[0034] Figure 9 The effect of enzymatic hydrolysis time on protoplast yield and viability of 'Musang King' grapes; among which... Figure 9 Figure A in the figure shows the changes in protoplast production in 'Musang King'. Figure 9 Figure B in the image shows changes in protoplast activity in 'Musang King' dinosaurs.

[0035] Figure 10 The effect of enzymatic hydrolysis time on protoplast yield and viability of 'Musang King' grapes; among which... Figure 10 Figure A in the diagram: 2h; Figure 10 Figure B in the diagram: 4h; Figure 10 Figure C in the diagram: 6h; Figure 10 D diagram in the image: 8h; Figure 10 E diagram in the image: 10h;

[0036] Figure 11 The effects of enzyme concentration combinations on the yield and viability of 'Golden Pillow' protoplasts; among which... Figure 11 Figure A in the figure shows the changes in protoplast production in 'Golden Pillow' genus. Figure 11 Figure B in the figure shows the changes in protoplast activity in 'Golden Pillow' genus.

[0037] Figure 12 Extraction efficiency of protoplasts using a combination of 2.0% cellulase and 0.2% dissociation enzyme; among which, Figure 12 Figure A in the diagram: Bright field; Figure 12 Figure B in the diagram: Fluorescence field;

[0038] Figure 13 The effects of enzyme concentration combinations on protoplast yield and viability in 'Musang King' grapes; among which... Figure 13 Figure A in the figure shows the changes in protoplast production in 'Musang King'. Figure 13 Figure B in the image shows changes in protoplast activity in 'Musang King' dinosaurs.

[0039] Figure 14 The extraction effect of protoplasts using a combination of 2.0% cellulase and 0.4% dissociation enzyme; among which, Figure 14 Figure A in the diagram: Bright field; Figure 14 Figure B in the diagram: Fluorescence field;

[0040] Figure 15 The effect of oscillation rate on the yield and viability of 'Golden Pillow' protoplasts; among which... Figure 15 Figure A in the figure shows the changes in protoplast production in 'Golden Pillow' genus. Figure 15 Figure B in the figure shows the changes in protoplast activity in 'Golden Pillow' genus.

[0041] Figure 16 The effect of oscillation rate on the yield and viability of 'Golden Pillow' protoplasts; among which... Figure 16 Figure A in the diagram: 0 rpm; Figure 16 Figure B in the diagram: 30 rpm; Figure 16 C in the diagram: 60 rpm; Figure 16 D-graph in the image: 90 rpm; Figure 16 E-graph in the image: 120 rpm;

[0042] Figure 17 The effect of oscillation rate on protoplast yield and viability in 'Musang King' varietals; among which... Figure 17 Figure A in the figure shows the changes in protoplast production in 'Musang King'. Figure 17 Figure B in the image shows changes in protoplast activity in 'Musang King' dinosaurs.

[0043] Figure 18 The effect of oscillation rate on protoplast yield and viability in 'Musang King' varietals; among which... Figure 18 Figure A in the diagram: 0 rpm; Figure 18 Figure B in the diagram: 30 rpm; Figure 18 C in the diagram: 60 rpm; Figure 18 D-graph in the image: 90 rpm; Figure 18 E-graph in the image: 120 rpm;

[0044] Figure 19 The effect of centrifugation rate on the yield and viability of 'Golden Pillow' protoplasts; among which... Figure 19 Figure A in the figure shows the changes in protoplast production in 'Golden Pillow' genus. Figure 19 Figure B in the figure shows the changes in protoplast activity in 'Golden Pillow' genus.

[0045] Figure 20 The effect of centrifugation rate on the yield and viability of 'Golden Pillow' protoplasts; among which... Figure 20 Figure A in the diagram: 200 rpm; Figure 20 Figure B in the diagram: 400 rpm; Figure 20 C in the diagram: 600 rpm; Figure 20 D-graph in the image: 800rpm; Figure 20 E-graph in the image: 1000rpm;

[0046] Figure 21 The effect of centrifugation rate on protoplast yield and viability in 'Musang King' varietals; among which, Figure 21 Figure A in the figure shows the changes in protoplast production in 'Musang King'. Figure 21 Figure B in the image shows changes in protoplast activity in 'Musang King' dinosaurs.

[0047] Figure 22The effect of centrifugation rate on protoplast yield and viability in 'Musang King' varietals; among which, Figure 22 Figure A in the diagram: 200 rpm; Figure 22 Figure B in the diagram: 400 rpm; Figure 22 C in the diagram: 600 rpm; Figure 22 D-graph in the image: 800rpm; Figure 22 E-graph in the image: 1000rpm;

[0048] Figure 23 The effect of centrifugation time on the yield and viability of 'Golden Pillow' protoplasts; among which... Figure 23 Figure A in the figure shows the changes in protoplast production in 'Golden Pillow' genus. Figure 23 Figure B in the figure shows the changes in protoplast activity in 'Golden Pillow' genus.

[0049] Figure 24 The effect of centrifugation time on the yield and viability of 'Golden Pillow' protoplasts; among which... Figure 24 Figure A in the diagram: 3 minutes; Figure 24 Figure B in the diagram: 5 minutes; Figure 24 Figure C in the chart: 7 minutes; Figure 24 D graph in the image: 9min;

[0050] Figure 25 The effect of centrifugation time on protoplast yield and viability in 'Musang King' grapes; among which... Figure 25 Figure A in the figure shows the changes in protoplast production in 'Musang King'. Figure 25 Figure B in the image shows changes in protoplast activity in 'Musang King' dinosaurs.

[0051] Figure 26 The effect of centrifugation time on protoplast yield and viability in 'Musang King' grapes; among which... Figure 26 Figure A in the diagram: 3 minutes; Figure 26 Figure B in the diagram: 5 minutes; Figure 26 Figure C in the chart: 7 minutes; Figure 26 D graph in the image: 9min;

[0052] Figure 27 The effect of pretreatment time on the yield and viability of 'Golden Pillow' protoplasts; among which, Figure 27 Figure A in the figure shows the changes in protoplast production in 'Golden Pillow' genus. Figure 27 Figure B in the figure shows the changes in protoplast activity in 'Golden Pillow' genus.

