Preparation method and application of an enzymatic solution of red radish protoplasts and a preparation method of red radish protoplasts

Abstract

The application provides a kind of preparation of red radish protoplast enzymatic hydrolysate and the preparation method and application of red radish protoplast, belong to the technical field of plant cytology.The enzymatic hydrolysate includes:1.0~1.5w / v% cellulase, 0.3~0.5w / v% dissociation enzyme and 0.3~0.5w / v% hemicellulase.The cellulase has the effect of degrading cellulose in plant cell wall, dissociation enzyme is used with cellulase, which is beneficial to separate plant tissue into single cell, and the main effect of hemicellulase is to degrade hemicellulose in plant cell wall.The enzymatic hydrolysate can synergistically promote the effect of red radish tissue cell wall lysis through the comprehensive effect of three enzymes, can better and faster destroy cell wall skeleton, is more conducive to the preparation of red radish protoplast and further maximally release protoplast, is conducive to improving the yield of protoplast, and can also keep the protoplast with high activity.

Description

Technical Field

[0001] This invention belongs to the field of plant cell biology technology, specifically relating to an enzymatic hydrolysate for preparing carrot protoplasts, a method for preparing carrot protoplasts, and their applications. Background Technology

[0002] carrots ( Raphanus sativus Radish (L.) is a biennial or annual herbaceous plant belonging to the genus Raphanus in the family Brassicaceae. Among them, Nujiang Red Radish is a unique variety from Nujiang Prefecture, Yunnan Province. Its fleshy root has a skin and flesh color that ranges from red to purplish-red. It has a crisp and sweet taste, and its flesh is tender and juicy. It is of great significance for the breeding and production of radish varieties.

[0003] Plant protoplasts are plant cells with their cell walls removed, possessing a certain reproductive capacity and capable of developing into complete plants under appropriate culture conditions. Because protoplasts lack cell walls, they more easily overcome incompatibility barriers in distant hybridization and readily absorb exogenous macromolecules such as organelles and proteins. They have been widely used in subcellular localization, gene expression analysis, protein-protein interactions, and gene editing, and are considered ideal materials for developmental biology, cell biology, and cytogenetics research. The three main components of plant cell walls are cellulose, hemicellulose, and pectin. These components vary significantly depending on the plant species and the part of the plant from which they are obtained. The genotype and tissue type of the suitable material are crucial factors influencing the isolation, preparation, and regeneration of plant protoplasts. Currently, there is no research on the preparation of protoplasts from Nujiang carrots. Establishing and optimizing a protoplast preparation system for carrots is of great significance for achieving cell fusion of protoplasts from different radish varieties and for the efficient introduction and expression of exogenous genes. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide an application of a compound enzyme in the preparation of carrot protoplasts. The compound enzyme can enzymatically hydrolyze the plant tissue of Nujiang carrots to achieve the preparation of carrot protoplasts, thereby improving the yield and viability of carrot protoplasts.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] This invention provides an application of a compound enzyme in the preparation of carrot protoplasts. The compound enzyme comprises, by weight, 1.0-1.5 parts of cellulase, 0.3-0.5 parts of dissociative enzyme and 0.3-0.5 parts of hemicellulase.

[0007] Preferably, the cellulase includes cellulase RS; the analyte includes analyte R-10.

[0008] Preferably, by weight, the complex enzyme comprises: 1.0 part cellulase, 0.5 part dissociation enzyme, and 0.3 part hemicellulase;

[0009] Alternatively, the complex enzyme comprises: 1.5 parts cellulase, 0.3 parts ionase, and 0.5 parts hemicellulase;

[0010] Alternatively, the complex enzyme comprises: 1.0 part cellulase, 0.5 part dissociation enzyme, and 0.5 part hemicellulase.

[0011] This invention provides an enzymatic hydrolysate for preparing carrot protoplasts, comprising, by total volume: 1.0–1.5 w / v% cellulase, 0.3–0.5 w / v% dissociative enzyme, 0.3–0.5 w / v% hemicellulase, 0.50–0.60 mol / L mannitol, 18–22 mmol / L MES, 18–22 mmol / L KCl, 8–12 mmol / L CaCl2, 0.08–0.12 w / v% BSA, and 0.03–0.07 mmol / L β-mercaptoethanol.

[0012] This invention provides the application of the enzymatic hydrolysate described in the above technical solution in any one or more of the following: preparing carrot protoplasts, improving protoplast activity, and improving protoplast yield.

[0013] This invention provides a method for preparing carrot protoplasts, comprising:

[0014] The carrot plant tissue was enzymatically hydrolyzed using the enzymatic hydrolysate described in the above technical solution to obtain carrot protoplasts.

[0015] Preferably, the enzymatic hydrolysis temperature is 28~30℃; and the enzymatic hydrolysis time is 2.5~4h.

[0016] Preferably, the carrot plant tissue includes carrot seedling plant tissue; the carrot seedling plant tissue includes any one or more of leaves, cotyledons, and petiolate cotyledons.

[0017] Preferably, the method for cultivating carrot seedlings includes culturing carrot seeds using plant tissue culture to obtain carrot seedlings.

[0018] Preferably, the temperature for plant tissue culture is 23-25℃; the photoperiod for plant tissue culture is 15-17 h / d; the light intensity for plant tissue culture is 2500-3200 Lx; and the culture time is 4-6 days.

[0019] Beneficial effects:

[0020] This invention provides an application of a compound enzyme in the preparation of carrot protoplasts. The compound enzyme, by weight, comprises: 1.0-1.5 parts cellulase, 0.3-0.5 parts dissociative enzyme, and 0.3-0.5 parts hemicellulase. In the compound enzyme provided by this invention, cellulase degrades cellulose in plant cell walls; the dissociative enzyme, used in conjunction with cellulase, facilitates the separation of plant tissue into single cells; and the hemicellulase primarily degrades hemicellulose in plant cell walls. Through the combined action of these three enzymes, the compound enzyme synergistically promotes the lysis of carrot plant tissue cell walls, better and faster disrupting the cell wall skeleton, thus facilitating the preparation of carrot protoplasts, maximizing protoplast release, increasing protoplast yield, and maintaining high protoplast viability. The results of the embodiments of this invention show that using an enzymatic hydrolysate containing the compound enzyme to prepare protoplasts from Nujiang carrots can significantly improve the protoplast yield and viability. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The image shows the observation results of carrot protoplasts prepared in Example 1; the right image shows the FDA staining results, and the left image shows the corresponding bright field protoplast image. The scale bar in the image is 100µm.

[0023] Figure 2 The image shows the observation results of carrot protoplasts prepared in Example 2; the right image shows the FDA staining results, and the left image shows the corresponding bright field protoplast image. The scale bar in the image is 100µm.

[0024] Figure 3 The image shows the observation results of carrot protoplasts prepared in Comparative Example 1; the right image shows the FDA staining results, and the left image shows the corresponding bright field protoplast image. The scale bar in the image is 100µm.

[0025] Figure 4 The image shows the observation results of carrot protoplasts prepared in Comparative Example 2; the right image shows the FDA staining results, and the left image shows the corresponding bright-field protoplast image. The scale bar in the image is 100µm. Detailed Implementation

[0026] This invention provides an application of a compound enzyme in the preparation of carrot protoplasts. The compound enzyme comprises, by weight, 1.0-1.5 parts of cellulase, 0.3-0.5 parts of dissociative enzyme and 0.3-0.5 parts of hemicellulase.

[0027] Unless otherwise specified, the present invention does not have any special limitations on the source of each raw material in the following technical solutions, and any commercially available products in the field can be used.

[0028] As an optional embodiment of the present invention, the composite enzyme comprises 1.0 to 1.5 parts by weight of cellulase, which can be 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 parts. In the present invention, the cellulase can be cellulase RS. In the present invention, the cellulase acts on the cell wall and has the function of degrading cellulose in the cell wall of carrots.

[0029] As an optional embodiment of the present invention, based on the mass fraction of cellulase, the complex enzyme includes 0.3 to 0.5 parts of a dissociative enzyme, which can be 0.3, 0.4, or 0.5 parts. In the present invention, the dissociative enzyme can be dissociative enzyme R-10. In the present invention, the combined use of the dissociative enzyme and cellulase is beneficial for separating plant tissues into single cells, thereby facilitating the acquisition of protoplasts.

[0030] As an optional embodiment of the present invention, based on the mass fraction of cellulase, the composite enzyme includes 0.3 to 0.5 parts of hemicellulase, which can be 0.3, 0.4, or 0.5 parts. In the present invention, the main function of the hemicellulase is to degrade hemicellulose in plant cell walls. When used in combination with cellulase and dissociation enzyme, it can degrade the cell wall skeleton better and faster, which is beneficial to improving the protoplast yield.

[0031] The composite enzyme provided by this invention, through the combined action of three enzymes, can synergistically promote the lysis of cell walls in carrot plant tissues, thereby better and faster disrupting the cell wall skeleton. This is more conducive to the preparation of carrot protoplasts, maximizing the release of protoplasts and improving protoplast yield, while also maintaining high protoplast viability. The results of the embodiments of this invention show that using an enzymatic hydrolysate containing the aforementioned composite enzyme to prepare protoplasts from Nujiang carrots can significantly improve the protoplast yield and viability.

[0032] As an optional embodiment of the present invention, the composite enzyme comprises, by weight, 1.0 part of cellulase, 0.5 part of dissociative enzyme and 0.3 part of hemicellulase.

[0033] As an optional embodiment of the present invention, the composite enzyme comprises, by weight, 1.5 parts cellulase, 0.3 parts dissociation enzyme and 0.5 parts hemicellulase;

[0034] As an optional embodiment of the present invention, the composite enzyme comprises, by weight, 1.0 part of cellulase, 0.5 part of dissociative enzyme and 0.5 part of hemicellulase.

[0035] This invention provides an enzymatic hydrolysate for preparing carrot protoplasts, comprising, by total volume: 1.0–1.5 w / v% cellulase, 0.3–0.5 w / v% dissociative enzyme, 0.3–0.5 w / v% hemicellulase, 0.50–0.60 mol / L mannitol, 18–22 mmol / L MES, 18–22 mmol / L KCl, 8–12 mmol / L CaCl2, 0.08–0.12 w / v% BSA, and 0.03–0.07 mmol / L β-mercaptoethanol.

[0036] As an optional embodiment of the present invention, the enzymatic hydrolysate comprises 1.0~1.5 w / v% cellulase, based on the total volume of the enzymatic hydrolysate, or may be 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5 w / v.

[0037] As an optional embodiment of the present invention, the enzymatic hydrolysate comprises 0.3~0.5 w / v% of the isolating enzyme, which can be 0.3, 0.4 or 0.5 w / v, based on the total volume of the enzymatic hydrolysate.

[0038] As an optional embodiment of the present invention, the enzymatic hydrolysate includes 0.3~0.5 w / v% hemicellulase, which can be 0.3, 0.4 or 0.5 w / v, based on the total volume of the enzymatic hydrolysate.

