Enzymatic hydrolysate for preparing carrot protoplast and preparation method and application of carrot protoplast

Abstract

The invention provides enzymatic hydrolysate for preparing carrot protoplast and a preparation method and application of the carrot protoplast, and belongs to the technical field of plant cytology. The enzymatic hydrolysate is prepared from 1.0 to 1.5 w / v percent of cellulase, 0.3 to 0.5 w / v percent of macerozyme and 0.3 to 0.5 w / v percent of hemicellulase. Wherein the cellulase has the effect of degrading cellulose in plant cell walls, the macerozyme and the cellulase are matched for use, so that plant tissues can be separated into single cells, and the hemicellulase is mainly used for degrading hemicellulose in the plant cell walls. The enzymatic hydrolysate can play a synergistic role in promoting splitting decomposition of carrot tissue cell walls through the comprehensive action of three enzymes, can better and faster destroy cell wall skeletons, is more beneficial to preparation of carrot protoplasts, further releases the protoplasts to the maximum extent, and is beneficial to improvement of the protoplast yield; meanwhile, the protoplast can keep relatively high activity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant cytology, and particularly relates to an enzyme solution for preparing red radish protoplasts and a preparation method and application of red radish protoplasts. BACKGROUND

[0002] Red radish (Raphanus sativus L.) is a biennial or annual herbaceous plant of the Raphanus genus in the Brassicaceae family. Raphanus sativus The Nujiang red radish is a unique variety in Nujiang Prefecture, Yunnan Province, with the skin and flesh of the fleshy root being red to purple red, a crisp and sweet taste, and tender and juicy flesh, which is of great significance for radish variety breeding and production.

[0003] Plant protoplast refers to a plant cell from which the cell wall is removed, and has a certain reproductive capacity and can develop into a complete plant under appropriate culture conditions. Because protoplasts do not have cell walls, they are more likely to overcome distant hybridization incompatibility barriers and easily take up organelles, proteins and other exogenous macromolecules, and have been widely used in subcellular localization, gene expression analysis, protein-protein interaction, gene editing and other aspects, and are considered to be an ideal material for developmental biology, cell biology and cytogenetics research. The three main components of plant cell walls are cellulose, hemicellulose and pectin. Each component varies greatly depending on the plant species and the part of the material. The genotype of the suitable material and the tissue type of the material are one of the important factors affecting the isolation and preparation of plant protoplasts and the regeneration of plants. At present, there is no related research on the preparation of Nujiang red radish protoplasts, and the establishment and optimization of the preparation system of red radish protoplasts is of great significance for realizing the fusion of radish protoplasts from different varieties, and the efficient introduction and expression of exogenous genes. SUMMARY

[0004] In view of the defects in the prior art, the purpose of the present application is to provide an application of a composite enzyme in the preparation of red radish protoplasts, which can enzymatically hydrolyze the plant tissues of the Nujiang red radish, realize the preparation of red radish protoplasts, and improve the yield and activity of red radish protoplasts.

[0005] The purpose of the present application is achieved by the following technical solutions: The present application provides an application of a composite enzyme in the preparation of red radish protoplasts, which comprises, by mass fraction, 1.0-1.5 parts of cellulase, 0.3-0.5 parts of dissociation enzyme, and 0.3-0.5 parts of hemicellulase.

[0006] Preferably, the cellulase comprises cellulase RS, and the dissociation enzyme comprises dissociation enzyme R-10.

[0007] Preferably, the complex enzyme comprises cellulase 1.0 parts by mass, dissociation enzyme 0.5 parts by mass, and hemicellulase 0.3 parts by mass; Alternatively, the complex enzyme comprises cellulase 1.5 parts by mass, dissociation enzyme 0.3 parts by mass, and hemicellulase 0.5 parts by mass. Alternatively, the complex enzyme comprises cellulase 1.0 parts by mass, dissociation enzyme 0.5 parts by mass, and hemicellulase 0.5 parts by mass.

[0008] The application provides an enzyme solution for preparing red radish protoplasts, comprising, in terms of total volume of the enzyme solution, 1.0-1.5 w / v% cellulase, 0.3-0.5 w / v% dissociation 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.

[0009] The application provides application of the enzyme solution in any one or two or more of preparation of red radish protoplasts, improvement of protoplast activity, and improvement of protoplast yield.

[0010] The application provides a method for preparing red radish protoplasts, comprising: The enzyme solution is used to perform enzymolysis on red radish plant tissues to obtain red radish protoplasts.

[0011] Preferably, the temperature of the enzymolysis is 28-30°C, and the time of the enzymolysis is 2.5-4 h.

[0012] Preferably, the red radish plant tissues comprise red radish seedling plant tissues, and the red radish seedling plant tissues comprise any one or two or more of leaves, cotyledons, and cotyledons with petioles.

[0013] Preferably, the method for culturing red radish seedlings comprises culturing seeds of red radish by using a plant tissue culture method to obtain red radish seedlings.

[0014] Preferably, the temperature of the plant tissue culture is 23-25°C, the photoperiod of the plant tissue culture is 15-17 h / d, the light intensity of the plant tissue culture is 2500-3200 Lx, and the time of the plant tissue culture is 4-6 d.

[0015] Beneficial effects: The application provides application of a composite enzyme in preparation of red radish protoplasts, and the composite enzyme comprises cellulase 1.0-1.5 parts by mass, dissociative enzyme 0.3-0.5 parts by mass and hemicellulase 0.3-0.5 parts by mass. In the composite enzyme, the cellulase has the effect of degrading cellulose in the plant cell wall, the dissociative enzyme is used in cooperation with the cellulase to facilitate separation of the plant tissue into single cells, and the hemicellulase mainly has the effect of degrading hemicellulose in the plant cell wall. The composite enzyme can synergistically promote the effect of promoting lysis of the red radish plant tissue cell wall through the comprehensive effect of the three enzymes, can better and faster destroy the cell wall skeleton, is more conducive to preparation of the red radish protoplasts, and then maximally releases the protoplasts, is conducive to improving the protoplast yield, and can also make the protoplasts maintain high activity. The results of the examples show that the protoplasts of Nujiang red radish are prepared by using the enzyme solution containing the composite enzyme, and the yield and activity of the protoplasts can be significantly improved. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0017] Figure 1 The figure is an observation result of the red radish protoplasts prepared in Example 1; the right figure is an FDA staining result, and the left figure is a protoplast image under a corresponding bright field view, and the size of the scale in the figure is 100 µm; Figure 2 The figure is an observation result of the red radish protoplasts prepared in Example 2; the right figure is an FDA staining result, and the left figure is a protoplast image under a corresponding bright field view, and the size of the scale in the figure is 100 µm; Figure 3 The figure is an observation result of the red radish protoplasts prepared in Comparative Example 1; the right figure is an FDA staining result, and the left figure is a protoplast image under a corresponding bright field view, and the size of the scale in the figure is 100 µm; Figure 4 The figure is an observation result of the red radish protoplasts prepared in Comparative Example 2; the right figure is an FDA staining result, and the left figure is a protoplast image under a corresponding bright field view, and the size of the scale in the figure is 100 µm. DETAILED DESCRIPTION

[0018] The application provides application of a compound enzyme in preparation of red radish protoplasts, and the compound enzyme comprises cellulase 1.0-1.5 parts by mass, dissociation enzyme 0.3-0.5 parts by mass and hemicellulase 0.3-0.5 parts by mass.