[0053] Figure 28 The effect of pretreatment time on the yield and viability of 'Golden Pillow' protoplasts; among which, Figure 28 Figure A in the diagram: 0h; Figure 28 Figure B in the diagram: 0.5h; Figure 28 Figure C in the diagram: 1.0h; Figure 28Figure D in the diagram: 1.5h;

[0054] Figure 29 The effect of pretreatment time on protoplast yield and viability in 'Musang King' varietals; among which, Figure 29 Figure A in the figure shows the changes in protoplast production in 'Musang King'. Figure 29 Figure B in the image shows changes in protoplast activity in 'Musang King' dinosaurs.

[0055] Figure 30 The effect of pretreatment time on protoplast yield and viability in 'Musang King' varietals; among which, Figure 30 Figure A in the diagram: 0h; Figure 30 Figure B in the diagram: 0.5h; Figure 30 Figure C in the diagram: 1.0h; Figure 30 D diagram in the image: 1.5h. Detailed Implementation

[0056] The present invention will be further described in detail below with reference to the embodiments. Reagents or instruments used without a specified manufacturer are considered to be conventional products that can be purchased on the market.

[0057] 1. Materials and Methods

[0058] 1.1 Plant materials

[0059] The callus tissue of 'Golden Pillow' and 'Musang King' durians, obtained through multiple generations of subculture and propagation, is stable in a light yellow and loose state. Unopened young leaves from healthy 4-5 year old durian seedlings.

[0060] 1.2 Main Instruments, Equipment and Reagents

[0061] 50mL sterile centrifuge tubes, 70μm cell sieve, pure water system, pH meter, autoclave, fluorescence upright microscope (ZEISS Axioscope 5), electronic balance, constant temperature shaker, 50mL conical flask, hemocytometer, glass slides, coverslips, 0.22μm filter, scalpel, forceps, centrifuge.

[0062] Table 1 Test Reagents

[0063]

[0064] 1.3 Test Methods

[0065] 1.3.1 Enzymatic hydrolysis material treatment method

[0066] After callus tissue was propagated according to the optimal subculture hormone ratio for the corresponding variety, it was sterilized in a sterile laminar flow hood after 30 minutes of ultraviolet irradiation. Using sterile forceps, vigorous callus tissue that had grown for about 20 days and was free of browning contamination was selected. At this time, the callus appeared loose and was pale yellow in color. The selected callus tissue was placed on weighing paper and weighed 1g in an analytical balance. Larger callus pieces were gently cut with a sharp blade to avoid crushing the material. The callus tissue was cut into small pieces with a diameter of about 3mm. After cutting, the small callus pieces were quickly placed into the prepared enzymatic hydrolysis solution. The container was gently shaken to ensure that the enzymatic hydrolysis material was completely submerged, preventing the material from being exposed to air for a long time, which would cause water loss and reduce cell viability.

[0067] Leaf material was selected from the tender new leaves that have just sprouted from vigorous durian shoots, prioritizing those that are not fully unfolded. Leaves should be bright green, clean, and free from pests and diseases. Cut the leaves at the petiole and immediately place them in a resealable bag containing moistened filter paper to prevent wilting during transport. Transfer the leaves to a glass plate and use a sharp, sterile blade to remove the main vein. Weigh 1g of the cut leaves on a balance. Then, shred the leaves into thin strips 1mm-2mm wide, keeping the blade perpendicular to the leaf surface and cutting quickly to avoid creating tissue debris. Immediately immerse the shredded leaves in the enzymatic hydrolysis solution to ensure the material remains moist. To prevent crushing of the tender tissue during cutting, replace the blade as soon as it becomes dull to minimize debris formation.

[0068] 1.3.2 Enzymatic hydrolysis and purification methods

[0069] Before material processing, an enzymatic hydrolysate needs to be prepared. Add 5 mL of sterile water to a sterile, enzyme-free centrifuge tube, then add 20 mmol / L 2-(N-morpholino)ethanesulfonic acid (MES) and 10 mmol / L potassium chloride solution as the base components of the hydrolysate. Then, according to the experimental design, add mannitol powder, dissociative enzyme, and cellulase. Dissolve thoroughly using a vortex mixer, then incubate at 55°C for 10 min. After removal, add 10 mmol / L calcium chloride solution and 0.1% bovine serum albumin. Adjust the volume to 10 mL with sterile water, adjust the pH to 5.7, and filter sterilize using a 0.22 μm filter. Transfer the solution to a sterile 50 mL Erlenmeyer flask for later use. After cutting the material, quickly place it into the hydrolysate, gently shake to submerge the material, seal the flask with sealing film, and wrap the entire Erlenmeyer flask with aluminum foil. Place it in a 22°C constant temperature shaker for enzymatic hydrolysis, protected from light.

[0070] To prepare W5 solution, add 80 mL of sterile water to the bottle, then add glucose, MES, potassium chloride, calcium chloride, and sodium chloride to make the final concentrations 5 mmol / L glucose, 2 mmol / L MES, 5 mmol / L potassium chloride, 125 mmol / L calcium chloride, and 154 mmol / L sodium chloride, respectively. Adjust the volume to 100 mL based on the solution volume.

[0071] After the enzymatic hydrolysis reaction is complete, add pre-cooled W5 solution. When adding, tilt the conical flask to allow the liquid to flow in gradually, preventing excessive flow rate from causing protoplast rupture. Then, place a 70μm sterile cell sieve onto a 50mL centrifuge tube. First, rinse the sieve several times with W5 solution to wet it. Then, slowly pour in the enzymatic hydrolysis solution, using W5 solution to rinse any remaining material in the conical flask and transfer it to the cell sieve. Gently squeeze the hydrolyzed material in the cell sieve using the tip of a pipette. After squeezing and observing a small amount of liquid dripping, add more W5 solution to wet the material and squeeze again, repeating this process three times. Centrifuge the tube. After centrifugation, discard the supernatant. Slowly add W5 solution dropwise, gently aspirate and mix, then centrifuge again, repeating this process twice. Finally, discard the supernatant and bring the volume to 10mL with W5 solution for subsequent counting and viability testing.