[0039] As an optional embodiment of the present invention, the enzymatic hydrolysate contains 0.55~0.60 mol / L mannitol, which can be 0.55, 0.56, 0.57, 0.58, 0.59 or 0.60 mol / L; the enzymatic hydrolysate contains 19~21 mmol / L LME S, which can be 19, 20 or 21 mmol / L; the enzymatic hydrolysate contains 19~21 mmol / L KCl, which can be 19, 20 or 21 mmol / L; the enzymatic hydrolysate contains 9~11 mmol / L CaCl2, which can be 9, 10 or 11 mmol / L; the enzymatic hydrolysate contains 0.09~0.11 w / v% BSA, which can be 0.09, 0.10 or 0.11 w / v%; the enzymatic hydrolysate contains 0.04~0.06 mmol / L β-mercaptoethanol, which can be 0.04, 0.05 or 0.06 mmol / L.

[0040] The enzymatic hydrolysate provided by this invention contains three enzyme preparations: cellulase, dissociative enzyme, and hemicellulase. These three enzymes synergistically enhance the degradation of plant tissue cell walls during the preparation of carrot protoplasts, thereby maximizing the release of protoplasts and improving their activity. The mannitol, MES, KCl, CaCl2, BSA, and β-mercaptoethanol components in the hydrolysate stabilize osmotic pressure, maintain protoplast activity, and regulate pH, thus facilitating the preparation of carrot protoplasts and improving their yield and activity.

[0041] This invention provides the application of the enzymatic hydrolysate described in the above technical solution in any one or more of the following: preparing carrot protoplasts, improving protoplast activity, and improving protoplast yield.

[0042] This invention provides a method for preparing carrot protoplasts, comprising: enzymatically hydrolyzing carrot plant tissue using the enzymatic hydrolysate described in the above technical solution to obtain carrot protoplasts.

[0043] As an optional embodiment of the present invention, the carrot plant tissue includes carrot seedling plant tissue. The present invention does not specifically limit the cultivation method of the carrot seedlings; any conventional seedling cultivation method in the art can be used. As an optional embodiment of the present invention, the method for cultivating carrot seedlings includes culturing carrot seeds using plant tissue culture to obtain carrot seedlings. The present invention does not specifically limit the method of plant tissue culture; any conventional plant tissue culture method in the art can be used. In the present invention, the temperature for plant tissue culture can be 23~25℃, or 23, 24, or 25℃; the photoperiod for plant tissue culture is 15~17 h / d, or 16 h / d; the light intensity for plant tissue culture is 2500~3200 Lx, or 3000 Lx; and the cultivation time for plant tissue culture is 4~6 days, or 5 days.

[0044] After obtaining carrot seedlings, this invention allows for the preparation of protoplasts from different tissues of the seedlings using the enzymatic hydrolysate. As an optional embodiment of this invention, after obtaining the seedlings, it is preferable to treat the seedlings in the dark before preparing protoplasts from different tissues using the enzymatic hydrolysate. The dark treatment time can be 24-48 hours, or 24, 25, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 hours. In this invention, the different tissues can be any one or more of leaves, cotyledons, and petiolate cotyledons.

[0045] This invention utilizes the aforementioned enzymatic hydrolysate to enzymatically hydrolyze carrot plant tissues to obtain carrot protoplasts. As an optional embodiment of this invention, the cotyledons and / or petiolate cotyledons of carrots are enzymatically hydrolyzed using the aforementioned enzymatic hydrolysate to obtain carrot protoplasts. When performing enzymatic hydrolysis in this invention, the ratio of the number of cotyledons and / or stalked cotyledons to the volume of the enzymatic hydrolysate can be (20~30) cotyledons:15mL, or it can be 20 cotyledons:15mL, 21 cotyledons:15mL, 22 cotyledons:15mL, 23 cotyledons:15mL, 24 cotyledons:15mL, 25 cotyledons:15mL, 26 cotyledons:15mL, 27 cotyledons:15mL, 28 cotyledons:15mL, 29 cotyledons:15mL, or 30 cotyledons:15mL; the cotyledons and / or stalked cotyledons can be cut into thin filaments with a width of 0.5~1mm for enzymatic hydrolysis, or they can be cut into thin filaments with a width of 0.5, 0.6, 0.7, 0.8, 0.9, or 1mm for enzymatic hydrolysis.

[0046] As an optional embodiment of the present invention, the enzymatic hydrolysis temperature is 28~30℃, or 28, 29, or 30℃; the enzymatic hydrolysis time is 2.5~4h, or 2.5, 3, 3.5, or 4h; the enzymatic hydrolysis is accompanied by oscillation; the oscillation speed is 45~50rpm, or 45, 46, 47, 48, 49, or 50rpm; the enzymatic hydrolysis is carried out in the dark. The enzymatic hydrolysis conditions described in the present invention can ensure optimal enzymatic hydrolysis conditions, and at the same time, can maintain higher activity of protoplasts.

[0047] As an optional embodiment of the present invention, the enzymatic hydrolysis product can be gently blown with a cut sterile pipette tip to fully release the protoplasts during or after the enzymatic hydrolysis process.

[0048] After enzymatic hydrolysis, the present invention yields protoplasts of carrots. Alternatively, as an optional embodiment of the present invention, after obtaining the enzymatic hydrolysis product, the present invention preferably further includes washing and resuspending the enzymatic hydrolysis product to obtain protoplasts. The present invention does not have a particular limitation on the washing and resuspending method; any conventional protoplast washing and resuspending method in the art can be used. In the embodiments of the present invention, W5 solution and MMG solution are used to wash and resuspend the protoplasts.

[0049] The method for preparing carrot protoplasts described in this invention is simple and efficient, shortens the enzymatic hydrolysis time, and can obtain a large number of carrot protoplasts that maintain their viability for a long time.

[0050] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0051] Cellulase RS, Macerozyme R-10, and Pectolyase Y-23 were purchased from YAKULT PHARMACEUTICAL IND. CO., LTD.; MS medium was purchased from PhytoTech LABS; cellulase R-10 was purchased from YAKULT PHARMACEUTICAL IND. CO., LTD.; hemicellulase was purchased from Shanghai Yuanye Company, catalog number: S10045; MES, mannitol, KCl, CaCl2, BSA, β-mercaptoethanol, agar, sucrose, etc. were purchased from Sangon Biotech (Shanghai) Co., Ltd.; 0.45μm filter membranes were purchased from Sartorius (Sartorius Stedim Biotech GmbH, Germany); disposable sterile cell filters were purchased from Biosharp (Biosharp Life Sciences, Beijing Labgic Technology Co., Ltd.).

[0052] The carrots used in the following technical solutions are: carrots produced in the Nujiang area; a unique variety of Nujiang Prefecture, Yunnan Province, whose fleshy root skin and flesh are both red to purplish-red, with a crisp and sweet taste and tender and juicy flesh.

[0053] The experimental equipment used in the following protocol, such as forceps, scalpel blades, glass culture dishes, centrifuge tubes, pipette tips, etc., must be sterilized at 121°C for 25 minutes. The cell filter sieve is a disposable sterile cell filter sieve, and the filter membrane is a 0.45µm sterile microporous filter membrane. The laminar flow hood is wiped with 75% alcohol, irradiated with ultraviolet light for 30 minutes, and ventilated for 10 minutes.

[0054] Prepare W5 solution, which consists of: 2 mmol / L MES, 154 mmol / L NaCl solution, 125 mmol / L CaCl2 solution, 5 mmol / L KCl solution, 5 mmol / L glucose, and a pH of 5.7.

[0055] Prepare an MMG solution with the following composition: 0.4 mol / L mannitol, 15 mmol / L MgCl2 solution, 4 mmol / L MES, and a pH of 5.7.

[0056] The following method describes the preparation of carrot seedlings used for protoplast preparation in the following scheme:

[0057] Approximately 100 plump, intact carrot seeds were selected and placed in sterilized 50mL centrifuge tubes. Seed sterilization and inoculation were performed in a clean bench. The seeds were first sterilized with 75% ethanol for 30 seconds, then soaked in 0.2% HgCl2 solution for 6 minutes, followed by rinsing five times with 45mL sterile water for approximately 1 minute each time. The seeds were then gently blotted dry with sterile filter paper and cultured on 1 / 2 MS medium containing 1.5% sucrose and 0.7% agar (pH=5.8±0.05). Culture conditions were: temperature 24℃±1℃, photoperiod 16h light / 8h dark, and light intensity approximately 3000 Lx. Carrot seedlings were obtained after 4-6 days of culture. These seedlings were used for subsequent protoplast preparation.

[0058] Example 1

[0059] 1. A complex enzyme for preparing carrot protoplasts, comprising, by weight: 1.0 part of cellulase RS, 0.5 part of dissociative enzyme R-10 and 0.3 part of hemicellulase.

[0060] 2. An enzymatic hydrolysate containing one of the complex enzymes, comprising, by total volume of the hydrolysate: 1.0 w / v% cellulase RS, 0.5 w / v% analyte R-10, 0.3 w / v% hemicellulase, a MES acid-base buffer solution with a final mannitol concentration of 0.55 mol / L and a pH of 5.7, a final MES concentration of 20 mmol / L, a final KCl concentration of 20 mmol / L, a final CaCl2 concentration of 10 mmol / L, a final BSA concentration of 0.1%, and a final β-mercaptoethanol concentration of 0.05 mmol / L.

[0061] The preparation method of the enzymatic hydrolysate is as follows: Calculate the amount of each substance to be added based on the composition of the enzymatic hydrolysate. Then, mix cellulase RS, analyte R-10, hemicellulase, mannitol, MES acid-base buffer, and KCl, and incubate in a water bath at 55°C for 10 min. After the water bath, cool to room temperature, add CaCl2, BSA, and β-mercaptoethanol, and then add ultrapure water to bring the volume to 15 mL to obtain the enzymatic hydrolysate.

[0062] After preparing the enzymatic hydrolysate, filter the prepared enzymatic hydrolysate through a 0.45 μm filter membrane to remove bacteria, and then pour it into a 50 mL centrifuge tube for later use.

[0063] 3. The method for preparing carrot protoplasts using the two enzymatic hydrolysates is as follows:

[0064] (1) Carrot seedlings that have been cultured for 4-6 days were treated in the dark for 24 hours. Then, 30 cotyledons with petioles were taken, with the petiole length being about 3-5 mm. The cotyledons with petioles were cut into thin strips 1 mm wide. The strips (about 1 mm wide) were quickly placed into 50 mL centrifuge tubes containing 15 mL of enzymatic hydrolysate. The centrifuge tubes were wrapped with aluminum foil and placed on a flat plate shaker in the dark and shaken (28℃, 45 rpm) for 2.5 h.

[0065] (2) Place the 70µm cell filter sieve on a new 50mL sterile centrifuge tube and add about 1mL of W5 solution to rinse the cell filter sieve by rotation. Gently blow the enzymatic hydrolysate obtained in step (1) about 6-8 times with a cut sterile 1mL pipette tip to fully release the protoplasts. Then transfer the enzymatic hydrolysate in batches to the rinsed cell filter sieve for filtration. Gently press the undigested tissue in the cell filter sieve 3-5 times with the piston of a 5mL sterile syringe. Then slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinsing twice. Collect the filtrate, which is the filtered enzymatic hydrolysate.