[0019] In the following technical solutions, the source of each raw material is not specially limited in the application, and conventional commercially available products in the art can be used.

[0020] As an optional embodiment of the application, the compound enzyme comprises cellulase 1.0-1.5 parts by mass, which can be 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5 parts by mass. In the application, the cellulase can be cellulase RS. In the application, the cellulase acts on the cell wall and has the effect of degrading cellulose in the cell wall of red radish.

[0021] As an optional embodiment of the application, the compound enzyme comprises dissociation enzyme 0.3-0.5 parts by mass based on the mass of cellulase, which can be 0.3, 0.4 or 0.5 parts by mass. In the application, the dissociation enzyme can be dissociation enzyme R-10. In the application, the dissociation enzyme is used in combination with cellulase to facilitate the separation of plant tissues into single cells, and then facilitate the obtaining of protoplasts.

[0022] As an optional embodiment of the application, the compound enzyme comprises hemicellulase 0.3-0.5 parts by mass based on the mass of cellulase, which can be 0.3, 0.4 or 0.5 parts by mass. In the application, the main effect of the hemicellulase is to degrade hemicellulose in the cell wall of plants, and the hemicellulase is used in combination with cellulase and dissociation enzyme to better and faster degrade the cell wall skeleton, which is conducive to improving the yield of protoplasts.

[0023] The compound enzyme provided in the application can synergistically promote the lysis of the cell wall of red radish plant tissues through the comprehensive action of the three enzymes, better and faster destroy the cell wall skeleton, more conducive to the preparation of red radish protoplasts and the maximum release of protoplasts, conducive to improving the yield of protoplasts, and can also maintain high activity of the protoplasts. The results of the examples show that the preparation of protoplasts of Nujiang red radish using the enzyme solution containing the compound enzyme can significantly improve the yield and activity of the protoplasts.

[0024] As an optional embodiment of the application, the compound enzyme comprises cellulase 1.0 parts by mass, dissociation enzyme 0.5 parts by mass and hemicellulase 0.3 parts by mass.

[0025] As an optional embodiment of the application, the compound enzyme comprises cellulase 1.5 parts by mass, dissociation enzyme 0.3 parts by mass and hemicellulase 0.5 parts by mass. As an optional embodiment of the present application, the composite enzyme comprises, in parts by mass: 1.0 parts of cellulase, 0.5 parts of dissociative enzyme, and 0.5 parts of hemicellulase.

[0026] The present application provides an enzymatic hydrolysate of red radish protoplasts, comprising, in total volume of the enzymatic hydrolysate: 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.

[0027] As an optional embodiment of the present application, the enzymatic hydrolysate comprises 1.0-1.5 w / v% cellulase, which can be 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 w / v%, in total volume of the enzymatic hydrolysate.

[0028] As an optional embodiment of the present application, the enzymatic hydrolysate comprises 0.3-0.5 w / v% dissociative enzyme, which can be 0.3, 0.4, or 0.5 w / v%, in total volume of the enzymatic hydrolysate.

[0029] As an optional embodiment of the present application, the enzymatic hydrolysate comprises 0.3-0.5 w / v% hemicellulase, which can be 0.3, 0.4, or 0.5 w / v%, in total volume of the enzymatic hydrolysate.

[0030] As an optional embodiment of the present application, the enzymatic hydrolysate comprises 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; 19-21 mmol / L MES, which can be 19, 20, or 21 mmol / L; 19-21 mmol / L KCl, which can be 19, 20, or 21 mmol / L; 9-11 mmol / L CaCl2, which can be 9, 10, or 11 mmol / L; 0.09-0.11 w / v% BSA, which can be 0.09, 0.10, or 0.11 w / v%; or 0.04-0.06 mmol / L β-mercaptoethanol, which can be 0.04, 0.05, or 0.06 mmol / L, in total volume of the enzymatic hydrolysate.

[0031] The enzyme solution provided by the application adds cellulase, isolation enzyme and hemicellulase, and the three enzymes can synergistically degrade the cell wall of the plant tissue, thereby releasing the protoplast to the greatest extent, and also improving the activity of the protoplast. The components of the enzyme solution, such as mannitol, MES, KCl, CaCl2, BSA and β-mercaptoethanol, can stabilize the osmotic pressure, maintain the activity of the protoplast and adjust the pH, thereby facilitating the preparation of the red radish protoplast, and also improving the yield and activity of the red radish protoplast.

[0032] The application provides the application of the enzyme solution in any one or two or more of the preparation of the red radish protoplast, the improvement of the activity of the protoplast and the improvement of the yield of the protoplast.

[0033] The application provides a method for preparing a red radish protoplast, which comprises: using the enzyme solution to enzymatically hydrolyze the plant tissue of the red radish, so as to obtain the red radish protoplast.

[0034] As an optional embodiment of the application, the plant tissue of the red radish comprises the plant tissue of the seedling of the red radish. The method for culturing the seedling of the red radish is not particularly limited in the application, and a conventional seedling culturing method in the art can be used. As an optional embodiment of the application, the method for culturing the seedling of the red radish comprises culturing the seed of the red radish by using a plant tissue culturing method, so as to obtain the seedling of the red radish. The method for culturing the plant tissue is not particularly limited in the application, and a conventional plant tissue culturing method in the art can be used. In the application, the temperature for culturing the plant tissue can be 23-25 DEG C, or 23 DEG C, 24 DEG C or 25 DEG C; the photoperiod for culturing the plant tissue can be 15-17 h / d, or 16 h / d; the light intensity for culturing the plant tissue can be 2500-3200 Lx, or 3000 Lx; and the culturing time for culturing the plant tissue can be 4-6 d, or 5 d.

[0035] After obtaining the seedling of the red radish, the application can use different tissues of the seedling to prepare the protoplast by using the enzyme solution. As an optional embodiment of the application, after obtaining the seedling, the application preferably performs light shielding treatment on the seedling, and then uses different tissues of the seedling to prepare the protoplast by using the enzyme solution. The light shielding treatment time can be 24-48 h, or 24 h, 25 h, 26 h, 28 h, 30 h, 32 h, 34 h, 36 h, 38 h, 40 h, 42 h, 44 h, 46 h or 48 h. In the application, the different tissues can be any one or two or more of the leaf, the cotyledon and the cotyledon with petiole.

[0036] As an optional embodiment of the present application, the enzyme solution is used to enzymolysis cotyledon and / or cotyledon with petiole of red radish to obtain red radish protoplast. When the present application is used to enzymolysis, the ratio of the number of cotyledon and / or cotyledon with petiole to the volume of enzyme solution can be (20-30) pieces: 15 mL, or 20 pieces: 15 mL, 21 pieces: 15 mL, 22 pieces: 15 mL, 23 pieces: 15 mL, 24 pieces: 15 mL, 25 pieces: 15 mL, 26 pieces: 15 mL, 27 pieces: 15 mL, 28 pieces: 15 mL, 29 pieces: 15 mL or 30 pieces: 15 mL; the cotyledon and / or cotyledon with petiole can be cut into filaments with a width of 0.5-1 mm for enzymolysis, or cut into filaments with a width of 0.5, 0.6, 0.7, 0.8, 0.9 or 1 mm for enzymolysis.