[0072] Use a hemocytometer to count protoplasts. Before use, wipe the hemocytometer clean with anhydrous ethanol and let it air dry on the lab bench. Gently invert the purified protoplast suspension several times to ensure uniform dispersion of the protoplasts and avoid local aggregation that could affect the counting results. Use a pipette tip to draw 10 μL of the well-mixed protoplast solution and drop it into the center of the hemocytometer. Smoothly cover the counting area of ​​the hemocytometer with a coverslip. Select the four large squares in the corners of the counting plate as the counting area to calculate the number of protoplasts, following the counting principle of "counting the top but not the bottom, counting the left but not the right": only individuals located on the top and left edges of the squares are counted, while those on the bottom and right edges are not counted. Exclude broken protoplast fragments, impurities, and other contaminants. The calculation formula is: Protoplast concentration (cells / mL) = average number of protoplasts in the four large squares × 10 4 ×Dilution factor. Protoplast samples treated with the same method were counted in 5 parallel runs, and the average of the 5 counts was taken as the final protoplast concentration for this treatment.

[0073] Add 100 μL of purified protoplast suspension to a 2 mL centrifuge tube, then add 2.4 μL of 5 mg / mL fluorescein diacetate (FDA). Gently pipette several times to ensure the staining solution is fully in contact with the protoplast suspension. Incubate on ice for 5 min in the dark. Take 10 μL of protoplast suspension and place it under an upright fluorescence microscope. Use 488 nm as the excitation wavelength and 520 nm as the emission wavelength to count protoplast viability. Count 12 fields of view and calculate the average value. Protoplast viability = number of cells emitting green fluorescence / total number of protoplasts in the field of view × 100%.

[0074] 1.3.3 Screening of different explant materials

[0075] After preliminary screening of enzymatic hydrolysis conditions, callus tissue and tender leaves of field seedlings were selected as enzymatic hydrolysis materials. Under the same conditions of enzymatic hydrolysis solution composition, enzymatic hydrolysis temperature, shaking rate, and purification method, the separation effect of the two materials was compared, with three replicates.

[0076] 1.3.4 Screening of Mannitol Concentration

[0077] Using callus tissue as the enzymatic hydrolysis material, with other enzymatic hydrolysis and purification conditions remaining unchanged, after preliminary screening, mannitol concentration gradients of 0.2 mol / L, 0.4 mol / L, 0.6 mol / L, 0.8 mol / L, and 1.0 mol / L were set up, with 3 replicates, and the yield and activity were calculated to determine the optimal mannitol concentration.

[0078] 1.3.5 Screening of enzymatic hydrolysis time

[0079] Using callus tissue as the enzymatic hydrolysis material, the optimal mannitol concentration was used, and the remaining enzymatic hydrolysis and purification conditions were kept unchanged. The enzymatic hydrolysis time was set to 2h, 4h, 6h, 8h, and 10h, and three replicates were set. The yield and activity were calculated, and the effect of enzymatic hydrolysis time on protoplasts was compared based on the results.

[0080] 1.3.6 Screening of enzyme concentration combinations

[0081] Using callus tissue as the enzymatic hydrolysis material, the optimal mannitol concentration and hydrolysis time were employed, while keeping other hydrolysis and purification conditions unchanged. Cellulase R-10 and Macerozyme R-10 were selected for concentration combinations, with concentration gradients set at 1.0%, 1.5%, 2.0% and 0.2%, 0.4%, 0.6%, respectively. Three replicates were performed, and the yield and activity were calculated. Based on the results, the effects of enzyme concentration combinations on protoplast extraction were compared.

[0082] 1.3.7 Screening of Enzymatic Digestion Oscillation Rate

[0083] Using callus tissue as the enzymatic hydrolysis material, the optimal combination of mannitol concentration, hydrolysis time, and enzyme concentration was used, while keeping other hydrolysis and purification conditions unchanged. The shaking rates were set to 0 rpm, 30 rpm, 60 rpm, 90 rpm, and 120 rpm, with three replicates for each treatment. The yield and viability were calculated, and the effects of different shaking rates on protoplast extraction were compared.

[0084] 1.3.8 Screening of Centrifugation Rate

[0085] Callus tissue was used as the enzymatic hydrolysis material, and optimal enzymatic hydrolysis conditions were employed. Protoplasts were then purified after hydrolysis. Centrifugation times were kept consistent, and centrifugation rates were set at 200 rpm, 400 rpm, 600 rpm, 800 rpm, and 1000 rpm. Each treatment was performed in triplicate. Yield and viability were calculated, and the effect of different centrifugation rates on protoplast purification was compared.

[0086] 1.3.9 Selection of Centrifugation Time

[0087] Callus tissue was used as the enzymatic hydrolysis material. Optimal hydrolysis conditions were employed, and centrifugation rates were kept consistent. Centrifugation times were set to 3 min, 5 min, 7 min, and 9 min. Each treatment was repeated three times. Yield and viability were calculated, and the effects of different centrifugation times on protoplast purification were compared.

[0088] 1.3.10 Selection of Material Pretreatment Time

[0089] After determining the optimal enzymatic hydrolysis and purification conditions, the effect of pretreatment on the enzymatic hydrolysis material was explored. Well-grown callus tissue was used and gently cut. The callus tissue was then placed in the selected optimal mannitol solution for soaking times of 0 h, 0.5 h, 1.0 h, 1.5 h, and 2.0 h, with three replicates to compare the effect of pretreatment on the enzymatic hydrolysis efficiency.