[0066] (3) Centrifuge 100g of the enzyme hydrolysate obtained in step (2) for 7 min. After centrifugation, discard the supernatant. Tilt the centrifuge tube and slowly add 3 mL of W5 solution along the tube wall to resuspend the precipitate. Filter the resuspended liquid through a 40µm cell filter sieve. Wash the residual tissue on the cell sieve with 1 mL of W5 solution. Repeat this process once more (i.e., wash the residual tissue on the cell sieve with 1 mL of W5 solution, the same below), and collect the filtrate. Centrifuge 100g of the filtrate for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain carrot protoplasts.

[0067] (4) Protoplast viability detection using FDA (fluorescein diacetate). Take 100 μL of protoplast suspension and place it in a 1.5 mL centrifuge tube pre-wrapped in aluminum foil. Add 3 μL of 1 mg / mL FDA and stain in the dark for 5 min. After staining, aspirate 50 μL onto a glass slide and observe in the dark using an inverted fluorescence microscope (Thermo Fisher EVOS M3000). Viable protoplasts will emit yellow-green fluorescence; count the number of yellow-green protoplasts. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field of view is the protoplast viability value. The prepared protoplast viability value was 86.11%. Figure 1 As shown, Figure 1 The right image shows the FDA staining results, and the left image shows the corresponding bright-field protoplast image. The scale bar in the image is 100µm.

[0068] Protoplast viability: Protoplast viability = (Number of protoplasts emitting yellow-green fluorescence / Total number of protoplasts) × 100%.

[0069] To determine the protoplast concentration: A drop of the prepared protoplasts was placed on one end of a coverslip in a hemocytometer. The droplet was allowed to fill the entire coverslip until no air bubbles appeared. After standing for two minutes, the protoplasts were counted. In this example, the density of the prepared protoplasts was approximately 39.78 × 10⁻⁶. 5 per mL.

[0070] Protoplast density (units / mL) = Number of protoplasts in 25 square cells × 10 4 × Dilution factor.

[0071] Example 2

[0072] 1. A complex enzyme for preparing carrot protoplasts, comprising, by weight: 1.5 parts of cellulase RS, 0.3 parts of dissociative enzyme R-10, and 0.5 parts of hemicellulase.

[0073] 2. An enzymatic hydrolysate containing one of the complex enzymes, comprising, by total volume, 1.5 w / v% cellulase RS, 0.3 w / v% analyte R-10, 0.5 w / v% hemicellulase, a MES acid-base buffer solution with a final mannitol concentration of 0.55 mol / L and a pH of 5.7, a final MES concentration of 20 mmol / L, a final KCl concentration of 20 mmol / L, a final CaCl2 concentration of 10 mmol / L, a final BSA concentration of 0.1%, and a final β-mercaptoethanol concentration of 0.05 mmol / L.

[0074] The preparation method of the enzymatic hydrolysate is the same as in Example 1.

[0075] 3. The method for preparing carrot protoplasts using the two enzymatic hydrolysates is as follows:

[0076] (1) Carrot seedlings that have been cultured for 4-6 days were treated in the dark for 24 hours. Then, 30 cotyledons with petioles were taken, with the petiole length being about 3-5 mm. The cotyledons with petioles were cut into thin strips 1 mm wide. The strips (about 1 mm wide) were quickly placed into 50 mL centrifuge tubes containing 15 mL of enzymatic hydrolysate. The centrifuge tubes were wrapped with aluminum foil and placed on a flat plate shaker in the dark and shaken (28℃, 45 rpm) for 3 hours.

[0077] (2) Place the 70µm cell filter sieve on a new 50mL sterile centrifuge tube and add about 1mL of W5 solution to rinse the cell filter sieve by rotation. Gently blow the enzymatic hydrolysate obtained in step (1) about 6-8 times with a cut sterile 1mL pipette tip to fully release the protoplasts. Then transfer the enzymatic hydrolysate in batches to the rinsed cell filter sieve for filtration. Gently press the undigested tissue in the cell filter sieve 3-5 times with the piston of a 5mL sterile syringe. Then slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinsing twice. Collect the filtrate, which is the filtered enzymatic hydrolysate.

[0078] (3) Centrifuge 100g of the enzyme hydrolysate obtained in step (2) for 7 min. After centrifugation, discard the supernatant. Tilt the centrifuge tube and slowly add 3 mL of W5 solution along the tube wall to resuspend the precipitate. Filter the resuspended liquid through a 40µm cell filter sieve. Wash the residual tissue on the cell sieve with 1 mL of W5 solution. Repeat this process once more (i.e., wash the residual tissue on the cell sieve with 1 mL of W5 solution) and collect the filtrate. Centrifuge 100g of the filtrate for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain carrot protoplasts.

[0079] (4) Protoplast viability detection using FDA (fluorescein diacetate). Take 100 μL of protoplast suspension and place it in a 1.5 mL centrifuge tube pre-wrapped in aluminum foil. Add 3 μL of 1 mg / mL FDA and stain in the dark for 5 min. After staining, aspirate 50 μL onto a glass slide and observe in the dark using an inverted fluorescence microscope (Thermo Fisher EVOS M3000). Viable protoplasts will emit yellow-green fluorescence; count the number of yellow-green protoplasts. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field of view is the protoplast viability value. The prepared protoplast viability value was 84.77%. Figure 2 As shown, Figure 2 The right image shows the FDA staining results, and the left image shows the corresponding bright-field protoplast image. The scale bar in the image is 100µm.

[0080] Protoplast viability: Protoplast viability = (Number of protoplasts emitting yellow-green fluorescence / Total number of protoplasts) × 100%.

[0081] To determine the protoplast concentration: A drop of the prepared protoplasts was placed on one end of a coverslip in a hemocytometer. The droplet was allowed to fill the entire coverslip until no air bubbles appeared. After standing for two minutes, the protoplasts were counted. In this example, the density of the prepared protoplasts was approximately 38.18 × 10⁻⁶. 5 per mL.

[0082] Protoplast density (units / mL) = Number of protoplasts in 25 square cells × 10 4 × Dilution factor.

[0083] Example 3

[0084] 1. A complex enzyme for preparing carrot protoplasts, comprising, by weight, 1.0 part of cellulase RS, 0.5 part of dissociative enzyme R-10 and 0.5 part of hemicellulase.

[0085] 2. An enzymatic hydrolysate containing one of the complex enzymes, comprising, by total volume of the hydrolysate: 1.0 w / v% cellulase RS, 0.5 w / v% analyte R-10, 0.5 w / v% hemicellulase, a MES acid-base buffer solution with a final mannitol concentration of 0.55 mol / L and a pH of 5.7, a final MES concentration of 20 mmol / L, a final KCl concentration of 20 mmol / L, a final CaCl2 concentration of 10 mmol / L, a final BSA concentration of 0.1%, and a final β-mercaptoethanol concentration of 0.05 mmol / L.

[0086] The preparation method of the enzymatic hydrolysate is the same as in Example 1.

[0087] 3. The method for preparing carrot protoplasts using the two enzymatic hydrolysates is as follows:

[0088] (1) Carrot seedlings that have been cultured for 4-6 days were treated in the dark for 24 hours. Then, 30 cotyledons with petioles were taken, with the petiole length being about 3-5 mm. The cotyledons with petioles were cut into thin strips 1 mm wide. The strips (about 1 mm wide) were quickly placed into 50 mL centrifuge tubes containing 15 mL of enzymatic hydrolysate. The centrifuge tubes were wrapped with aluminum foil and placed on a flat plate shaker in the dark and shaken (28℃, 45 rpm) for 4 hours.

[0089] (2) Place the 70µm cell filter sieve on a new 50mL sterile centrifuge tube and add about 1mL of W5 solution to rinse the cell filter sieve by rotation. Gently blow the enzymatic hydrolysate obtained in step (1) about 6-8 times with a cut sterile 1mL pipette tip to fully release the protoplasts. Then transfer the enzymatic hydrolysate in batches to the rinsed cell filter sieve for filtration. Gently press the undigested tissue in the cell filter sieve 3-5 times with the piston of a 5mL sterile syringe. Then slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinsing twice. Collect the filtrate, which is the filtered enzymatic hydrolysate.

[0090] (3) Centrifuge 100g of the enzyme hydrolysate obtained in step (2) for 7 min. After centrifugation, discard the supernatant. Tilt the centrifuge tube and slowly add 3 mL of W5 solution along the tube wall to resuspend the precipitate. Filter the resuspended liquid through a 40µm cell filter sieve. Wash the residual tissue on the cell sieve with 1 mL of W5 solution. Repeat this process once more (i.e., wash the residual tissue on the cell sieve with 1 mL of W5 solution) and collect the filtrate. Centrifuge 100g of the filtrate for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain carrot protoplasts.

[0091] (4) Protoplast viability was detected using FDA (fluorescein diacetate). 100 μL of protoplast suspension was placed in a 1.5 mL centrifuge tube pre-wrapped in aluminum foil, and 3 μL of 1 mg / mL FDA was added. The tube was stained in the dark for 5 min. After staining, 50 μL was transferred to a glass slide and observed in the dark using an inverted fluorescence microscope (Thermo Fisher EVOS M3000). Viable protoplasts emitted a yellow-green fluorescence; the number of yellow-green protoplasts was counted. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field of view is the protoplast viability value. The prepared protoplast viability value was 85.78%.

[0092] Protoplast viability: Protoplast viability = (Number of protoplasts emitting yellow-green fluorescence / Total number of protoplasts) × 100%.

[0093] To determine the protoplast concentration: A drop of the prepared protoplasts was placed on one end of a coverslip in a hemocytometer. The droplet was allowed to fill the entire coverslip until no air bubbles appeared. After standing for two minutes, the protoplasts were counted. In this example, the protoplast density was approximately 38.02 × 10⁻⁶. 5 per mL.

[0094] Protoplast density (units / mL) = Number of protoplasts in 25 square cells × 10 4 × Dilution factor.

[0095] Comparative Example 1

[0096] 1. A compound enzyme, by weight, comprising: 2.0 parts of cellulase R-10 and 0.2 parts of pectinase Y-23.

[0097] 2. An enzymatic hydrolysate containing one of the complex enzymes, comprising, by total volume, 2.0 w / v% cellulase R-10, 0.2 w / v% pectinase Y-23, a MES acid-base buffer solution with a final concentration of 0.55 mol / L mannitol and a pH of 5.7, a final concentration of 20 mmol / L MES, a final concentration of 20 mmol / L KCl, a final concentration of 10 mmol / L CaCl2, a final concentration of 0.1% BSA, and a final concentration of 0.05 mmol / L β-mercaptoethanol.

[0098] The preparation method of the enzymatic hydrolysate is as follows: Calculate the amount of each substance to be added based on the composition of the enzymatic hydrolysate. Then, mix cellulase R-10, pectinase Y-23, mannitol, MES acid-base buffer, and KCl, and incubate at 55°C for 10 minutes. After the water bath, cool to room temperature, add CaCl2, BSA, and β-mercaptoethanol, and then add ultrapure water to a final volume of 15 mL to obtain the enzymatic hydrolysate.