[0037] As an optional embodiment of the present application, the temperature of enzymolysis can be 28-30℃, or 28℃, 29℃ or 30℃; the time of enzymolysis can be 2.5-4 h, or 2.5 h, 3 h, 3.5 h or 4 h; the enzymolysis is accompanied by oscillation; the rotation speed of oscillation can be 45-50 rpm, or 45 rpm, 46 rpm, 47 rpm, 48 rpm, 49 rpm or 50 rpm; the enzymolysis is carried out in the dark. The enzymolysis conditions of the present application can ensure the optimal enzymolysis conditions, and at the same time, can make the protoplast maintain higher activity.

[0038] As an optional embodiment of the present application, the enzymolysis product can be gently blown and beaten with a sterile pipette tip with a cut end during or after the completion of enzymolysis to fully release the protoplast.

[0039] After the completion of enzymolysis, the present application obtains the protoplast of red radish. Alternatively, as an optional embodiment of the present application, after obtaining the enzymolysis product, the present application preferably further comprises washing and resuspending the enzymolysis product to obtain the protoplast. The method of washing and resuspending the protoplast is not particularly limited in the present application, and any conventional method of washing and resuspending protoplast in the art can be used. In the examples of the present application, W5 solution and MMG solution are used to wash and resuspend the protoplast.

[0040] The preparation method of the red radish protoplast of the present application is simple and efficient, shortens the enzymolysis time, and can obtain a large amount of red radish protoplast which maintains activity for a long time.

[0041] In order to further illustrate the present application, the technical solutions provided by the present application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0042] 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 Yuan Ye Company, item number: S10045; MES, mannitol, KCl, CaCl2, BSA, β-mercaptoethanol, agar, sucrose, etc. were purchased from Shengong (Shanghai Shengong Bioengineering Co., Ltd.); 0.45 μm filter membrane was purchased from Sartorius (Sartorius Stedim Biotech GmbH, Germany); disposable sterile cell filter screen was purchased from Biosharp (Biosharp Life Sciences, Beijing Labgic Technology Co., Ltd.).

[0043] The red radish used in the following technical solutions is a red radish produced in the Nujiang region; it is a unique variety in Nujiang Prefecture, Yunnan Province, with the skin and flesh of the fleshy root being red to purple red, a crisp and sweet taste, and tender and juicy flesh.

[0044] The experimental tools such as forceps, scalpel blades, glass culture dishes, centrifuge tubes, suction tips, etc. used in the following solutions need to be sterilized at 121°C for 25 minutes, the cell filter screen is a disposable sterile cell filter screen, and the filter membrane is a 0.45 µm sterile microporous filter membrane; the ultraclean workbench is wiped with 75% alcohol, irradiated with a UV lamp for 30 min, and ventilated for 10 min.

[0045] Prepare W5 solution, and the composition of the W5 solution is: 2 mmol / L of MES, 154 mmol / L of NaCl solution, 125 mmol / L of CaCl2 solution, 5 mmol / L of KCl solution, 5 mmol / L of glucose, and the solution pH is 5.7.

[0046] Prepare MMG solution, and the composition is: 0.4 mol / L of mannitol, 15 mmol / L of MgCl2 solution, 4 mmol / L of MES, and the solution pH is 5.7.

[0047] The preparation method of red radish seedlings used in the preparation of protoplasts in the following solutions is as follows: About 100 seeds of red radish with full grains and complete epidermis were selected and placed in a sterilized 50 mL centrifuge tube to complete the steps of seed disinfection and inoculation in a clean bench. First, the seeds were sterilized with 75% ethanol for 30 s, then soaked in 0.2% HgCl2 solution for 6 min, and then washed with 45 mL of sterile water for 5 times, about 1 min each time, and then the surface moisture of the seeds was absorbed with sterile filter paper, and then placed on 1 / 2MS+1.5% sucrose+0.7% agar (pH=5.8±0.05) medium for culture. The culture conditions were as follows: temperature 24℃±1℃, light cycle 16h light / 8h dark, light intensity about 3000Lx. Red radish seedlings were obtained after 4-6 days of culture. The obtained red radish seedlings were used for subsequent protoplast preparation.

[0048] Example 1 1. A complex enzyme for preparing red radish protoplasts, comprising, in parts by mass: 1.0 parts of cellulase RS, 0.5 parts of isolated enzyme R-10, and 0.3 parts of hemicellulase.

[0049] 2. An enzyme hydrolysate containing the complex enzyme in 1, comprising, in terms of total volume of the enzyme hydrolysate: 1.0 w / v% of cellulase RS, 0.5 w / v% of isolated enzyme R-10, 0.3 w / v% of hemicellulase, a final concentration of 0.55 mol / L of mannitol, a pH of 5.7 of MES acid-base buffer, a final concentration of 20 mmol / L of MES, a final concentration of 20 mmol / L of KCl, a final concentration of 10 mmol / L of CaCl2, a final concentration of 0.1% of BSA, and a final concentration of 0.05 mmol / L of β-mercaptoethanol.

[0050] The preparation method of the enzyme hydrolysate is as follows: according to the composition of the enzyme hydrolysate, the addition amount of each substance is calculated, then the cellulase RS, isolated enzyme R-10, hemicellulase, mannitol, MES acid-base buffer and KCl are mixed, and then 55℃ water bath is performed for 10 min. After the water bath is completed, it is cooled to room temperature, CaCl2, BSA and β-mercaptoethanol are added, and then ultrapure water is added to make up to 15 mL to obtain the enzyme hydrolysate.

[0051] After the enzyme hydrolysate is prepared, the prepared enzyme hydrolysate is filtered with a 0.45 μm filter membrane to remove bacteria, and then poured into a 50 mL centrifuge tube for standby use.

[0052] 3. The method for preparing red radish protoplasts using the enzyme hydrolysate in 2, the steps are as follows: (1) The 4-6 day old red radish seedlings were treated with light for 24 h, and then 30 cotyledon petioles with cotyledon petioles of about 3-5 mm long were collected. The cotyledon petioles were cut into 1 mm wide filaments, and the cut filaments were quickly placed in a 50 mL centrifuge tube containing 15 mL enzyme solution. The centrifuge tube was wrapped with tin foil and placed in a dark place to avoid light and shaken (28°C, 45 rpm) for 2.5 h.

[0053] (2) A 70 µm cell filter screen was placed on a new 50 mL sterile centrifuge tube, and about 1 mL of W5 solution was added to rotate and rinse the cell filter screen. The enzyme solution obtained in step (1) was gently blown about 6-8 times with a sterile 1 mL pipette to fully release the protoplasts, and then the enzyme solution was transferred to the rinsed cell screen in batches. The un-enzymolysis tissue in the cell filter screen was gently pressed 3-5 times with the piston of a 5 mL sterile syringe, and then 3 mL of W5 solution was slowly added to rinse the un-enzymolysis tissue, and the rinsing was repeated twice. The filtrate was collected, which was the filtered enzyme solution.