[0090] 1.4 Results Analysis

[0091] Data was entered using Excel 2016, and between-group difference analysis was performed using GraphPad Prism 10 software. One-way ANOVA was performed, and multiple comparisons were conducted using the Tukey method to indicate significant differences between groups and to complete the charts.

[0092] 2 Results

[0093] 2.1 Screening of enzymatic hydrolysis materials

[0094] A comparison of the protoplast extraction effects using callus tissue and young leaves, with separation effects as follows: Figure 2As shown, the protoplasts isolated from callus tissue are large in volume and quantity. The protoplast solution after enzymatic hydrolysis is normal, and the protoplast suspension is clear and transparent without abnormal viscosity, precipitation or turbidity. Subsequent sieving, centrifugation and purification steps can be performed. The entire purification process is smooth and requires no additional adjustments.

[0095] The separation effect using young leaves was generally poor, with small and few isolated protoplasts, and subsequent purification was significantly hindered. Due to the species' characteristics, the intercellular spaces and cytoplasm of leaf tissue contain a large amount of water-soluble mucilage, mainly pectin-like polysaccharides. During the enzymatic hydrolysis reaction, this mucilage is released in large quantities into the hydrolysate along with tissue decomposition and cell wall degradation, resulting in an extremely viscous, gel-like hydrolysate that drastically reduces the fluidity of the hydrolysate. To improve the viscosity, subsequent cell filtration and purification were performed, and the hydrolysate was diluted multiple times with W5 buffer during purification. However, the viscosity of the solution was not effectively improved; a large amount of viscous material remained, making dispersion difficult. Only a very small amount of solution could pass through the cell sieve. During subsequent centrifugation, the viscous liquid settled at the bottom of the tube, causing stratification and preventing complete purification. Therefore, it is not suitable as a material for enzymatic hydrolysis.

[0096] 2.2 Screening of mannitol concentration

[0097] 2.2.1 Screening of mannitol concentration in 'Golden Pillow' cultivar

[0098] Using vigorously growing callus tissue as enzymatic hydrolysis material, the effect of mannitol concentration on protoplasts was screened. Table 2 shows that the protoplast yield of *Golden Pillow* var. *golden pillow* initially increased and then decreased with increasing mannitol concentration, and differences in protoplast yield and viability existed among different treatments. The highest protoplast yield (2.49 ± 0.12 × 10⁻⁶) was observed when the mannitol concentration was 0.6 mol / L. 5 cells / mL, compared with 0.2 mol / L (1.60 ± 0.17 × 10⁻⁶). 5 (1.59 ± 0.26 × 10⁻⁶) and 1.0 mol / L (1.59 ± 0.26 × 10⁻⁶). 5 There was a significant difference compared to the treatment with 0.4 mol / L (1.98 ± 0.14 × 10⁻⁶ cells / mL), compared to 0.4 mol / L (1.98 ± 0.14 × 10⁻⁶ cells / mL). 5 0.8 mol / L (2.10 ± 0.23 × 10⁻⁶ cells / mL) and 0.8 mol / L (2.10 ± 0.23 × 10⁻⁶ cells / mL). 5 There were no significant differences between treatments (number of cells / mL).

[0099] The trend of protoplast viability was similar to that of yield, initially increasing and then decreasing with changes in mannitol concentration, reaching a peak of 76.81±2.94% at 0.6 mol / L. This was significantly higher than the 0.2 mol / L (47.34±3.03%) and 0.4 mol / L (60.46±2.26%) treatments, but not significantly different from the 0.8 mol / L (67.66±3.78%) and 1.0 mol / L (67.56±1.98%) treatments. Considering both protoplast yield and viability, 0.6 mol / L is the optimal mannitol concentration for protoplast isolation from *Gynostemma pentaphyllum* using callus tissue.

[0100] Table 2. Effects of mannitol concentration on protoplast yield and viability of 'Golden Pillow' genus.

[0101]

[0102] 2.2.2 Screening of mannitol concentration in 'Musang King' grapes

[0103] Vigorous callus tissue was used as the enzymatic hydrolysis material. Mannitol concentration was screened, and protoplast yield and viability were statistically analyzed. The results are shown in Table 3. In terms of yield, the protoplast yield of 'Musang King' protoplasts first increased and then decreased with increasing mannitol concentration. The highest yield was observed at 0.8 mol / L, reaching 2.71 ± 0.22 × 10⁻⁶. 5 The yield per mL was significantly higher than that of the 0.2 mol / L, 0.4 mol / L, and 1.0 mol / L treatments. The yield of the 0.6 mol / L treatment was not significantly different from that of the 0.8 mol / L treatment, at 2.53 ± 0.09 × 10⁻⁶. 5 The concentration of [number] cells / mL was significantly different from the remaining three treatments, with the 0.2 mol / L treatment showing the lowest yield at 1.58 ± 0.17 × 10⁻⁶. 5 per mL.

[0104] Regarding protoplast viability, the highest viability was observed at a mannitol concentration of 0.8 mol / L (77.84 ± 2.02%), significantly higher than that at 0.2 mol / L and 0.4 mol / L. The viability at 0.6 mol / L and 1.0 mol / L was 71.83 ± 3.15% and 71.08 ± 2.46%, respectively, with no significant difference from the 0.8 mol / L treatment. The lowest viability was observed at 0.2 mol / L (51.03 ± 3.08%).

[0105] Based on the yield and vigor test results, a mannitol concentration of 0.8 mol / L is beneficial for the separation of 'Musang King' protoplasts.

[0106] Table 3. Effects of mannitol concentration on protoplast yield and viability of 'Musang King' cultivar.