[0099] After preparing the enzymatic hydrolysate, filter the prepared enzymatic hydrolysate through a 0.45 μm filter membrane to remove bacteria, and then pour it into a 50 mL centrifuge tube for later use.

[0100] 3. The method for preparing carrot protoplasts using the two enzymatic hydrolysates is as follows:

[0101] (1) Carrot seedlings that have been cultured for 4-6 days were treated in the dark for 24 hours. Then, 30 cotyledons with petioles were taken, with the petiole length being about 3-5 mm. The cotyledons with petioles were cut into thin strips 1 mm wide. The strips (about 1 mm wide) were quickly placed into 50 mL centrifuge tubes containing 15 mL of enzymatic hydrolysate. The centrifuge tubes were wrapped with aluminum foil and placed on a flat plate shaker in the dark and shaken (28℃, 45 rpm) for 4 hours.

[0102] (2) Place the 70µm cell filter sieve on a new 50mL sterile centrifuge tube and add about 1mL of W5 solution to rinse the cell filter sieve by rotation. Gently blow the enzymatic hydrolysate obtained in step (1) about 6-8 times with a cut sterile 1mL pipette tip to fully release the protoplasts. Then transfer the enzymatic hydrolysate in batches to the rinsed cell filter sieve for filtration. Gently press the undigested tissue in the cell filter sieve 3-5 times with the piston of a 5mL sterile syringe. Then slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinsing twice. Collect the filtrate, which is the filtered enzymatic hydrolysate.

[0103] (3) Centrifuge 100g of the enzyme hydrolysate obtained in step (2) for 7 min. After centrifugation, discard the supernatant. Tilt the centrifuge tube and slowly add 3 mL of W5 solution along the tube wall to resuspend the precipitate. Filter the resuspended liquid through a 40µm cell filter sieve. Wash the residual tissue on the cell sieve with 1 mL of W5 solution. Repeat this process once more (i.e., wash the residual tissue on the cell sieve with 1 mL of W5 solution, the same below), and collect the filtrate. Centrifuge 100g of the filtrate for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain carrot protoplasts.

[0104] (4) Protoplast viability detection using FDA (fluorescein diacetate). Take 100 μL of protoplast suspension and place it in a 1.5 mL centrifuge tube pre-wrapped in aluminum foil. Add 3 μL of 1 mg / mL FDA and stain in the dark for 5 min. After staining, aspirate 50 μL onto a glass slide and observe in the dark using an inverted fluorescence microscope (Thermo Fisher EVOS M3000). Viable protoplasts will emit yellow-green fluorescence; count the number of yellow-green protoplasts. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field of view is the protoplast viability value. The prepared protoplast viability value was 70.58%. Figure 3 As shown, Figure 3The right image shows the FDA staining results, and the left image shows the corresponding bright-field protoplast image. The scale bar in the image is 100µm.

[0105] Protoplast viability: Protoplast viability = (Number of protoplasts emitting yellow-green fluorescence / Total number of protoplasts) × 100%.

[0106] To determine the protoplast concentration: A drop of the prepared protoplasts was placed on one end of a coverslip in a hemocytometer. The droplet was allowed to fill the entire coverslip until no air bubbles appeared. After standing for two minutes, the protoplasts were counted. The density of the protoplasts prepared in this comparative example was approximately 1.44 × 10⁻⁶. 5 per mL.

[0107] Protoplast density (units / mL) = Number of protoplasts in 25 square cells × 10 4 × Dilution factor.

[0108] Comparative Example 2

[0109] 1. A complex enzyme, by mass parts, comprising: 1.5 parts of cellulase RS and 0.5 parts of dissociative enzyme R-10.

[0110] 2. An enzymatic hydrolysate containing one of the complex enzymes, comprising, by total volume of the hydrolysate: 1.5 w / v% cellulase RS, 0.5 w / v% analyte R-10, a MES acid-base buffer solution with a final concentration of 0.55 mol / L mannitol and a pH of 5.7, a final concentration of 20 mmol / L MES, a final concentration of 20 mmol / L KCl, a final concentration of 10 mmol / L CaCl2, a final concentration of 0.1% BSA, and a final concentration of 0.05 mmol / L β-mercaptoethanol.

[0111] The preparation method of the enzymatic hydrolysate is as follows: Calculate the amount of each substance to be added based on the composition of the enzymatic hydrolysate. Then, mix cellulase RS, analyte R-10, mannitol, MES acid-base buffer, and KCl, and incubate in a water bath at 55°C for 10 min. After the water bath, cool to room temperature, add CaCl2, BSA, and β-mercaptoethanol, and then add ultrapure water to bring the volume to 15 mL to obtain the enzymatic hydrolysate.

[0112] After preparing the enzymatic hydrolysate, filter the prepared enzymatic hydrolysate through a 0.45 μm filter membrane to remove bacteria, and then pour it into a 50 mL centrifuge tube for later use.

[0113] 3. The method for preparing carrot protoplasts using the two enzymatic hydrolysates is as follows:

[0114] (1) Carrot seedlings that have been cultured for 4-6 days were treated in the dark for 24 hours. Then, 30 cotyledons with petioles were taken, with the petiole length being about 3-5 mm. The cotyledons with petioles were cut into thin strips 1 mm wide. The strips (about 1 mm wide) were quickly placed into 50 mL centrifuge tubes containing 15 mL of enzymatic hydrolysate. The centrifuge tubes were wrapped with aluminum foil and placed on a flat plate shaker in the dark and shaken (28℃, 45 rpm) for 4 hours.

[0115] (2) Place the 70µm cell filter sieve on a new 50mL sterile centrifuge tube and add about 1mL of W5 solution to rinse the cell filter sieve by rotation. Gently blow the enzymatic hydrolysate obtained in step (1) about 6-8 times with a cut sterile 1mL pipette tip to fully release the protoplasts. Then transfer the enzymatic hydrolysate in batches to the rinsed cell filter sieve for filtration. Gently press the undigested tissue in the cell filter sieve 3-5 times with the piston of a 5mL sterile syringe. Then slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinsing twice. Collect the filtrate, which is the filtered enzymatic hydrolysate.

[0116] (3) Centrifuge 100g of the enzyme hydrolysate obtained in step (2) for 7 min. After centrifugation, discard the supernatant. Tilt the centrifuge tube and slowly add 3 mL of W5 solution along the tube wall to resuspend the precipitate. Filter the resuspended liquid through a 40µm cell filter sieve. Wash the residual tissue on the cell sieve with 1 mL of W5 solution. Repeat this process once more (i.e., wash the residual tissue on the cell sieve with 1 mL of W5 solution, the same below), and collect the filtrate. Centrifuge 100g of the filtrate for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain carrot protoplasts.

[0117] (4) Protoplast viability detection using FDA (fluorescein diacetate). Take 100 μL of protoplast suspension and place it in a 1.5 mL centrifuge tube pre-wrapped in aluminum foil. Add 3 μL of 1 mg / mL FDA and stain in the dark for 5 min. After staining, aspirate 50 μL onto a glass slide and observe in the dark using an inverted fluorescence microscope (Thermo Fisher EVOS M3000). Viable protoplasts will emit yellow-green fluorescence; count the number of yellow-green protoplasts. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field of view is the protoplast viability value. The prepared protoplast viability value was 63.08%. Figure 4 As shown, Figure 4 The right image shows the FDA staining results, and the left image shows the corresponding bright-field protoplast image. The scale bar in the image is 100µm.

[0118] Protoplast viability: Protoplast viability = (Number of protoplasts emitting yellow-green fluorescence / Total number of protoplasts) × 100%.

[0119] To determine the protoplast concentration: A drop of the prepared protoplasts was placed on one end of a coverslip in a hemocytometer. The droplet was allowed to fill the entire coverslip until no air bubbles appeared. After standing for two minutes, the protoplasts were counted. The density of the protoplasts prepared in this comparative example was approximately 3.08 × 10⁻⁶. 5 per mL.

[0120] Protoplast density (units / mL) = Number of protoplasts in 25 square cells × 10 4 × Dilution factor.

[0121] Comparative Example 3

[0122] 1. A complex enzyme, by mass parts, comprising: 0.5 parts of analyte R-10 and 0.5 parts of hemicellulase.

[0123] 2. An enzymatic hydrolysate containing one of the complex enzymes, comprising, by total volume of the hydrolysate: 0.5 w / v% analyte R-10, 0.5 w / v% hemicellulase, a MES acid-base buffer solution with a final concentration of 0.55 mol / L mannitol and a pH of 5.7, a final concentration of 20 mmol / L MES, a final concentration of 20 mmol / L KCl, a final concentration of 10 mmol / L CaCl2, a final concentration of 0.1% BSA, and a final concentration of 0.05 mmol / L β-mercaptoethanol.

[0124] The preparation method of the enzymatic hydrolysate is as follows: Calculate the amount of each substance to be added based on the composition of the enzymatic hydrolysate. Then, mix the ionizing enzyme R-10, hemicellulase, mannitol, MES acid-base buffer, and KCl, and incubate in a water bath at 55°C for 10 min. After the water bath, cool to room temperature, add CaCl2, BSA, and β-mercaptoethanol, and then add ultrapure water to a final volume of 15 mL to obtain the enzymatic hydrolysate.

[0125] After preparing the enzymatic hydrolysate, filter the prepared enzymatic hydrolysate through a 0.45 μm filter membrane to remove bacteria, and then pour it into a 50 mL centrifuge tube for later use.

[0126] 3. The method for preparing carrot protoplasts using the two enzymatic hydrolysates is as follows:

[0127] (1) Carrot seedlings that have been cultured for 4-6 days were treated in the dark for 24 hours. Then, 30 cotyledons with petioles were taken, with the petiole length being about 3-5 mm. The cotyledons with petioles were cut into thin strips 1 mm wide. The strips (about 1 mm wide) were quickly placed into 50 mL centrifuge tubes containing 15 mL of enzymatic hydrolysate. The centrifuge tubes were wrapped with aluminum foil and placed on a flat plate shaker in the dark and shaken (28℃, 45 rpm) for 4 hours.

[0128] (2) Place the 70µm cell filter sieve on a new 50mL sterile centrifuge tube and add about 1mL of W5 solution to rinse the cell filter sieve by rotation. Gently blow the enzymatic hydrolysate obtained in step (1) about 6-8 times with a cut sterile 1mL pipette tip to fully release the protoplasts. Then transfer the enzymatic hydrolysate in batches to the rinsed cell filter sieve for filtration. Gently press the undigested tissue in the cell filter sieve 3-5 times with the piston of a 5mL sterile syringe. Then slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinsing twice. Collect the filtrate, which is the filtered enzymatic hydrolysate.

[0129] (3) Centrifuge 100g of the enzyme hydrolysate obtained in step (2) for 7 min. After centrifugation, discard the supernatant. Tilt the centrifuge tube and slowly add 3 mL of W5 solution along the tube wall to resuspend the precipitate. Filter the resuspended liquid through a 40µm cell filter sieve. Wash the residual tissue on the cell sieve with 1 mL of W5 solution. Repeat this process once more (i.e., wash the residual tissue on the cell sieve with 1 mL of W5 solution, the same below), and collect the filtrate. Centrifuge 100g of the filtrate for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain carrot protoplasts.