[0054] (3) The filtered enzyme solution obtained in step (2) was centrifuged at 100 g for 7 min, and the supernatant was discarded. The precipitate was resuspended with 3 mL of W5 solution by slowly adding it along the wall of the tube. The resuspended liquid was filtered through a 40 µm cell filter screen, and the residual tissue on the cell filter screen was washed with 1 mL of W5 solution. The washing was repeated once (i.e., the residual tissue on the cell filter screen was washed with 1 mL of W5 solution, and the same was repeated), and the filtrate was collected. Then the filtrate was centrifuged at 100 g for 7 min, and the supernatant was discarded. The precipitate was resuspended with 3 mL of MMG solution, and red radish protoplasts were obtained.

[0055] (4) The viability of red radish protoplasts was detected using FDA (fluorescein diacetate). 100 µL of protoplast suspension was taken and placed in a 1.5 mL centrifuge tube wrapped with tin foil. 3 µL of 1 mg / mL FDA was added, and the staining was performed in the dark for 5 min. After staining, 50 µL was taken and placed on a glass slide. The viability of the protoplasts was observed under a fluorescence microscope (Thermo Fisher EVOS M3000) in the dark. Viable protoplasts emitted yellow-green fluorescence, and 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 was the protoplast viability value. The prepared protoplast viability value was 86.11%, as shown in Figure 1 Figure 1 The right panel of the figure is the FDA staining result, and the left panel is the protoplast image under the corresponding bright field. The scale bar in the figure is 100 µm.

[0056] The protoplast viability was calculated as follows: protoplast viability = (number of yellow-green fluorescent protoplasts / total number of protoplasts) x 100%. ​

[0057] Statistical protoplast concentration: The prepared protoplasts were taken and added to one end of the cover glass of a hemocytometer, and the liquid drop was allowed to fill the entire cover glass without air bubbles. After two minutes of standing, the count was observed. The protoplast density prepared in this example was about 39.78 x 10 5 individuals / mL.

[0058] Protoplast density (individuals / mL) = 25 x protoplast number in a square grid x 10 4 x dilution factor.

[0059] Example 2 1. A complex enzyme for preparing red radish protoplasts, comprising, by mass fraction, 1.5 parts of cellulase RS, 0.3 parts of isolated enzyme R-10, and 0.5 parts of hemicellulase.

[0060] 2. An enzymatic hydrolysate containing 1 complex enzyme, comprising, by volume of the total enzymatic hydrolysate, 1.5 w / v% of cellulase RS, 0.3 w / v% of isolated enzyme R-10, 0.5 w / v% of hemicellulase, a final concentration of 0.55 mol / L of mannitol, a pH of 5.7 of MES acid-base buffer, a final concentration of 20 mmol / L of MES, a final concentration of 20 mmol / L of KCl, a final concentration of 10 mmol / L of CaCl2, a final concentration of 0.1% of BSA, and a final concentration of 0.05 mmol / L of β-mercaptoethanol.

[0061] The preparation method of the enzymatic hydrolysate is the same as that of Example 1.

[0062] 3. A method for preparing red radish protoplasts using the enzymatic hydrolysate of 2, comprising the following steps: (1) 30 cotyledon petioles of red radish seedlings cultured for 4-6 days were treated with light shielding for 24 h, and then the cotyledon petioles with a length of about 3-5 mm were cut into thin filaments with a width of about 1 mm. The thin filaments were quickly placed in a 50 mL centrifuge tube containing 15 mL of the enzymatic hydrolysate, the centrifuge tube was wrapped with tin foil paper, and the tube was placed in a dark place for light shielding and shaking (28°C, 45 rpm) for 3 h.

[0063] (2) A 70 µm cell filter screen was placed on a new 50 mL sterile centrifuge tube, about 1 mL of W5 solution was added to the cell filter screen, and the cell filter screen was rotated and rinsed. A sterile 1 mL pipette tip with a cut end was used to gently blow the enzymatic hydrolysate obtained in step (1) about 6-8 times to release the protoplasts. Then, the enzymatic hydrolysate was transferred to the rinsed cell filter screen in batches, and the piston of a 5 mL sterile syringe was gently pressed on the unhydrolyzed tissue in the cell filter screen 3-5 times, and then 3 mL of W5 solution was slowly added to rinse the unhydrolyzed tissue, and the rinsing was repeated twice. The filtrate was collected, which was the filtered enzymatic hydrolysate.

[0064] (3) Centrifuge 100 g of the filtrate obtained in step (2) for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of W5 solution along the wall of the centrifuge tube. The resuspended liquid is filtered through a 40 pm cell filter screen, and the residual tissue on the cell screen is washed with 1 mL of W5 solution, and the washing is repeated once (i.e., the residual tissue on the cell screen is washed with 1 mL of W5 solution), and the filtrate is collected. Then, centrifuge 100 g of the filtrate for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution, to obtain the red radish protoplasts.

[0065] (4) The viability of the red radish protoplasts is detected by using FDA (fluorescein diacetate). 100 μL of the protoplast suspension is taken and placed in a 1.5 mL centrifuge tube wrapped with tin foil in advance, 3 μL of 1 mg / mL FDA is added, and the staining is performed in the dark for 5 min. After the staining is completed, 50 μL is taken and placed on a glass slide, and the observation is performed under a dark environment of an inverted fluorescence microscope (Thermo Fisher EVOS M3000). The viable protoplasts emit yellow-green fluorescence, and the number of the yellow-green protoplasts is counted. The ratio of the number of the viable protoplasts to the total number of the protoplasts in the same field of view is the protoplast viability value. The prepared protoplast viability value is 84.77%, as shown in FIG. 2. Figure 2 Figure 2 The right image in FIG. 2 is the FDA staining result, and the left image is the protoplast image under the corresponding bright field, and the size of the scale in the image is 100 pm.

[0066] The protoplast viability is calculated as follows: protoplast viability = (number of protoplasts emitting yellow-green fluorescence / total number of protoplasts) x 100%.

[0067] The protoplast concentration is calculated as follows: the prepared protoplasts are dropped on one end of a hemocytometer cover glass, the liquid drop is allowed to fill the entire cover glass without air bubbles, and the observation and counting are performed after the standing for two minutes. The prepared protoplast density in this embodiment is about 38.18 x 10 5

[0068] The protoplast density (number / mL) = 25 x 10 4 x dilution factor.

[0069] Example 3 1. A complex enzyme for preparing red radish protoplasts, which comprises, by mass fraction, 1.0 parts of cellulase RS, 0.5 parts of isolated enzyme R-10, and 0.5 parts of hemicellulase.