[0107]

[0108] 2.3 Screening of enzymatic hydrolysis time

[0109] 2.3.1 Screening of 'Golden Pillow' enzymatic hydrolysis time

[0110] Callus tissue was used as the enzymatic hydrolysis material, and the mannitol concentration was set at 0.6 mol / L, with other conditions kept constant. The effect of enzymatic hydrolysis time on protoplast extraction was investigated, and the results are shown in Table 4. During the extraction of 'Golden Pillow' protoplasts, the protoplast yield generally increased with the extension of enzymatic hydrolysis time, from 1.23 ± 0.29 × 10⁻⁶. 5 The concentration of cells / mL increased to 2.57 ± 0.10 × 10⁻⁶. 5 The yield was [number] cells / mL, reaching its peak at 6 hours, showing a significant difference from other treatment groups; however, the yield began to decline after 6 hours, decreasing to 2.29 ± 0.11 × 10⁻⁶ at 8 hours. 5 The concentration of cells / mL continued to decrease to 1.64 ± 0.30 × 10⁻⁶ at 10 h. 5 The result of 1 cell / mL indicates that too short an enzymatic hydrolysis time will lead to insufficient cell wall degradation and insufficient release of protoplasts from the tissue, while long-term enzymatic hydrolysis will cause protoplast rupture and a decrease in yield.

[0111] Regarding protoplast viability, within the time range of 2 to 6 hours, the viability remained at 72% to 78%, with no significant difference among treatments. However, as the enzymatic hydrolysis time increased, when the time was extended to 8 to 10 hours, the protoplast viability gradually decreased, reaching a minimum of 57.64 ± 4.74%. Considering both yield and viability, 6 hours is the optimal enzymatic hydrolysis time for 'Golden Pillow' protoplast extraction.

[0112] Table 4. Effects of enzymatic hydrolysis time on the yield and viability of 'Golden Pillow' protoplasts.

[0113]

[0114] 2.3.2 Screening of 'Musang King' enzymatic hydrolysis time

[0115] Callus tissue was used as the enzymatic hydrolysis material, and the mannitol concentration was set to 0.8 mol / L. All other conditions remained consistent. The results are shown in Table 5. The protoplast yield first increased and then decreased with increasing hydrolysis time, with the lowest yield being 1.30 ± 0.14 × 10⁻⁶ at 2 hours. 5 The concentration of cells / mL reached a peak of 2.73 ± 0.15 × 10⁻⁶ over a period of 6 hours. 5 The yield was 2.13 ± 0.17 × 10⁻⁶ cells / mL; after 8 hours, the yield decreased to 2.13 ± 0.17 × 10⁻⁶ cells / mL. 5 The concentration of cells / mL increased slightly at 10 hours, reaching 2.25 ± 0.23 × 10⁻⁶.5 The number of cells / mL was still lower than that of the 6-hour treatment, and there was no significant difference between the multiple treatment groups.

[0116] Regarding protoplast viability, the highest viability was observed at 2 hours (78.53±3.51%). There was no significant difference in viability between 4 and 6 hours. Viability decreased slightly at 8 hours, falling below 70%, and reached its lowest point at 10 hours (61.37±3.28%). Both excessively short and excessively long enzymatic hydrolysis times negatively impact protoplast yield and viability. To ensure high protoplast yield and viability, 6 hours of enzymatic hydrolysis is the optimal time for protoplast isolation from 'Musang King' grapes.

[0117] Table 5. Effects of enzymatic hydrolysis time on protoplast yield and viability of 'Musang King' grapes.

[0118]

[0119] 2.4 Screening of enzyme concentration combinations

[0120] 2.4.1 Screening of 'Golden Pillow' enzyme concentration combinations

[0121] Using callus tissue as the enzymatic hydrolysis material, the optimal mannitol concentration and hydrolysis time were selected and screened, keeping other experimental conditions constant. The results of the effect of enzyme concentration combinations on protoplast extraction are shown in Table 6. Statistical analysis of protoplast yield revealed that when the concentration of the dissociative enzyme (M) was fixed at 0.2%, the protoplast yield showed a continuous increasing trend with the concentration of cellulase (C), with the highest yield being 2.0C + 0.2M, yielding 2.63 ± 0.16 × 10⁻⁶. 5 Cells / mL; when the concentration of cellulase was increased to 0.4%, the yield showed a trend of first increasing and then decreasing with the change in cellulase concentration, with the yield of the 1.5C + 0.4M combination being 2.27 ± 0.22 × 10⁻⁶. 5 The yield was slightly higher than that of 1.0C+0.4M and 2.0C+0.4M, but there was no significant difference among the combinations. When the concentration of the cellulase was further increased to 0.6%, the yield also showed a trend of first increasing and then decreasing with the concentration of cellulase. The 1.5C+0.6M combination had the highest yield, which was 2.37±0.27×10⁻⁶. 5 The yield was slightly higher than that at 1.0C+0.6M and 2.0C+0.6M, and the overall yield level was roughly similar to that of the combinations at a 0.4% analyte concentration, and none of them were significantly higher than the 2.0C+0.2M combination.

[0122] Regarding protoplast viability, when the cleavage enzyme concentration was 0.2%, protoplast viability fluctuated slightly between 70.72% and 72.00%, with no significant difference. When the cleavage enzyme concentration was 0.4%, the viability showed a trend of first increasing and then decreasing with the change in cellulase concentration. The 1.5C + 0.4M combination showed the highest viability at 75.32 ± 2.09%, with no significant difference compared to other treatments with the same cleavage enzyme concentration. When the cleavage enzyme concentration increased to 0.6%, the viability decreased with increasing cellulase concentration, reaching a minimum of 57.14 ± 4.43%, which was the lowest viability treatment in this experiment, indicating that increasing enzyme concentration leads to a decrease in protoplast viability. Considering both yield and viability, 2.0% cellulase + 0.2% cleavage enzyme was considered the optimal enzyme concentration combination for 'Golden Pillow' protoplast extraction.

[0123] Table 6. Effects of enzyme concentration combinations on the yield and viability of 'Golden Pillow' protoplasts.