[0130] (4) Protoplast viability was detected using FDA (fluorescein diacetate). 100 μL of protoplast suspension was placed in a 1.5 mL centrifuge tube pre-wrapped in aluminum foil, and 3 μL of 1 mg / mL FDA was added. The tube was stained in the dark for 5 min. After staining, 50 μL was transferred to a glass slide and observed in the dark using an inverted fluorescence microscope (Thermo Fisher EVOS M3000). Viable protoplasts emitted a yellow-green fluorescence; the number of yellow-green protoplasts was counted. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field of view is the protoplast viability value. The prepared protoplast viability value was 60.50%.

[0131] Protoplast viability: Protoplast viability = (Number of protoplasts emitting yellow-green fluorescence / Total number of protoplasts) × 100%.

[0132] To determine the protoplast concentration: A drop of the prepared protoplasts was placed on one end of a coverslip in a hemocytometer. The droplet was allowed to fill the entire coverslip until no air bubbles appeared. After standing for two minutes, the protoplasts were counted. The density of the protoplasts prepared in this comparative example was approximately 0.48 × 10⁻⁶. 5 per mL.

[0133] Protoplast density (units / mL) = Number of protoplasts in 25 square cells × 10 4 × Dilution factor.

[0134] Comparative Example 4

[0135] 1. A complex enzyme, by mass parts, comprising: 1.5 parts of cellulase RS and 0.5 parts of hemicellulase.

[0136] 2. An enzymatic hydrolysate containing one of the complex enzymes, comprising, by total volume of the hydrolysate: 1.5 w / v% cellulase RS, 0.5 w / v% hemicellulase, a MES acid-base buffer solution with a final concentration of 0.55 mol / L mannitol and a pH of 5.7, a final concentration of 20 mmol / L MES, a final concentration of 20 mmol / L KCl, a final concentration of 10 mmol / L CaCl2, a final concentration of 0.1% BSA, and a final concentration of 0.05 mmol / L β-mercaptoethanol.

[0137] The preparation method of the enzymatic hydrolysate is as follows: Calculate the amount of each substance to be added based on the composition of the enzymatic hydrolysate. Then, mix the ionizing enzyme R-10, hemicellulase, mannitol, MES acid-base buffer, and KCl, and incubate in a water bath at 55°C for 10 min. After the water bath, cool to room temperature, add CaCl2, BSA, and β-mercaptoethanol, and then add ultrapure water to a final volume of 15 mL to obtain the enzymatic hydrolysate.

[0138] After preparing the enzymatic hydrolysate, filter the prepared enzymatic hydrolysate through a 0.45 μm filter membrane to remove bacteria, and then pour it into a 50 mL centrifuge tube for later use.

[0139] 3. The method for preparing carrot protoplasts using the two enzymatic hydrolysates is as follows:

[0140] (1) Carrot seedlings that have been cultured for 4-6 days were treated in the dark for 24 hours. Then, 30 cotyledons with petioles were taken, with the petiole length being about 3-5 mm. The cotyledons with petioles were cut into thin strips 1 mm wide. The strips (about 1 mm wide) were quickly placed into 50 mL centrifuge tubes containing 15 mL of enzymatic hydrolysate. The centrifuge tubes were wrapped with aluminum foil and placed on a flat plate shaker in the dark and shaken (28℃, 45 rpm) for 4 hours.

[0141] (2) Place the 70µm cell filter sieve on a new 50mL sterile centrifuge tube and add about 1mL of W5 solution to rinse the cell filter sieve by rotation. Gently blow the enzymatic hydrolysate obtained in step (1) about 6-8 times with a cut sterile 1mL pipette tip to fully release the protoplasts. Then transfer the enzymatic hydrolysate in batches to the rinsed cell filter sieve for filtration. Gently press the undigested tissue in the cell filter sieve 3-5 times with the piston of a 5mL sterile syringe. Then slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinsing twice. Collect the filtrate, which is the filtered enzymatic hydrolysate.

[0142] (3) Centrifuge 100g of the enzyme hydrolysate obtained in step (2) for 7 min. After centrifugation, discard the supernatant. Tilt the centrifuge tube and slowly add 3 mL of W5 solution along the tube wall to resuspend the precipitate. Filter the resuspended liquid through a 40µm cell filter sieve. Wash the residual tissue on the cell sieve with 1 mL of W5 solution. Repeat this process once more (i.e., wash the residual tissue on the cell sieve with 1 mL of W5 solution, the same below), and collect the filtrate. Centrifuge 100g of the filtrate for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain carrot protoplasts.

[0143] (4) Protoplast viability was detected using FDA (fluorescein diacetate). 100 μL of protoplast suspension was placed in a 1.5 mL centrifuge tube pre-wrapped in aluminum foil, and 3 μL of 1 mg / mL FDA was added. The tube was stained in the dark for 5 min. After staining, 50 μL was transferred to a glass slide and observed in the dark using an inverted fluorescence microscope (Thermo Fisher EVOS M3000). Viable protoplasts emitted a yellow-green fluorescence; the number of yellow-green protoplasts was counted. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field of view is the protoplast viability value. The prepared protoplast viability value was 66.05%.

[0144] Protoplast viability: Protoplast viability = (Number of protoplasts emitting yellow-green fluorescence / Total number of protoplasts) × 100%.

[0145] To determine the protoplast concentration: A drop of the prepared protoplasts was placed on one end of a coverslip in a hemocytometer. The droplet was allowed to fill the entire coverslip until no air bubbles appeared. After standing for two minutes, the protoplasts were counted. The density of the protoplasts prepared in this comparative example was approximately 0.64 × 10⁻⁶. 5 per mL.

[0146] Protoplast density (units / mL) = Number of protoplasts in 25 square cells × 10 4 × Dilution factor.

[0147] Comparative Example 5

[0148] 1. A compound enzyme, by weight, comprises: 2.0 parts of cellulase RS, 1.0 parts of dissociative enzyme R-10, and 0.1 parts of pectinase Y-23.

[0149] 2. An enzymatic hydrolysate containing one of the complex enzymes, comprising, by total volume, 2.0 w / v% cellulase RS, 1.0 w / v% analyte R-10, 0.1 w / v% pectinase Y-23, a MES acid-base buffer solution with a final mannitol concentration of 0.55 mol / L and a pH of 5.7, wherein the final concentrations of MES, KCl, CaCl2, BSA, and β-mercaptoethanol are 20 mmol / L, 20 mmol / L, 10 mmol / L, 0.1%, and 0.05 mmol / L, respectively.

[0150] The preparation method of the enzymatic hydrolysate is as follows: Based on the composition of the hydrolysate, calculate the amount of each substance to be added. Then, mix cellulase RS, analyte R-10, pectinase Y-23, mannitol, MES acid-base buffer, and KCl, and incubate at 55°C for 10 minutes. After the water bath, cool to room temperature, add CaCl2, BSA, and β-mercaptoethanol, and then add ultrapure water to a final volume of 15 mL to obtain the enzymatic hydrolysate.

[0151] After preparing the enzymatic hydrolysate, filter the prepared enzymatic hydrolysate through a 0.45 μm filter membrane to remove bacteria, and then pour it into a 50 mL centrifuge tube for later use.

[0152] 3. The method for preparing carrot protoplasts using the two enzymatic hydrolysates is as follows:

[0153] (1) Carrot seedlings that have been cultured for 4-6 days were treated in the dark for 24 hours. Then, 30 cotyledons with petioles were taken, with the petiole length being about 3-5 mm. The cotyledons with petioles were cut into thin strips 1 mm wide. The strips (about 1 mm wide) were quickly placed into 50 mL centrifuge tubes containing 15 mL of enzymatic hydrolysate. The centrifuge tubes were wrapped with aluminum foil and placed on a flat plate shaker in the dark and shaken (28℃, 45 rpm) for 4 hours.

[0154] (2) Place the 70µm cell filter sieve on a new 50mL sterile centrifuge tube and add about 1mL of W5 solution to rinse the cell filter sieve by rotation. Gently blow the enzymatic hydrolysate obtained in step (1) about 6-8 times with a cut sterile 1mL pipette tip to fully release the protoplasts. Then transfer the enzymatic hydrolysate in batches to the rinsed cell filter sieve for filtration. Gently press the undigested tissue in the cell filter sieve 3-5 times with the piston of a 5mL sterile syringe. Then slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinsing twice. Collect the filtrate, which is the filtered enzymatic hydrolysate.

[0155] (3) Centrifuge 100g of the enzyme hydrolysate obtained in step (2) for 7 min. After centrifugation, discard the supernatant. Tilt the centrifuge tube and slowly add 3 mL of W5 solution along the tube wall to resuspend the precipitate. Filter the resuspended liquid through a 40µm cell filter sieve. Wash the residual tissue on the cell sieve with 1 mL of W5 solution. Repeat once more (i.e., wash the residual tissue on the cell sieve with 1 mL of W5 solution and collect the filtrate). Then centrifuge 100g of the filtrate for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain carrot protoplasts.

[0156] (4) Protoplast viability was detected using FDA (fluorescein diacetate). 100 μL of protoplast suspension was placed in a 1.5 mL centrifuge tube pre-wrapped in aluminum foil, and 3 μL of 1 mg / mL FDA was added. The tube was stained in the dark for 5 min. After staining, 50 μL was transferred to a glass slide and observed in the dark using an inverted fluorescence microscope (Thermo Fisher EVOS M3000). Viable protoplasts emitted a yellow-green fluorescence; the number of yellow-green protoplasts was counted. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field of view is the protoplast viability value. The prepared protoplast viability value was 80.95%.

[0157] Protoplast viability: Protoplast viability = (Number of protoplasts emitting yellow-green fluorescence / Total number of protoplasts) × 100%.

[0158] To determine the protoplast concentration: A drop of the prepared protoplasts was placed on one end of a coverslip in a hemocytometer. The droplet was allowed to fill the entire coverslip until no air bubbles appeared. After standing for two minutes, the protoplasts were counted. The density of the protoplasts prepared in this comparative example was approximately 13.08 × 10⁻⁶. 5 per mL.

[0159] Protoplast density (units / mL) = Number of protoplasts in 25 square cells × 10 4 × Dilution factor.

[0160] Comparative Example 6

[0161] 1. A complex enzyme, by mass parts, comprising: 1.0 part of cellulase R-10, 0.5 part of dissociative enzyme R-10, and 0.5 part of hemicellulase.

[0162] 2. An enzymatic hydrolysate containing one of the complex enzymes, comprising, by total volume of the hydrolysate: 1.0 w / v% cellulase R-10, 0.5 w / v% analyte R-10, 0.5 w / v% hemicellulase, a MES acid-base buffer solution with a final mannitol concentration of 0.55 mol / L and a pH of 5.7, a final MES concentration of 20 mmol / L, a final KCl concentration of 20 mmol / L, a final CaCl2 concentration of 10 mmol / L, a final BSA concentration of 0.1%, and a final β-mercaptoethanol concentration of 0.05 mmol / L.