[0070] ​​2. An enzymatic solution containing the complex enzyme of claim 1, consisting of: 1.0 w / v% cellulase RS, 0.5 w / v% isolated enzyme R-10, 0.5 w / v% hemicellulase, mannitol with a final concentration of 0.55 mol / L, MES acid-base buffer with a pH of 5.7, a final concentration of 20 mmol / L of MES, a final concentration of 20 mmol / L of KCl, a final concentration of 10 mmol / L of CaCl2, a final concentration of 0.1% of BSA, and a final concentration of 0.05 mmol / L of β-mercaptoethanol, based on the total volume of the enzymatic solution.

[0071] The preparation method of the enzymatic solution is the same as that of Example 1.

[0072] 3. A method for preparing red radish protoplasts using the enzymatic solution of claim 2, comprising the following steps: (1) 30 cotyledon petioles with a length of about 3-5 mm were obtained from red radish seedlings cultured for 4-6 days, and the seedlings were subjected to light-avoiding treatment for 24 h. The cotyledon petioles were cut into filaments with a width of about 1 mm, and the filaments were quickly placed in a 50 mL centrifuge tube containing 15 mL of the enzymatic solution. The centrifuge tube was wrapped with tin foil and placed in a dark place for enzymatic hydrolysis (28°C, 45 rpm) for 4 h.

[0073] (2) A 70 µm cell filter screen was placed on a new 50 mL sterile centrifuge tube, and about 1 mL of W5 solution was added to rotate and rinse the cell filter screen. The enzymatic hydrolysis product obtained in step (1) was gently blown about 6-8 times with a sterile 1 mL pipette tip with a cut end to fully release the protoplasts, and then the enzymatic hydrolysis product was transferred to the rinsed cell screen in batches. The piston of a 5 mL sterile syringe was gently pressed 3-5 times on the unhydrolyzed tissue in the cell filter screen, and then 3 mL of W5 solution was slowly added to rinse the unhydrolyzed tissue, and the rinsing was repeated twice. The filtrate was collected, which was the filtered enzymatic hydrolysis solution.

[0074] (3) The filtered enzymatic hydrolysis solution obtained in step (2) was centrifuged at 100 g for 7 min, and the supernatant was discarded. The precipitate was resuspended with 3 mL of W5 solution by slowly adding the solution along the wall of the inclined centrifuge tube. The resuspended liquid was filtered through a 40 µm cell filter screen, and the residual tissue on the cell filter screen was washed with 1 mL of W5 solution, and the washing was repeated once (i.e., the residual tissue on the cell filter screen was washed with 1 mL of W5 solution). The filtrate was collected. Then the filtrate was centrifuged at 100 g for 7 min, the supernatant was discarded, and the precipitate was resuspended with 3 mL of MMG solution, thereby obtaining the red radish protoplasts.

[0075] (4) Red radish protoplast viability detection using FDA (fluorescein diacetate). 100 μL of protoplast suspension was taken and placed in a 1.5 mL centrifuge tube wrapped with tin foil in advance, 3 μL of 1 mg / mL FDA was added, and staining was performed in the dark for 5 min. After staining, 50 μL was taken and placed on a glass slide, and observation was performed under a dark environment using an inverted fluorescence microscope (Thermo Fisher EVOS M3000). Viable protoplasts emit yellow-green fluorescence, and 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 was the protoplast viability value. The prepared protoplast viability value was 85.78%.

[0076] Protoplast viability was calculated as follows: protoplast viability = (number of yellow-green fluorescent protoplasts / total number of protoplasts) x 100%.

[0077] Protoplast concentration was calculated as follows: the prepared protoplasts were added to one end of a hemocytometer cover glass, and the liquid drop was allowed to fill the entire cover glass without air bubbles. After standing for two minutes, the number of protoplasts was counted. The prepared protoplast density in this example was about 38.02 x 10 5 protoplasts / mL.

[0078] Protoplast density (protoplasts / mL) = 25 x 10 4 x dilution factor.

[0079] Comparative Example 1 1. A composite enzyme, comprising, by mass parts: 2.0 parts of cellulase R-10 and 0.2 parts of pectinase Y-23.

[0080] 2. An enzyme solution containing the composite enzyme in Example 1, comprising, by volume of the total enzyme solution: 2.0 w / v% of cellulase R-10, 0.2 w / v% of pectinase Y-23, 0.55 mol / L of mannitol, 5.7 of pH of MES acid-base buffer, 20 mmol / L of MES, 20 mmol / L of KCl, 10 mmol / L of CaCl2, 0.1% of BSA, and 0.05 mmol / L of β-mercaptoethanol.

[0081] The preparation method of the enzyme solution is as follows: according to the composition of the enzyme solution, the addition amount of each substance is calculated, then the cellulase R-10, the pectinase Y-23, the mannitol, the MES acid-base buffer and the KCl are mixed, and then the mixture is subjected to water bath at 55°C for 10 min. After the water bath is completed, the mixture is cooled to room temperature, and then the CaCl2, the BSA and the β-mercaptoethanol are added, and then the ultrapure water is added to make up to 15 mL to obtain the enzyme solution.

[0082] After the prepared enzymatic hydrolysate, the prepared enzymatic hydrolysate was filtered with a 0.45 μm filter membrane to remove bacteria, and then poured into a 50 mL centrifuge tube for standby.

[0083] 3. A method for preparing red radish protoplasts using the enzymatic hydrolysate of 2, the steps are as follows: (1) The 4-6 day old red radish seedlings were treated with light for 24 h, and then 30 cotyledon petioles with a length of about 3-5 mm were collected. The cotyledon petioles were cut into thin filaments with a width of 1 mm, and the cut thin filaments (about 1 mm wide) were quickly placed in a 50 mL centrifuge tube containing 15 mL of enzymatic hydrolysate. The centrifuge tube was wrapped with tin foil and placed in a dark place on a flat shaker (28°C, 45 rpm) for enzymatic hydrolysis for 4 h.

[0084] (2) A 70 μm cell filter screen was placed on a new 50 mL sterile centrifuge tube, and about 1 mL of W5 solution was added to rotate and rinse the cell filter screen. The cut end of a sterile 1 mL suction head was used to gently blow the enzymatic hydrolysate obtained in step (1) about 6-8 times to release the protoplasts, and then the enzymatic hydrolysate was transferred to the rinsed cell screen in batches. The piston of a 5 mL sterile syringe was gently pressed on the unhydrolyzed tissue in the cell filter screen 3-5 times, and then 3 mL of W5 solution was slowly added to rinse the unhydrolyzed tissue, and the rinsing was repeated twice. The filtrate was collected, which was the filtered enzymatic hydrolysate.

[0085] (3) The filtered enzymatic hydrolysate obtained in step (2) was centrifuged at 100 g for 7 min, and the supernatant was discarded. The inclined centrifuge tube was slowly added with 3 mL of W5 solution along the tube wall to resuspend the precipitate. The resuspended liquid was filtered through a 40 μm cell filter screen, and the residual tissue on the cell filter screen was washed with 1 mL of W5 solution again. The washing was repeated once (i.e., the residual tissue on the cell filter screen was washed with 1 mL of W5 solution, and the same below), and the filtrate was collected. Then the filtrate was centrifuged at 100 g for 7 min, the supernatant was discarded, and the precipitate was resuspended with 3 mL of MMG solution, and the red radish protoplasts were obtained.