[0124]

[0125] 2.4.2 Screening of 'Musang King' enzyme concentration combinations

[0126] Using callus tissue as the enzymatic hydrolysis material, the optimal mannitol concentration and hydrolysis time were selected and screened, keeping other experimental conditions constant. The results of the effect of enzyme concentration combinations on protoplast extraction are shown in Table 7. Regarding protoplast yield, when the concentration of the separating enzyme was 0.2%, the yield increased from 1.20 ± 0.16 × 10⁻⁶ when the cellulase concentration increased. 5 The concentration of cells / mL increased to 2.36 ± 0.17 × 10⁻⁶. 5 The highest yield was achieved with a concentration of 2.0% cellulase + 0.2% chromogenic enzyme. When the chromogenic enzyme concentration increased to 0.4%, protoplast production increased with increasing cellulase concentration, reaching a maximum of 2.80 ± 0.23 × 10⁻⁶ cells / mL at the 2.0% cellulase + 0.4% chromogenic enzyme combination. 5 The yield was significantly higher than that of the other two groups; when the enzyme concentration was 0.6%, the yield was 2.13 × 10⁻⁶. 5 cells / mL ~2.35×10 5 The number of cells / mL was between [number] and [number], and there was no significant difference between the combinations.

[0127] Regarding protoplast viability, the overall viability was high at a 0.2% chromogenic enzyme concentration, with all groups exceeding 70%, and the 1.5% cellulase + 0.2% chromogenic enzyme combination exhibiting the highest viability. At a 0.4% chromogenic enzyme concentration, there was no significant difference between treatments, with the 2.0% cellulase + 0.4% chromogenic enzyme combination showing the highest viability. At a 0.6% chromogenic enzyme concentration, viability decreased with increasing concentration, with the 2.0% cellulase + 0.6% chromogenic enzyme combination showing the lowest viability. Considering both yield and viability, the 2.0% cellulase + 0.4% chromogenic enzyme combination is considered the best enzyme concentration combination for protoplast extraction from 'Musang King' grapes.

[0128] Table 7. Effects of enzyme concentration combinations on protoplast yield and viability of 'Musang King' grapes.

[0129]

[0130] 2.5 Screening of Enzymatic Digestion Shaking Rate

[0131] 2.5.1 Screening of the enzymatic hydrolysis shaking rate of 'Golden Pillow' granules

[0132] Protoplasts were extracted using callus tissue. The mannitol concentration was set at 0.6 mol / L, the enzymatic hydrolysis time was 6 h, and the enzyme concentration combination was 2.0% cellulase + 0.2% dissociative enzyme, with other conditions remaining constant. The effect of shaking rate on protoplast extraction was investigated, and the results are shown in Table 8.

[0133] The overall trend of protoplast yield was first increased and then decreased, with the lowest yield at 0 rpm, only 0.97 ± 0.25 × 10⁻⁶. 5 The yield was significantly different from other treatments at 1.98 ± 0.24 × 10⁻⁶ cells / mL; when the rate was increased to 30 rpm, the yield was 1.98 ± 0.24 × 10⁻⁶ cells / mL. 5 The concentration was highest at 60 rpm, at 2.70 ± 0.13 × 10⁻⁶ cells / mL. 5 The concentrations at 90 rpm and 120 rpm were 2.42 ± 0.22 × 10⁻⁶ cells / mL, respectively. 5 cells / mL and 2.06±0.34×10 5 The number of cells / mL was not significantly different among the treatments except for 0 rpm.

[0134] In terms of activity, the activity ranged from 68% to 74% within the range of 0 rpm to 90 rpm, with the highest activity at 60 rpm (73.48 ± 1.63%), which was not significantly different from the other treatments. When the oscillation rate increased to 120 rpm, the activity decreased to 54.88 ± 2.43%, which was significantly lower than all other treatments.

[0135] The combined yield and viability results show that when the oscillation rate is too low, the protoplast release rate is slow, while when it is too high, the yield and viability will decrease. 60 rpm was determined to be the optimal enzymatic oscillation rate for 'Golden Pillow', which can maintain good yield and viability.

[0136] Table 8. Effects of oscillation rate on protoplast yield and viability of 'Golden Pillow' genus.

[0137]

[0138] 2.5.2 Screening of 'Musang King' enzymatic hydrolysis shaking rate

[0139] Using callus tissue as the enzymatic hydrolysis material, the mannitol concentration was set at 0.8 mol / L, the hydrolysis time was 6 h, and the enzyme concentration combination was 2.0% cellulase + 0.4% ionizing enzyme. All other experimental conditions were kept unchanged. The results are shown in Table 9.

[0140] In terms of protoplast yield, the yield at 0 rpm was 1.13 ± 0.30 × 10⁻⁶. 5 The lowest value was 2.87 ± 0.19 × 10⁻⁶ cells / mL; as the rate gradually increased, the yield gradually increased, reaching a maximum value of 2.87 ± 0.19 × 10⁻⁶ cells / mL at 60 rpm. 5 The yield was significantly higher than that at 0 rpm and 30 rpm; however, the yield began to decline when the rate exceeded 60 rpm, decreasing to 2.16 ± 0.22 × 10⁻⁶ at 90 rpm and 120 rpm, respectively. 5 cells / mL and 1.79±0.25×10 5 per mL.

[0141] Regarding protoplast activity, there was no significant difference between 0 rpm, 30 rpm, and 60 rpm. The highest activity was 71.30 ± 2.45% at 60 rpm; the activity was moderate at 90 rpm, at 62.74 ± 5.62%; and the lowest was at 120 rpm, at 53.53 ± 2.05%.

[0142] Considering both protoplast yield and viability, 60 rpm yields protoplasts with high yield and moderate viability, and this oscillation rate is considered to be the optimal rate for 'Musang King'.

[0143] Table 9. Effects of oscillation rate on protoplast yield and viability in 'Musang King' cultivars.