[0163] The preparation method of the enzymatic hydrolysate is as follows: Calculate the amount of each substance to be added based on the composition of the enzymatic hydrolysate. Then, mix the analyte R-10, hemicellulase, mannitol, MES acid-base buffer, and KCl, and incubate at 55°C for 10 minutes. After the water bath, cool to room temperature, add CaCl2, BSA, and β-mercaptoethanol, and then add ultrapure water to a final volume of 15 mL to obtain the enzymatic hydrolysate.

[0164] After preparing the enzymatic hydrolysate, filter the prepared enzymatic hydrolysate through a 0.45 μm filter membrane to remove bacteria, and then pour it into a 50 mL centrifuge tube for later use.

[0165] 3. The method for preparing carrot protoplasts using the two enzymatic hydrolysates is as follows:

[0166] (1) Carrot seedlings that have been cultured for 4-6 days were treated in the dark for 24 hours. Then, 30 cotyledons with petioles were taken, with the petiole length being about 3-5 mm. The cotyledons with petioles were cut into thin strips 1 mm wide. The strips (about 1 mm wide) were quickly placed into 50 mL centrifuge tubes containing 15 mL of enzymatic hydrolysate. The centrifuge tubes were wrapped with aluminum foil and placed on a flat plate shaker in the dark and shaken (28℃, 45 rpm) for 4 hours.

[0167] (2) Place the 70µm cell filter sieve on a new 50mL sterile centrifuge tube and add about 1mL of W5 solution to rinse the cell filter sieve by rotation. Gently blow the enzymatic hydrolysate obtained in step (1) about 6-8 times with a cut sterile 1mL pipette tip to fully release the protoplasts. Then transfer the enzymatic hydrolysate in batches to the rinsed cell filter sieve for filtration. Gently press the undigested tissue in the cell filter sieve 3-5 times with the piston of a 5mL sterile syringe. Then slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinsing twice. Collect the filtrate, which is the filtered enzymatic hydrolysate.

[0168] (3) Centrifuge 100g of the enzyme hydrolysate obtained in step (2) for 7 min. After centrifugation, discard the supernatant. Tilt the centrifuge tube and slowly add 3 mL of W5 solution along the tube wall to resuspend the precipitate. Filter the resuspended liquid through a 40µm cell filter sieve. Wash the residual tissue on the cell sieve with 1 mL of W5 solution. Repeat this process once more (i.e., wash the residual tissue on the cell sieve with 1 mL of W5 solution) and collect the filtrate. Centrifuge 100g of the filtrate for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain carrot protoplasts.

[0169] (4) Protoplast viability was detected using FDA (fluorescein diacetate). 100 μL of protoplast suspension was placed in a 1.5 mL centrifuge tube pre-wrapped in aluminum foil, and 3 μL of 1 mg / mL FDA was added. The tube was stained in the dark for 5 min. After staining, 50 μL was transferred to a glass slide and observed in the dark using an inverted fluorescence microscope (Thermo Fisher EVOS M3000). Viable protoplasts emitted a yellow-green fluorescence; the number of yellow-green protoplasts was counted. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field of view is the protoplast viability value. The prepared protoplast viability value was 82.32%.

[0170] Protoplast viability: Protoplast viability = (Number of protoplasts emitting yellow-green fluorescence / Total number of protoplasts) × 100%.

[0171] To determine the protoplast concentration: A drop of the prepared protoplasts was placed on one end of a coverslip in a hemocytometer. The droplet was allowed to fill the entire coverslip until no air bubbles appeared. After standing for two minutes, the protoplasts were counted. The density of the protoplasts prepared in this comparative example was approximately 17.54 × 10⁻⁶. 5 per mL.

[0172] Protoplast density (units / mL) = Number of protoplasts in 25 square cells × 10 4 × Dilution factor.

[0173] Comparative Example 7

[0174] 1. A complex enzyme, by mass parts, comprising: 0.5 parts of cellulase RS, 0.5 parts of analyte R-10, and 0.3 parts of hemicellulase.

[0175] 2. An enzymatic hydrolysate containing one of the complex enzymes, comprising, by total volume, the following: 0.5 w / v% cellulase RS, 0.5 w / v% analyte R-10, 0.3 w / v% hemicellulase, a MES acid-base buffer solution with a final mannitol concentration of 0.55 mol / L and a pH of 5.7, a final MES concentration of 20 mmol / L, a final KCl concentration of 20 mmol / L, a final CaCl2 concentration of 10 mmol / L, a final BSA concentration of 0.1%, and a final β-mercaptoethanol concentration of 0.05 mmol / L.

[0176] The preparation method of the enzymatic hydrolysate is as follows: Calculate the amount of each substance to be added based on the composition of the enzymatic hydrolysate. Then, mix cellulase RS, analyte R-10, hemicellulase, mannitol, MES acid-base buffer, and KCl, and incubate at 55°C for 10 min. After the water bath, cool to room temperature, add CaCl2, BSA, and β-mercaptoethanol, and then add ultrapure water to a final volume of 15 mL to obtain the enzymatic hydrolysate.

[0177] After preparing the enzymatic hydrolysate, filter the prepared enzymatic hydrolysate through a 0.45 μm filter membrane to remove bacteria, and then pour it into a 50 mL centrifuge tube for later use.

[0178] 3. The method for preparing carrot protoplasts using the two enzymatic hydrolysates is as follows:

[0179] (1) Carrot seedlings that have been cultured for 4-6 days were treated in the dark for 24 hours. Then, 30 cotyledons with petioles were taken, with the petiole length being about 3-5 mm. The cotyledons with petioles were cut into thin strips 1 mm wide. The strips (about 1 mm wide) were quickly placed into 50 mL centrifuge tubes containing 15 mL of enzymatic hydrolysate. The centrifuge tubes were wrapped with aluminum foil and placed on a flat plate shaker in the dark and shaken (28℃, 45 rpm) for 4 hours.

[0180] (2) Place the 70µm cell filter sieve on a new 50mL sterile centrifuge tube and add about 1mL of W5 solution to rinse the cell filter sieve by rotation. Gently blow the enzymatic hydrolysate obtained in step (1) about 6-8 times with a cut sterile 1mL pipette tip to fully release the protoplasts. Then transfer the enzymatic hydrolysate in batches to the rinsed cell filter sieve for filtration. Gently press the undigested tissue in the cell filter sieve 3-5 times with the piston of a 5mL sterile syringe. Then slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinsing twice. Collect the filtrate, which is the filtered enzymatic hydrolysate.

[0181] (3) Centrifuge 100g of the enzyme hydrolysate obtained in step (2) for 7 min. After centrifugation, discard the supernatant. Tilt the centrifuge tube and slowly add 3 mL of W5 solution along the tube wall to resuspend the precipitate. Filter the resuspended liquid through a 40µm cell filter sieve. Wash the residual tissue on the cell sieve with 1 mL of W5 solution. Repeat this process once more (i.e., wash the residual tissue on the cell sieve with 1 mL of W5 solution, the same below), and collect the filtrate. Centrifuge 100g of the filtrate for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain carrot protoplasts.

[0182] (4) Protoplast viability was detected using FDA (fluorescein diacetate). 100 μL of protoplast suspension was placed in a 1.5 mL centrifuge tube pre-wrapped in aluminum foil, and 3 μL of 1 mg / mL FDA was added. The tube was stained in the dark for 5 min. After staining, 50 μL was transferred to a glass slide and observed in the dark using an inverted fluorescence microscope (Thermo Fisher EVOS M3000). Viable protoplasts emitted a yellow-green fluorescence; the number of yellow-green protoplasts was counted. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field of view is the protoplast viability value. The prepared protoplast viability value was 80.36%.

[0183] Protoplast viability: Protoplast viability = (Number of protoplasts emitting yellow-green fluorescence / Total number of protoplasts) × 100%.

[0184] To determine the protoplast concentration: A drop of the prepared protoplasts was placed on one end of a coverslip in a hemocytometer. The droplet was allowed to fill the entire coverslip until no air bubbles appeared. After standing for two minutes, the protoplasts were counted. The density of the protoplasts prepared in this comparative example was approximately 10.42 × 10⁻⁶. 5 per mL.

[0185] Protoplast density (units / mL) = Number of protoplasts in 25 square cells × 10 4 × Dilution factor.

[0186] Comparative Example 8

[0187] 1. A complex enzyme, by mass parts, comprising: 1.0 part of cellulase RS, 1.0 part of analyte R-10 and 0.3 part of hemicellulase.

[0188] 2. An enzymatic hydrolysate containing one of the complex enzymes, comprising, by total volume of the hydrolysate: 1.0 w / v% cellulase RS, 1.0 w / v% analyte R-10, 0.3 w / v% hemicellulase, a MES acid-base buffer solution with a final mannitol concentration of 0.55 mol / L and a pH of 5.7, a final MES concentration of 20 mmol / L, a final KCl concentration of 20 mmol / L, a final CaCl2 concentration of 10 mmol / L, a final BSA concentration of 0.1%, and a final β-mercaptoethanol concentration of 0.05 mmol / L.

[0189] The preparation method of the enzymatic hydrolysate is as follows: Calculate the amount of each substance to be added based on the composition of the enzymatic hydrolysate. Then, mix cellulase RS, analyte R-10, hemicellulase, mannitol, MES acid-base buffer, and KCl, and incubate in a water bath at 55°C for 10 min. After the water bath, cool to room temperature, add CaCl2, BSA, and β-mercaptoethanol, and then add ultrapure water to bring the volume to 15 mL to obtain the enzymatic hydrolysate.

[0190] After preparing the enzymatic hydrolysate, filter the prepared enzymatic hydrolysate through a 0.45 μm filter membrane to remove bacteria, and then pour it into a 50 mL centrifuge tube for later use.

[0191] 3. The method for preparing carrot protoplasts using the two enzymatic hydrolysates is as follows:

[0192] (1) Carrot seedlings that have been cultured for 4-6 days were treated in the dark for 24 hours. Then, 30 cotyledons with petioles were taken, with the petiole length being about 3-5 mm. The cotyledons with petioles were cut into thin strips 1 mm wide. The strips (about 1 mm wide) were quickly placed into 50 mL centrifuge tubes containing 15 mL of enzymatic hydrolysate. The centrifuge tubes were wrapped with aluminum foil and placed on a flat plate shaker in the dark and shaken (28℃, 45 rpm) for 4 hours.

[0193] (2) Place the 70µm cell filter sieve on a new 50mL sterile centrifuge tube and add about 1mL of W5 solution to rinse the cell filter sieve by rotation. Gently blow the enzymatic hydrolysate obtained in step (1) about 6-8 times with a cut sterile 1mL pipette tip to fully release the protoplasts. Then transfer the enzymatic hydrolysate in batches to the rinsed cell filter sieve for filtration. Gently press the undigested tissue in the cell filter sieve 3-5 times with the piston of a 5mL sterile syringe. Then slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinsing twice. Collect the filtrate, which is the filtered enzymatic hydrolysate.