[0086] (4) The viability of the red radish protoplasts was detected by FDA (fluorescein diacetate). 100 μL of the protoplast suspension was taken and placed in a 1.5 mL centrifuge tube wrapped with tin foil, and 3 μL of 1 mg / mL FDA was added. The staining was performed in the dark for 5 min. After the staining was completed, 50 μL was taken and placed on a glass slide, and the yellow-green fluorescent protoplasts were observed under an inverted fluorescence microscope (Thermo Fisher EVOS M3000) in the dark. The number of viable protoplasts was counted. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field was the protoplast viability value. The prepared protoplast viability value was 70.58%, as shown in Figure 3 . Figure 3The middle right picture is the FDA staining result, and the left picture is the protoplast image under the corresponding bright field view. The size of the scale bar in the picture is 100 pm.

[0087] Statistical protoplast viability: Protoplast viability = (number of yellow-green fluorescent protoplasts / total number of protoplasts) x 100%.

[0088] Statistical protoplast concentration: The prepared protoplasts were added to one end of the cover glass of the hemocytometer, and the liquid drop was allowed to fill the entire cover glass without air bubbles. After two minutes of standing, the number of protoplasts was observed and counted. The protoplast density prepared in the present comparative example was about 1.44 x 10 5 protoplasts / mL.

[0089] Protoplast density (protoplasts / mL) = 25 x number of protoplasts in the square grid x 10 4 x dilution factor.

[0090] Comparative Example 2 1. A composite enzyme, consisting of 1.5 parts by mass of cellulase RS and 0.5 parts by mass of isolated enzyme R-10.

[0091] 2. An enzymatic hydrolysate containing the composite enzyme in 1, consisting of 1.5 w / v% of cellulase RS, 0.5 w / v% of isolated enzyme R-10, mannitol at a final concentration of 0.55 mol / L, MES acid-base buffer at a pH of 5.7, MES at a final concentration of 20 mmol / L, KCl at a final concentration of 20 mmol / L, CaCl2 at a final concentration of 10 mmol / L, BSA at a final concentration of 0.1%, and β-mercaptoethanol at a final concentration of 0.05 mmol / L, based on the total volume of the enzymatic hydrolysate.

[0092] The preparation method of the enzymatic hydrolysate is as follows: based on the composition of the enzymatic hydrolysate, the addition amount of each substance is calculated, and then the cellulase RS, isolated enzyme R-10, mannitol, MES acid-base buffer, and KCl are mixed, and then subjected to water bath at 55°C for 10 min. After the water bath is completed, it is cooled to room temperature, and then CaCl2, BSA, and β-mercaptoethanol are added, and then ultrapure water is added to make up to 15 mL to obtain the enzymatic hydrolysate.

[0093] After the prepared enzymatic hydrolysate is obtained, the prepared enzymatic hydrolysate is filtered to remove bacteria using a 0.45 pm filter membrane, and then poured into a 50 mL centrifuge tube for standby.

[0094] 3. The method for preparing red radish protoplasts using the enzymatic hydrolysate in 2, the steps are as follows: (1) The 4-6 day old red radish seedlings were treated with light for 24 h, and then 30 cotyledon petioles with cotyledon petioles of about 3-5 mm long were collected. The cotyledon petioles were cut into 1 mm wide filaments, and the cut filaments were quickly placed in a 50 mL centrifuge tube containing 15 mL enzyme solution. The centrifuge tube was wrapped with tin foil and placed in a dark place on a flat plate shaker (28°C, 45 rpm) for enzyme hydrolysis for 4 h.

[0095] (2) A 70 µm cell filter screen was placed on a new 50 mL sterile centrifuge tube, and about 1 mL of W5 solution was added to rotate and rinse the cell filter screen. The enzyme hydrolysis product obtained in step (1) was gently blown about 6-8 times with a sterile 1 mL pipette with a cut end to release the protoplasts, and then the enzyme hydrolysis product was transferred to the rinsed cell screen in batches. The unhydrolyzed tissue in the cell filter screen was gently pressed 3-5 times with the piston of a 5 mL sterile syringe, and then 3 mL of W5 solution was slowly added to rinse the unhydrolyzed tissue, and the rinsing was repeated twice. The filtrate was collected, which was the filtered enzyme hydrolysis solution.

[0096] (3) The filtered enzyme hydrolysis solution obtained in step (2) was centrifuged at 100 g for 7 min, and the supernatant was discarded. The precipitate was resuspended with 3 mL of W5 solution by slowly adding it along the wall of the tube. The resuspended liquid was filtered through a 40 µm cell filter screen, and the residual tissue on the cell filter screen was washed with 1 mL of W5 solution. The washing was repeated once (i.e., the residual tissue on the cell filter screen was washed with 1 mL of W5 solution, and the same was repeated), and the filtrate was collected. Then the filtrate was centrifuged at 100 g for 7 min, and the supernatant was discarded. The precipitate was resuspended with 3 mL of MMG solution, and red radish protoplasts were obtained.

[0097] (4) The viability of red radish protoplasts was detected using FDA (fluorescein diacetate). 100 µL of the protoplast suspension was taken and placed in a 1.5 mL centrifuge tube wrapped with tin foil. 3 µL of 1 mg / mL FDA was added, and the staining was performed in the dark for 5 min. After staining, 50 µL was taken and placed on a glass slide. The viability of the protoplasts was observed under a fluorescence microscope (Thermo Fisher EVOS M3000) in the dark. Viable protoplasts emitted yellow-green fluorescence, and 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 was the protoplast viability value. The prepared protoplast viability value was 63.08%, as shown in Figure 4 Figure 4 The right panel of the figure is the FDA staining result, and the left panel is the protoplast image under the corresponding bright field. The scale bar in the figure is 100 µm.

[0098] The protoplast viability was calculated as follows: protoplast viability = (number of yellow-green fluorescent protoplasts / total number of protoplasts) x 100%. ​

[0099] Statistical protoplast concentration: prepared protoplast drops were taken and added to one end of a hemocytometer cover glass, the drops were allowed to fill the entire cover glass without air bubbles, and after two minutes of standing, the count was observed. The protoplast density prepared in the present comparative example was about 3.08 x 10 5 individuals / mL.

[0100] Protoplast density (individuals / mL) = 25 x protoplast number in a square grid x 10 4 x dilution factor.

[0101] Comparative Example 3 1. A composite enzyme, consisting of 0.5 parts by mass of dissociative enzyme R-10 and 0.5 parts by mass of hemicellulase.

[0102] 2. An enzymatic hydrolysate containing 1 composite enzyme, consisting of 0.5 w / v% dissociative enzyme R-10, 0.5 w / v% hemicellulase, 0.55 mol / L final concentration of mannitol, 5.7 pH of MES acid-base buffer, 20 mmol / L final concentration of MES, 20 mmol / L final concentration of KCl, 10 mmol / L final concentration of CaCl2, 0.1% final concentration of BSA, and 0.05 mmol / L final concentration of β-mercaptoethanol, based on the total volume of the enzymatic hydrolysate.