[0144]

[0145] 2.6 Screening of centrifugation rates

[0146] 2.6.1 Screening of centrifugation rates for 'Golden Pillow' cultivar

[0147] Protoplasts were extracted from callus tissue. The mannitol concentration was set at 0.6 mol / L, the enzymatic hydrolysis time was 6 h, the enzyme concentration combination was 2.0% cellulase + 0.2% ionizing enzyme, and the shaking rate was 60 rpm. After the enzymatic hydrolysis, a purification step was performed while keeping other conditions unchanged. The effect of centrifugation rate on protoplasts in the purification step was investigated.

[0148] In terms of protoplast yield, the lowest yield was observed at 200 rpm, at 1.35 ± 0.18 × 10⁻⁶. 5 The yield was significantly different from that at 400, 600, and 800 rpm. Subsequently, the yield gradually increased with increasing rate, reaching its highest value of 2.65 ± 0.21 × 10⁻⁶ at 600 rpm. 5 The yield per mL was [number] cells / mL; as the rate was further increased to 800 rpm to 1000 rpm, the yield gradually decreased, reaching a minimum of 1.48 ± 0.17 × 10⁻⁶ cells / mL at 1000 rpm. 5 per mL.

[0149] Regarding protoplast activity, within the speed range of 200 rpm to 600 rpm, the protoplast activity did not differ significantly, fluctuating around 70%, with the highest value reaching 74.08 ± 2.17%. When the speed exceeded 600 rpm, the activity began to decline, dropping to 62.95 ± 2.03% at 800 rpm, and further decreasing at 1000 rpm.

[0150] Based on the above results, protoplasts cannot settle effectively when the centrifugation rate is too low, but the difference in viability is not obvious. Therefore, 600 rpm was selected as the centrifugation rate for purifying 'Golden Pillow' protoplasts, which can ensure a high yield while maintaining a high viability.

[0151] Table 10 Effects of centrifugation rate on protoplast yield and viability of 'Golden Pillow' genus.

[0152]

[0153] 2.6.2 Screening of centrifugation rates for 'Musang King' cultivar

[0154] Using callus tissue as the enzymatic hydrolysis material, the mannitol concentration was set at 0.8 mol / L, the hydrolysis time was 6 h, the enzyme concentration combination was 2.0% cellulase + 0.4% dissociative enzyme, and the shaking rate was 60 rpm to extract protoplasts. Then, purification-related experiments were carried out to investigate the effect of centrifugation rate on protoplasts.

[0155] As the rotational speed increases, the output gradually increases, reaching its maximum value of 3.13 ± 0.15 × 10⁻⁶ at 600 rpm. 5The yield was significantly different from that at 200 rpm and 400 rpm; when the rate was increased to 800 rpm and 1000 rpm, the yield decreased, with the lowest being 1.91 ± 0.17 × 10⁻⁶ at 1000 rpm. 5 per mL.

[0156] In terms of activity, the activity range of 200, 400, 600 and 800 rpm was between 67% and 77%, with no significant difference among the treatments; however, when the rate increased to 1000 rpm, the activity decreased to 52.67±3.23%, which was significantly lower than other treatments.

[0157] Considering both yield and viability, a centrifugation rate of 600 rpm is considered to maintain high yield and viability, and can be used as the centrifugation rate for 'Musang King' protoplast purification.

[0158] Table 11 Effect of centrifugation rate on protoplast yield and viability of 'Musang King' cultivar

[0159]

[0160] 2.7 Selection of Centrifugation Time

[0161] 2.7.1 Screening of centrifugation time for 'Golden Pillow' cultivar

[0162] Callus tissue was used as the enzymatic hydrolysis material to extract protoplasts. The enzymatic hydrolysis step was carried out under the optimal conditions. The obtained protoplasts were then purified. The effect of different centrifugation times on the protoplasts was investigated. The results are shown in Table 12.

[0163] In terms of protoplast production, the lowest production was observed at 3 minutes, at 1.28 ± 0.09 × 10⁻⁶. 5 The yield increased to 1.88 ± 0.17 × 10⁻⁶ cells / mL as the time was extended to 5 min and 7 min. 5 cells / mL and 2.60±0.25×10 5 The yield was significantly different from other treatments at 7 min; when the time was extended to 9 min, the yield decreased to 1.90 ± 0.11 × 10⁻⁶. 5 per mL.

[0164] Regarding protoplast viability, the viability was 72.51±6.62% at 3 min, slightly decreased to 71.81±5.15% at 5 min, and 69.28±6.60% at 7 min; however, when the time was extended to 9 min, the viability decreased to 59.41±5.39%, with no significant difference between the treatment groups. Considering both yield and viability, a centrifugation time of 7 min was determined as the optimal centrifugation time for purifying 'Golden Pillow' callus tissue.

[0165] Table 12 Effects of centrifugation time on protoplast yield and viability of 'Golden Pillow' genus.

[0166]

[0167] 2.7.2 Screening of centrifugation time for 'Musang King'

[0168] Protoplasts were extracted from callus tissue using enzymatic hydrolysis. The optimal hydrolysis conditions using 'Musang King' hydrolysate were employed. The resulting protoplasts were then purified. The effect of different centrifugation times on the protoplast yield was investigated, and the results are shown in Table 13. Regarding yield, the yield gradually increased with increasing centrifugation time, reaching 1.73 ± 0.17 × 10⁻⁶ at 3 min. 5 The concentration of [number] cells / mL increased to 2.16 ± 0.24 × 10⁻⁶ at 5 min. 5 The concentration of cells / mL reached a peak of 2.91 ± 0.22 × 10⁻⁶ at 7 min. 5 The concentration of [number] cells / mL was significantly higher than that in the 3- and 5-minute treatment groups, but decreased to 2.45 ± 0.10 × 10⁻⁶ cells / mL at 9 minutes. 5 Protoplast viability was 75.94±7.07%, 71.15±2.14%, and 72.27±2.02% at 3, 5, and 7 min, respectively, with no significant difference among the three treatments, all maintaining a level above 70%. However, when the centrifugation time was extended to 9 min, the viability dropped sharply to 51.76±5.37%, showing a significant difference from the first three treatments. Overall, a centrifugation time of 7 min yielded the best results.