[0194] (3) Centrifuge 100g of the enzyme hydrolysate obtained in step (2) for 7 min. After centrifugation, discard the supernatant. Tilt the centrifuge tube and slowly add 3 mL of W5 solution along the tube wall to resuspend the precipitate. Filter the resuspended liquid through a 40µm cell filter sieve. Wash the residual tissue on the cell sieve with 1 mL of W5 solution. Repeat this process once more (i.e., wash the residual tissue on the cell sieve with 1 mL of W5 solution, the same below), and collect the filtrate. Centrifuge 100g of the filtrate for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain carrot protoplasts.

[0195] (4) Protoplast viability was detected using FDA (fluorescein diacetate). 100 μL of protoplast suspension was placed in a 1.5 mL centrifuge tube pre-wrapped in aluminum foil, and 3 μL of 1 mg / mL FDA was added. The tube was stained in the dark for 5 min. After staining, 50 μL was transferred to a glass slide and observed in the dark using an inverted fluorescence microscope (Thermo Fisher EVOS M3000). Viable protoplasts emitted a yellow-green fluorescence; the number of yellow-green protoplasts was counted. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field of view is the protoplast viability value. The prepared protoplast viability value was 82.09%.

[0196] Protoplast viability: Protoplast viability = (Number of protoplasts emitting yellow-green fluorescence / Total number of protoplasts) × 100%.

[0197] To determine the protoplast concentration: A drop of the prepared protoplasts was placed on one end of a coverslip in a hemocytometer. The droplet was allowed to fill the entire coverslip until no air bubbles appeared. After standing for two minutes, the protoplasts were counted. The density of the protoplasts prepared in this comparative example was approximately 32.48 × 10⁻⁶. 5 per mL.

[0198] Protoplast density (units / mL) = Number of protoplasts in 25 square cells × 10 4 × Dilution factor.

[0199] Comparative Example 9

[0200] 1. A complex enzyme, by mass parts, comprising: 1.0 part of cellulase RS, 0.5 part of dissociative enzyme R-10 and 1.0 part of hemicellulase.

[0201] 2. An enzymatic hydrolysate containing one of the complex enzymes, comprising, by total volume of the hydrolysate: 1.0 w / v% cellulase RS, 0.5 w / v% analyte R-10, 1.0 w / v% hemicellulase, a MES acid-base buffer solution with a final mannitol concentration of 0.55 mol / L and a pH of 5.7, a final MES concentration of 20 mmol / L, a final KCl concentration of 20 mmol / L, a final CaCl2 concentration of 10 mmol / L, a final BSA concentration of 0.1%, and a final β-mercaptoethanol concentration of 0.05 mmol / L.

[0202] The preparation method of the enzymatic hydrolysate is as follows: Calculate the amount of each substance to be added based on the composition of the enzymatic hydrolysate. Then, mix cellulase RS, analyte R-10, hemicellulase, mannitol, MES acid-base buffer, and KCl, and incubate in a water bath at 55°C for 10 min. After the water bath, cool to room temperature, add CaCl2, BSA, and β-mercaptoethanol, and then add ultrapure water to bring the volume to 15 mL to obtain the enzymatic hydrolysate.

[0203] After preparing the enzymatic hydrolysate, filter the prepared enzymatic hydrolysate through a 0.45 μm filter membrane to remove bacteria, and then pour it into a 50 mL centrifuge tube for later use.

[0204] 3. The method for preparing carrot protoplasts using the two enzymatic hydrolysates is as follows:

[0205] (1) Carrot seedlings that have been cultured for 4-6 days were treated in the dark for 24 hours. Then, 30 cotyledons with petioles were taken, with the petiole length being about 3-5 mm. The cotyledons with petioles were cut into thin strips 1 mm wide. The strips (about 1 mm wide) were quickly placed into 50 mL centrifuge tubes containing 15 mL of enzymatic hydrolysate. The centrifuge tubes were wrapped with aluminum foil and placed on a flat plate shaker in the dark and shaken (28℃, 45 rpm) for 4 hours.

[0206] (2) Place the 70µm cell filter sieve on a new 50mL sterile centrifuge tube and add about 1mL of W5 solution to rinse the cell filter sieve by rotation. Gently blow the enzymatic hydrolysate obtained in step (1) about 6-8 times with a cut sterile 1mL pipette tip to fully release the protoplasts. Then transfer the enzymatic hydrolysate in batches to the rinsed cell filter sieve for filtration. Gently press the undigested tissue in the cell filter sieve 3-5 times with the piston of a 5mL sterile syringe. Then slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinsing twice. Collect the filtrate, which is the filtered enzymatic hydrolysate.

[0207] (3) Centrifuge 100g of the enzyme hydrolysate obtained in step (2) for 7 min. After centrifugation, discard the supernatant. Tilt the centrifuge tube and slowly add 3 mL of W5 solution along the tube wall to resuspend the precipitate. Filter the resuspended liquid through a 40µm cell filter sieve. Wash the residual tissue on the cell sieve with 1 mL of W5 solution. Repeat this process once more (i.e., wash the residual tissue on the cell sieve with 1 mL of W5 solution, the same below), and collect the filtrate. Centrifuge 100g of the filtrate for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain carrot protoplasts.

[0208] (4) Protoplast viability was detected using FDA (fluorescein diacetate). 100 μL of protoplast suspension was placed in a 1.5 mL centrifuge tube pre-wrapped in aluminum foil, and 3 μL of 1 mg / mL FDA was added. The tube was stained in the dark for 5 min. After staining, 50 μL was transferred to a glass slide and observed in the dark using an inverted fluorescence microscope (Thermo Fisher EVOS M3000). Viable protoplasts emitted a yellow-green fluorescence; the number of yellow-green protoplasts was counted. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field of view is the protoplast viability value. The prepared protoplast viability value was 79.07%.

[0209] Protoplast viability: Protoplast viability = (Number of protoplasts emitting yellow-green fluorescence / Total number of protoplasts) × 100%.

[0210] To determine the protoplast concentration: A drop of the prepared protoplasts was placed on one end of a coverslip in a hemocytometer. The droplet was allowed to fill the entire coverslip until no air bubbles appeared. After standing for two minutes, the protoplasts were counted. The density of the protoplasts prepared in this comparative example was approximately 27.76 × 10⁻⁶. 5 per mL.

[0211] Protoplast density (units / mL) = Number of protoplasts in 25 square cells × 10 4 × Dilution factor.

[0212] Comparative Example 10

[0213] 1. A complex enzyme, by mass parts, comprises: 1.0 part of cellulase RS, 0.1 part of analyte R-10 and 0.3 part of hemicellulase.

[0214] 2. An enzymatic hydrolysate containing one of the complex enzymes, comprising, by total volume of the hydrolysate: 1.0 w / v% cellulase RS, 0.1 w / v% analyte R-10, 0.3 w / v% hemicellulase, a MES acid-base buffer solution with a final mannitol concentration of 0.55 mol / L and a pH of 5.7, a final MES concentration of 20 mmol / L, a final KCl concentration of 20 mmol / L, a final CaCl2 concentration of 10 mmol / L, a final BSA concentration of 0.1%, and a final β-mercaptoethanol concentration of 0.05 mmol / L.

[0215] The preparation method of the enzymatic hydrolysate is as follows: Calculate the amount of each substance to be added based on the composition of the enzymatic hydrolysate. Then, mix cellulase RS, analyte R-10, hemicellulase, mannitol, MES acid-base buffer, and KCl, and incubate in a water bath at 55°C for 10 min. After the water bath, cool to room temperature, add CaCl2, BSA, and β-mercaptoethanol, and then add ultrapure water to bring the volume to 15 mL to obtain the enzymatic hydrolysate.

[0216] After preparing the enzymatic hydrolysate, filter the prepared enzymatic hydrolysate through a 0.45 μm filter membrane to remove bacteria, and then pour it into a 50 mL centrifuge tube for later use.

[0217] 3. The method for preparing carrot protoplasts using the two enzymatic hydrolysates is as follows:

[0218] (1) Carrot seedlings that have been cultured for 4-6 days were treated in the dark for 24 hours. Then, 30 cotyledons with petioles were taken, with the petiole length being about 3-5 mm. The cotyledons with petioles were cut into thin strips 1 mm wide. The strips (about 1 mm wide) were quickly placed into 50 mL centrifuge tubes containing 15 mL of enzymatic hydrolysate. The centrifuge tubes were wrapped with aluminum foil and placed on a flat plate shaker in the dark and shaken (28℃, 45 rpm) for 4 hours.

[0219] (2) Place the 70µm cell filter sieve on a new 50mL sterile centrifuge tube and add about 1mL of W5 solution to rinse the cell filter sieve by rotation. Gently blow the enzymatic hydrolysate obtained in step (1) about 6-8 times with a cut sterile 1mL pipette tip to fully release the protoplasts. Then transfer the enzymatic hydrolysate in batches to the rinsed cell filter sieve for filtration. Gently press the undigested tissue in the cell filter sieve 3-5 times with the piston of a 5mL sterile syringe. Then slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinsing twice. Collect the filtrate, which is the filtered enzymatic hydrolysate.

[0220] (3) Centrifuge 100g of the enzyme hydrolysate obtained in step (2) for 7 min. After centrifugation, discard the supernatant. Tilt the centrifuge tube and slowly add 3 mL of W5 solution along the tube wall to resuspend the precipitate. Filter the resuspended liquid through a 40µm cell filter sieve. Wash the residual tissue on the cell sieve with 1 mL of W5 solution. Repeat this process once more (i.e., wash the residual tissue on the cell sieve with 1 mL of W5 solution, the same below), and collect the filtrate. Centrifuge 100g of the filtrate for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain carrot protoplasts.

[0221] (4) Protoplast viability was detected using FDA (fluorescein diacetate). 100 μL of protoplast suspension was placed in a 1.5 mL centrifuge tube pre-wrapped in aluminum foil, and 3 μL of 1 mg / mL FDA was added. The tube was stained in the dark for 5 min. After staining, 50 μL was transferred to a glass slide and observed in the dark using an inverted fluorescence microscope (Thermo Fisher EVOS M3000). Viable protoplasts emitted a yellow-green fluorescence; the number of yellow-green protoplasts was counted. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field of view is the protoplast viability value. The prepared protoplast viability value was 81.89%.

[0222] Protoplast viability: Protoplast viability = (Number of protoplasts emitting yellow-green fluorescence / Total number of protoplasts) × 100%.

[0223] To determine the protoplast concentration: A drop of the prepared protoplasts was placed on one end of a coverslip in a hemocytometer. The droplet was allowed to fill the entire coverslip until no air bubbles appeared. After standing for two minutes, the protoplasts were counted. The density of the protoplasts prepared in this comparative example was approximately 11.04 × 10⁻⁶. 5 per mL.

[0224] Protoplast density (units / mL) = Number of protoplasts in 25 square cells × 10 4 × Dilution factor.

[0225] Comparative Example 11

[0226] 1. A complex enzyme, by mass parts, comprising: 1.0 part of cellulase RS, 0.5 part of analyte R-10 and 0.1 part of hemicellulase.

[0227] 2. An enzymatic hydrolysate containing one of the complex enzymes, comprising, by total volume of the hydrolysate: 1.0 w / v% cellulase RS, 0.5 w / v% analyte R-10, 0.1 w / v% hemicellulase, a MES acid-base buffer solution with a final mannitol concentration of 0.55 mol / L and a pH of 5.7, a final MES concentration of 20 mmol / L, a final KCl concentration of 20 mmol / L, a final CaCl2 concentration of 10 mmol / L, a final BSA concentration of 0.1%, and a final β-mercaptoethanol concentration of 0.05 mmol / L.