[0103] The preparation method of the enzymatic hydrolysate is as follows: based on the composition of the enzymatic hydrolysate, the addition amount of each substance is calculated, then the dissociative enzyme R-10, hemicellulase, mannitol, MES acid-base buffer, and KCl are mixed, and then water bathed at 55°C for 10 min. After the water bath is completed, it is cooled to room temperature, CaCl2, BSA, and β-mercaptoethanol are added, and then ultrapure water is added to make up to 15 mL to obtain the enzymatic hydrolysate.

[0104] After the enzymatic hydrolysate is prepared, the prepared enzymatic hydrolysate is filtered to remove bacteria using a 0.45 μm filter membrane, and then poured into a 50 mL centrifuge tube for standby.

[0105] 3. A method for preparing red radish protoplasts using 2 enzymatic hydrolysate, the steps are as follows: (1) 30 cotyledon petioles of 4-6 day old red radish seedlings were treated with light for 24 h, and then the cotyledon petioles were cut into 1 mm wide filaments. The cut filaments were quickly placed in a 50 mL centrifuge tube containing 15 mL of enzymatic hydrolysate, the centrifuge tube was wrapped with tin foil paper, and then placed in a dark place to avoid light and shaken (28°C, 45 rpm) for 4 h.

[0106] (2) Put a 70-µm cell strainer on a new 50-mL sterile centrifuge tube, and add about 1 mL of W5 solution to spin-rinse the cell strainer. Gently pipette the enzymatic solution obtained in step (1) about 6-8 times with a sterile 1-mL pipette tip cut at the tip to release the protoplasts, and then transfer the enzymatic solution into the rinsed cell strainer in batches. Gently press the un-enzymatic tissue in the cell strainer with the plunger of a 5-mL sterile syringe for 3-5 times, and then slowly add 3 mL of W5 solution to rinse the un-enzymatic tissue, repeat the rinsing for 2 more times, and collect the filtrate, which is the filtered enzymatic solution.

[0107] (3) Centrifuge 100 g of the filtered enzymatic solution obtained in step (2) for 7 min, discard the supernatant, and slowly add 3 mL of W5 solution along the wall of the centrifuge tube to resuspend the precipitate. Filter the resuspended liquid through a 40-µm cell strainer, and then wash the residual tissue on the cell strainer with 1 mL of W5 solution, repeat the washing once (i.e., wash the residual tissue on the cell strainer with 1 mL of W5 solution, and the same applies hereinafter), and collect the filtrate. Then centrifuge 100 g of the filtrate for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain the red radish protoplasts.

[0108] (4) Use FDA (fluorescein diacetate) to detect the viability of the red radish protoplasts. Take 100 µL of the protoplast suspension and place it in a 1.5-mL centrifuge tube wrapped with tin foil in advance, add 3 µL of 1 mg / mL FDA, and stain for 5 min in the dark. After the staining is completed, take out 50 µL and place it on a glass slide, and observe it under an inverted fluorescence microscope (Thermo Fisher EVOS M3000) in a dark environment. The viable protoplasts will emit yellow-green fluorescence, and the number of yellow-green protoplasts is 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 is 60.50%.

[0109] Count the protoplast viability: Protoplast viability = (number of yellow-green fluorescent protoplasts / total number of protoplasts) × 100%.

[0110] Count the protoplast concentration: Take the prepared protoplasts and add them to one end of the cover glass of a hemocytometer, and wait for the liquid drop to fill the entire cover glass without air bubbles. After standing for two minutes, count and observe. The protoplast density prepared in this comparative example is about 0.48 × 10 5 individuals / mL.

[0111] Protoplast density (individuals / mL) = 25 individuals in a square cell × 10 4 × dilution factor.

[0112] Comparative Example 4 1. A composite enzyme, consisting of 1.5 parts of cellulase RS and 0.5 parts of hemicellulase by mass.

[0113] 2. An enzymatic hydrolysate containing the composite enzyme in claim 1, consisting of 1.5 w / v% of cellulase RS, 0.5 w / v% of hemicellulase, mannitol at a final concentration of 0.55 mol / L, MES acid-base buffer at a pH of 5.7, MES at a final concentration of 20 mmol / L, KCl at a final concentration of 20 mmol / L, CaCl2 at a final concentration of 10 mmol / L, BSA at a final concentration of 0.1%, and β-mercaptoethanol at a final concentration of 0.05 mmol / L, based on the total volume of the enzymatic hydrolysate.

[0114] The preparation method of the enzymatic hydrolysate is as follows: according to the composition of the enzymatic hydrolysate, the addition amount of each substance is calculated, then the isolated enzyme R-10, hemicellulase, mannitol, MES acid-base buffer and KCl are mixed, and then water bathed at 55°C for 10 min. After the water bath is completed, it is cooled to room temperature, CaCl2, BSA and β-mercaptoethanol are added, and then ultrapure water is added to make up to 15 mL to obtain the enzymatic hydrolysate.

[0115] After the prepared enzymatic hydrolysate is obtained, the prepared enzymatic hydrolysate is filtered to remove bacteria using a 0.45 μm filter membrane, and then poured into a 50 mL centrifuge tube for standby.

[0116] 3. A method for preparing red radish protoplasts using the enzymatic hydrolysate in claim 2, the steps being as follows: (1) 30 cotyledon petioles with a length of about 3-5 mm are taken from red radish seedlings cultured for 4-6 days after 24 h of light avoidance treatment. The cotyledon petioles are cut into thin filaments with a width of about 1 mm, and the cut thin filaments are quickly placed in a 50 mL centrifuge tube containing 15 mL of the enzymatic hydrolysate, the centrifuge tube is wrapped with tin foil paper, and the centrifuge tube is placed in a dark place for light avoidance and oscillation (28°C, 45 rpm) for 4 h.

[0117] (2) A 70 μm cell filter screen is placed on a new 50 mL sterile centrifuge tube, about 1 mL of W5 solution is added to rotate and rinse the cell filter screen. A sterile 1 mL pipette tip with a cut end is used to gently blow the enzymatic hydrolysate obtained in step (1) about 6-8 times to sufficiently release the protoplasts, and then the enzymatic hydrolysate is transferred to the rinsed cell filter screen in batches, and the piston of a 5 mL sterile syringe is gently pressed on the unhydrolyzed tissue in the cell filter screen for 3-5 times, and then 3 mL of W5 solution is slowly added to rinse the unhydrolyzed tissue, and the rinsing is repeated twice, and the filtrate is collected, which is the filtered enzymatic hydrolysate.

[0118] (3) Centrifuge 100 g of the filtrated enzymatic hydrolysate obtained in step (2) for 7 min, discard the supernatant after centrifugation, resuspend the precipitate by slowly adding 3 mL of W5 solution along the wall of the centrifuge tube, filter the resuspended liquid through a 40 pm cell filter screen, continue to wash the residual tissue on the cell screen with 1 mL of W5 solution, repeat once more (i.e., wash the residual tissue on the cell screen with 1 mL of W5 solution, and so on), and collect the filtrate. Then centrifuge 100 g of the filtrate for 7 min, discard the supernatant, resuspend the precipitate with 3 mL of MMG solution, and obtain the red radish protoplasts.