[0169] Table 13 Effects of centrifugation time on protoplast yield and viability of 'Musang King' cultivar

[0170]

[0171] 2.8 Selection of Material Pretreatment Time

[0172] 2.8.1 Screening of Pretreatment Time for 'Golden Pillow' Material

[0173] Protoplast extraction and purification were performed using the optimal enzymatic hydrolysis conditions of 'Golden Pillow'. Before enzymatic hydrolysis, the callus tissue was pretreated in a 0.6 mol / L mannitol solution to investigate whether pretreatment of the enzymatic hydrolysis material could improve protoplast yield and viability.

[0174] In terms of protoplast production, the yields at 0h and 0.5h were 2.60 ± 0.25 × 10⁻⁶. 5 cells / mL and 2.65±0.17×10 5The protoplast yield was not significantly different between the two treatments (1.0, 1.5, and 2.0 h) and was significantly different from other treatments (1.0, 1.5, and 2.0 h). Protoplast yield gradually decreased with increasing treatment time, reaching a minimum of 1.41 ± 0.34 × 10⁻⁶. 5 per mL.

[0175] Regarding viability, the highest viability was observed at 0 h (72.89±4.67%), which was not significantly different from the 0.5 h (68.35±5.04%) and 1.0 h (59.00±6.76%) treatments. When the time was extended to 1.5 h and 2.0 h, the viability decreased to 55.59±5.63% and 48.56±5.84%, respectively, which differed from the other treatments.

[0176] The results showed that the yield and vigor were not as expected after pretreatment. After 0.5 h of pretreatment, the yield increased slightly but the vigor decreased slightly. Pretreatment of 0.5 h can slightly improve the preparation effect.

[0177] Table 14 Effects of pretreatment time on protoplast yield and viability of 'Golden Pillow' cultivar.

[0178]

[0179] 2.8.2 Screening of 'Musang King' Material Pretreatment Time

[0180] Protoplast extraction and purification were performed using the optimal enzymatic hydrolysis conditions of 'Musang King'. Before the enzymatic hydrolysis began, the callus tissue was soaked in 0.8 mol / L mannitol solution to investigate the effect of pretreatment on protoplast extraction.

[0181] In terms of yield, the overall trend is a gradual decline, with the highest yield observed when soaking without pretreatment, at 2.95 ± 0.39 × 10⁻⁶. 5 The concentration of cells / mL was 2.75 ± 0.42 × 10⁻⁶ after pretreatment soaking, and the concentration was 2.75 ± 0.42 × 10⁻⁶ after 0.5 h. 5 The yield was significantly higher than other treatments, with no significant difference between the two treatments (number of cells / mL). When the pretreatment time reached 1.0 h, 1.5 h, and 2.0 h, the overall yield remained at 1.2 × 10⁻⁶. 5 cells / mL ~1.5×10 5 There was no significant difference between the number of cells / mL.

[0182] The viability also showed a consistent trend: the viability was highest without pretreatment (71.59±5.46%), and 62.45±3.72% after 0.5h, with no significant difference between the two. Viability gradually decreased after 1.5h (48.88±5.56%) and 2.0h (44.84±5.39%). In summary, for protoplast extraction from 'Musang King' grapes, no pretreatment yields better results.

[0183] Table 15 Effect of pretreatment time on protoplast yield and viability of 'Musang King' cultivars

[0184]

[0185] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A method for efficient separation of protoplasts adapted to durian callus tissue, characterized in that, The method includes the following steps: taking durian callus tissue in good condition, placing it in an enzymatic hydrolysate containing 0.4-0.8 mol / L mannitol, 1.0%-2.0% cellulase, and 0.2%-0.6% cleavage enzyme, and enzymatically hydrolyzing it horizontally with shaking at room temperature and 30-120 rpm in the dark for 2-8 hours. After the enzymatic hydrolysis is completed, adding W5 solution, centrifuging at 200-800 rpm for 5-7 minutes, and collecting the protoplasts; wherein the durian variety is 'Golden Pillow' or 'Musang King'.

2. The method for efficient separation of protoplasts adapted to durian callus tissue according to claim 1, characterized in that, The enzymatic hydrolysate also contains 20 mmol / L 2-(N-morpholino)ethanesulfonic acid, 10 mmol / L potassium chloride solution, 10 mmol / L calcium chloride solution, and 0.1% bovine serum albumin, with a pH of 5.7 ± 0.

1.

3. The method for efficient separation of protoplasts adapted to durian callus tissue according to claim 1, characterized in that, The W5 solution is prepared as follows: Add 80 mL of sterile water to the bottle, then add glucose, MES, potassium chloride, calcium chloride and sodium chloride to make the final concentrations 5 mmol / L glucose, 2 mmol / L MES, 5 mmol / L potassium chloride, 125 mmol / L calcium chloride and 154 mmol / L sodium chloride, respectively. Then, use sterile water to make up to 100 mL.

4. The method for efficient separation of protoplasts adapted to durian callus tissue according to claim 1, characterized in that, The enzymatic hydrolysis time is 2-6 hours.

5. The method for efficient separation of protoplasts adapted to durian callus tissue according to claim 1, characterized in that, The durian callus tissue was first pretreated in a 0.4-0.8 mol / L mannitol solution for a time greater than 0 h and less than or equal to 0.5 h.

6. The method for efficient separation of protoplasts adapted to durian callus tissue according to claim 1, characterized in that, The mannitol concentration is 0.6~0.8 mol / L.

7. The method for efficient separation of protoplasts adapted to durian callus tissue according to claim 1, characterized in that, Room temperature refers to 20~25℃.

8. The method for efficient separation of protoplasts adapted to durian callus tissue according to claim 1, characterized in that, The oscillation rate is 30~90 rpm.

9. The method for efficient separation of protoplasts adapted to durian callus tissue according to claim 1, characterized in that, The centrifugation rate is 200~600 rpm.

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