[0228] The preparation method of the enzymatic hydrolysate is as follows: Calculate the amount of each substance to be added based on the composition of the enzymatic hydrolysate. Then, mix cellulase RS, analyte R-10, hemicellulase, mannitol, MES acid-base buffer, and KCl, and incubate in a water bath at 55°C for 10 min. After the water bath, cool to room temperature, add CaCl2, BSA, and β-mercaptoethanol, and then add ultrapure water to bring the volume to 15 mL to obtain the enzymatic hydrolysate.

[0229] After preparing the enzymatic hydrolysate, filter the prepared enzymatic hydrolysate through a 0.45 μm filter membrane to remove bacteria, and then pour it into a 50 mL centrifuge tube for later use.

[0230] 3. The method for preparing carrot protoplasts using the two enzymatic hydrolysates is as follows:

[0231] (1) Carrot seedlings that have been cultured for 4-6 days were treated in the dark for 24 hours. Then, 30 cotyledons with petioles were taken, with the petiole length being about 3-5 mm. The cotyledons with petioles were cut into thin strips 1 mm wide. The strips (about 1 mm wide) were quickly placed into 50 mL centrifuge tubes containing 15 mL of enzymatic hydrolysate. The centrifuge tubes were wrapped with aluminum foil and placed on a flat plate shaker in the dark and shaken (28℃, 45 rpm) for 4 hours.

[0232] (2) Place the 70µm cell filter sieve on a new 50mL sterile centrifuge tube and add about 1mL of W5 solution to rinse the cell filter sieve by rotation. Gently blow the enzymatic hydrolysate obtained in step (1) about 6-8 times with a cut sterile 1mL pipette tip to fully release the protoplasts. Then transfer the enzymatic hydrolysate in batches to the rinsed cell filter sieve for filtration. Gently press the undigested tissue in the cell filter sieve 3-5 times with the piston of a 5mL sterile syringe. Then slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinsing twice. Collect the filtrate, which is the filtered enzymatic hydrolysate.

[0233] (3) Centrifuge 100g of the enzyme hydrolysate obtained in step (2) for 7 min. After centrifugation, discard the supernatant. Tilt the centrifuge tube and slowly add 3 mL of W5 solution along the tube wall to resuspend the precipitate. Filter the resuspended liquid through a 40µm cell filter sieve. Wash the residual tissue on the cell sieve with 1 mL of W5 solution. Repeat this process once more (i.e., wash the residual tissue on the cell sieve with 1 mL of W5 solution, the same below), and collect the filtrate. Centrifuge 100g of the filtrate for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain carrot protoplasts.

[0234] (4) Protoplast viability was detected using FDA (fluorescein diacetate). 100 μL of protoplast suspension was placed in a 1.5 mL centrifuge tube pre-wrapped in aluminum foil, and 3 μL of 1 mg / mL FDA was added. The tube was stained in the dark for 5 min. After staining, 50 μL was transferred to a glass slide and observed in the dark using an inverted fluorescence microscope (Thermo Fisher EVOS M3000). Viable protoplasts emitted a yellow-green fluorescence; the number of yellow-green protoplasts was counted. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field of view is the protoplast viability value. The prepared protoplast viability value was 82.25%.

[0235] Protoplast viability: Protoplast viability = (Number of protoplasts emitting yellow-green fluorescence / Total number of protoplasts) × 100%.

[0236] To determine the protoplast concentration: A drop of the prepared protoplasts was placed on one end of a coverslip in a hemocytometer. The droplet was allowed to fill the entire coverslip until no air bubbles appeared. After standing for two minutes, the protoplasts were counted. The density of the protoplasts prepared in this comparative example was approximately 14.18 × 10⁻⁶. 5 per mL.

[0237] Protoplast density (units / mL) = Number of protoplasts in 25 square cells × 10 4 × Dilution factor.

[0238] Comparative Example 12

[0239] 1. A complex enzyme, by mass parts, comprises: 1.0 part of cellulase RS, 0.5 part of analyte R-10 and 0.3 part of hemicellulase.

[0240] 2. An enzymatic hydrolysate containing one of the complex enzymes, comprising, by total volume of the hydrolysate: 1.0 w / v% cellulase RS, 0.5 w / v% analyte R-10, 0.3 w / v% hemicellulase, a MES acid-base buffer solution with a final mannitol concentration of 0.55 mol / L and a pH of 5.7, a final MES concentration of 20 mmol / L, a final KCl concentration of 20 mmol / L, a final CaCl2 concentration of 10 mmol / L, a final BSA concentration of 0.1%, and a final β-mercaptoethanol concentration of 0.05 mmol / L.

[0241] The preparation method of the enzymatic hydrolysate is as follows: Calculate the amount of each substance to be added based on the composition of the enzymatic hydrolysate. Then, mix cellulase RS, analyte R-10, hemicellulase, mannitol, MES acid-base buffer, and KCl, and incubate in a water bath at 55°C for 10 min. After the water bath, cool to room temperature, add CaCl2, BSA, and β-mercaptoethanol, and then add ultrapure water to bring the volume to 15 mL to obtain the enzymatic hydrolysate.

[0242] After preparing the enzymatic hydrolysate, filter the prepared enzymatic hydrolysate through a 0.45 μm filter membrane to remove bacteria, and then pour it into a 50 mL centrifuge tube for later use.

[0243] 3. The method for preparing carrot protoplasts using the two enzymatic hydrolysates is as follows:

[0244] (1) Carrot seedlings that have been cultured for 4-6 days were treated in the dark for 24 hours. Then, 30 cotyledons with petioles were taken, with the petiole length being about 3-5 mm. The cotyledons with petioles were cut into thin strips 1 mm wide. The strips (about 1 mm wide) were quickly placed into 50 mL centrifuge tubes containing 15 mL of enzymatic hydrolysate. The centrifuge tubes were wrapped with aluminum foil and placed on a flat plate shaker in the dark and shaken (28℃, 45 rpm) for 5 hours.

[0245] (2) Place the 70µm cell filter sieve on a new 50mL sterile centrifuge tube and add about 1mL of W5 solution to rinse the cell filter sieve by rotation. Gently blow the enzymatic hydrolysate obtained in step (1) about 6-8 times with a cut sterile 1mL pipette tip to fully release the protoplasts. Then transfer the enzymatic hydrolysate in batches to the rinsed cell filter sieve for filtration. Gently press the undigested tissue in the cell filter sieve 3-5 times with the piston of a 5mL sterile syringe. Then slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinsing twice. Collect the filtrate, which is the filtered enzymatic hydrolysate.

[0246] (3) Centrifuge 100g of the enzyme hydrolysate obtained in step (2) for 7 min. After centrifugation, discard the supernatant. Tilt the centrifuge tube and slowly add 3 mL of W5 solution along the tube wall to resuspend the precipitate. Filter the resuspended liquid through a 40µm cell filter sieve. Wash the residual tissue on the cell sieve with 1 mL of W5 solution. Repeat this process once more (i.e., wash the residual tissue on the cell sieve with 1 mL of W5 solution, the same below), and collect the filtrate. Centrifuge 100g of the filtrate for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain carrot protoplasts.

[0247] (4) Protoplast viability was detected using FDA (fluorescein diacetate). 100 μL of protoplast suspension was placed in a 1.5 mL centrifuge tube pre-wrapped in aluminum foil, and 3 μL of 1 mg / mL FDA was added. The tube was stained in the dark for 5 min. After staining, 50 μL was transferred to a glass slide and observed in the dark using an inverted fluorescence microscope (Thermo Fisher EVOS M3000). Viable protoplasts emitted a yellow-green fluorescence; the number of yellow-green protoplasts was counted. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field of view is the protoplast viability value. The prepared protoplast viability value was 79.54%.

[0248] Protoplast viability: Protoplast viability = (Number of protoplasts emitting yellow-green fluorescence / Total number of protoplasts) × 100%.

[0249] To determine the protoplast concentration: A drop of the prepared protoplasts was placed on one end of a coverslip in a hemocytometer. The droplet was allowed to fill the entire coverslip until no air bubbles appeared. After standing for two minutes, the protoplasts were counted. The density of the protoplasts prepared in this comparative example was approximately 35.74 × 10⁻⁶. 5 per mL.

[0250] Protoplast density (units / mL) = Number of protoplasts in 25 square cells × 10 4 × Dilution factor.

[0251] The yield and viability of carrot protoplasts in Examples 1-3 and Comparative Examples 1-12 are shown in Table 1.

[0252] Table 1. Yield and viability of carrot protoplasts in Examples 1-3 and Comparative Examples 1-12

[0253]

[0254] Note: Different letters in the same column in the table indicate significant differences at the P < 0.05 level.

[0255] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of an enzymatic hydrolysate in the preparation of carrot protoplasts, wherein the enzymatic hydrolysate comprises, by total volume: 1.0–1.5 w / v% cellulase, 0.3–0.5 w / v% dissociative enzyme, 0.3–0.5 w / v% hemicellulase, 0.50–0.60 mol / L mannitol, 18–22 mmol / L MES, 18–22 mmol / L KCl, 8–12 mmol / L CaCl2, 0.08–0.12 w / v% BSA, and 0.03–0.07 mmol / L β-mercaptoethanol; The cellulase is cellulase RS; the analyte is analyte R-10.

2. The application according to claim 1, characterized in that, Based on the total volume of the enzymatic hydrolysate, the hydrolysate comprises 1.0 w / v of cellulase, 0.5 w / v of ionizing enzyme, and 0.3 w / v of hemicellulase. Alternatively, the enzymatic hydrolysate may comprise 1.5 w / v cellulase, 0.3 w / v ionase, and 0.5 w / v hemicellulase. Alternatively, the enzymatic hydrolysate may contain 1.0 w / v cellulase, 0.5 w / v ionase, and 0.5 w / v hemicellulase.

3. The application according to claim 1, wherein the effect of preparing carrot protoplasts includes improving protoplast activity and / or increasing protoplast yield.

4. A method for preparing carrot protoplasts, characterized in that, include: The carrot plant tissue was enzymatically hydrolyzed using the enzymatic hydrolysate described in claim 1 to obtain carrot protoplasts.

5. The method according to claim 4, characterized in that, The enzymatic hydrolysis temperature is 28~30℃; the enzymatic hydrolysis time is 2.5~4h.

6. The method according to claim 4, characterized in that, The carrot plant tissue includes carrot seedling plant tissue.

7. The method according to claim 6, characterized in that, The carrot seedling plant tissue includes petiolate cotyledons.

8. The method according to claim 6, characterized in that, The method of cultivating carrot seedlings includes using plant tissue culture to cultivate carrot seeds to obtain carrot seedlings.

9. The method according to claim 8, characterized in that, The temperature for plant tissue culture is 23-25℃; the photoperiod for plant tissue culture is 15-17 h / d; the light intensity for plant tissue culture is 2500-3200 Lx; and the culture time is 4-6 days.

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