[0119] (4) Use FDA (fluorescein diacetate) to detect the viability of the red radish protoplasts. Take 100 μL of the protoplast suspension and place it in a 1.5 mL centrifuge tube wrapped with tin foil in advance, add 3 μL of 1 mg / mL FDA, and stain for 5 min in the dark. After staining, take out 50 μL and place it on a glass slide, and observe it under an inverted fluorescence microscope (Thermo Fisher EVOS M3000) in a dark environment. Viable protoplasts will emit yellow-green fluorescence, and the number of yellow-green protoplasts is counted. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field is the protoplast viability value. The prepared protoplast viability value is 66.05%.

[0120] Count the protoplast viability: Protoplast viability = (number of yellow-green fluorescent protoplasts / total number of protoplasts) x 100%.

[0121] Count the protoplast concentration: Take the prepared protoplasts and add them to one end of the cover glass of a hemocytometer, wait for the liquid drop to fill the entire cover glass without air bubbles, and observe and count after standing for two minutes. The protoplast density prepared in this comparative example is about 0.64 x 10 5 individuals / mL.

[0122] Protoplast density (individuals / mL) = 25 individuals in a square cell x 10 4 x dilution factor.

[0123] Comparative Example 5 1. A composite enzyme, consisting of, in parts by mass, cellulase RS 2.0 parts, isolated enzyme R-10 1.0 part, and pectinase Y-23 0.1 part.

[0124] 2. The enzymatic hydrolysate containing the complex enzyme in 1, consisting of 2.0 w / v% cellulase RS, 1.0 w / v% isolated enzyme R-10, 0.1 w / v% pectinase Y-23, mannitol with a final concentration of 0.55 mol / L, MES acid-base buffer with a pH of 5.7, a final concentration of 20 mmol / L of MES, a final concentration of 20 mmol / L of KCl, a final concentration of 10 mmol / L of CaCl2, a final concentration of 0.1% of BSA, and a final concentration of 0.05 mmol / L of β-mercaptoethanol, based on the total volume of the enzymatic hydrolysate.

[0125] The preparation method of the enzymatic hydrolysate is as follows: according to the composition of the enzymatic hydrolysate, the addition amount of each substance is calculated, then the cellulase RS, isolated enzyme R-10, pectinase Y-23, mannitol, MES acid-base buffer and KCl are mixed, and then water bathed at 55°C for 10 min. After the water bath is completed, it is cooled to room temperature, CaCl2, BSA and β-mercaptoethanol are added, and then ultrapure water is added to make up to 15 mL to obtain the enzymatic hydrolysate.

[0126] After the enzymatic hydrolysate is prepared, the prepared enzymatic hydrolysate is filtered to remove bacteria using a 0.45 μm filter membrane, and then poured into a 50 mL centrifuge tube for standby use.

[0127] 3. The method for preparing red radish protoplasts using the enzymatic hydrolysate in 2, the steps are as follows: (1) 30 cotyledon petiole leaves of 4-6 day-old red radish seedlings are taken after 24 h of light avoidance treatment, and the cotyledon petiole length is about 3-5 mm. The cotyledon petiole leaves are cut into thin filaments with a width of about 1 mm, and the cut thin filaments (about 1 mm wide) are quickly placed in a 50 mL centrifuge tube containing 15 mL of the enzymatic hydrolysate, the centrifuge tube is wrapped with tin foil paper and placed in a dark place for light avoidance and shaking (28°C, 45 rpm) for 4 h.

[0128] (2) A 70 μm cell filter screen is placed on a new 50 mL sterile centrifuge tube, about 1 mL of W5 solution is added to rotate and rinse the cell filter screen. The cut end of a sterile 1 mL suction head is used to gently blow the enzymatic hydrolysate obtained in step (1) about 6-8 times to fully release the protoplasts, and then the enzymatic hydrolysate is transferred to the rinsed cell filter screen in batches, and the piston of a 5 mL sterile syringe is gently pressed on the unhydrolyzed tissue in the cell filter screen for 3-5 times, and then 3 mL of W5 solution is slowly added to rinse the unhydrolyzed tissue, and the rinsing is repeated twice, and the filtrate is collected, which is the filtered enzymatic hydrolysate.

[0129] (3) Centrifuge 100 g of the filtrated enzymatic hydrolysate obtained in step (2) for 7 min, discard the supernatant after centrifugation, and resuspend the precipitate with 3 mL of W5 solution along the wall of the inclined centrifuge tube. The resuspended liquid is filtered through a 40 pm cell filter screen, and the residual tissue on the cell screen is washed with 1 mL of W5 solution, and the process is repeated once (i.e., the residual tissue on the cell screen is washed with 1 mL of W5 solution, and the filtrate is collected. Then, 100 g of the filtrate is centrifuged for 7 min, the supernatant is discarded, and the precipitate is resuspended with 3 mL of MMG solution, thereby obtaining red radish protoplasts.

[0130] (4) The viability of the red radish protoplasts is detected using FDA (fluorescein diacetate). 100 μL of the protoplast suspension is taken and placed in a 1.5 mL centrifuge tube wrapped with tin foil in advance, 3 μL of 1 mg / mL FDA is added, and the staining is performed in the dark for 5 min. After the staining is completed, 50 μL is taken and placed on a glass slide, and observed under an inverted fluorescence microscope (Thermo Fisher EVOS M3000) in a dark environment. The protoplasts that are alive will emit yellow-green fluorescence, and the number of yellow-green protoplasts is 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 is 80.95%.

[0131] The protoplast viability is calculated as follows: protoplast viability = (number of yellow-green fluorescent protoplasts / total number of protoplasts) x 100%.

[0132] The protoplast concentration is calculated as follows: the prepared protoplasts are dropped onto one end of the cover glass of a hemocytometer, and the liquid drop is allowed to fill the entire cover glass without air bubbles, and then observed and counted after standing for two minutes. The protoplast density prepared in the present comparative example is about 13.08 x 10 5 individuals / mL.

[0133] The protoplast density (individuals / mL) = 25 x 10 4 x dilution factor.

[0134] Comparative Example 6 1. A composite enzyme, which comprises, by mass parts: cellulase R-10 1.0 part, isolated enzyme R-10 0.5 part, and hemicellulase 0.5 part.

[0135] 2. The enzymatic solution containing the complex enzyme in claim 1, which comprises 1.0 w / v% cellulase R-10, 0.5 w / v% isolated enzyme R-10, 0.5 w / v% hemicellulase, mannitol with a final concentration of 0.55 mol / L, MES acid-base buffer with a pH of 5.7, a final concentration of 20 mmol / L of MES, a final concentration of 20 mmol / L of KCl, a final concentration of 10 mmol / L of CaCl2, a final concentration of 0.1% of BSA, and a final concentration of 0.05 mmol / L of β-mercaptoethanol.

[0136] The preparation method of the enzymatic solution is as follows: according to the composition of the enzymatic solution, the addition amount of each substance is calculated, then the isolated enzyme R-10, the isolated enzyme R-10, the hemicellulase, the mannitol, the MES acid-base buffer and the KCl are mixed, and then the mixture is subjected to water bath at 55°C for 10 min. After the water bath is completed, the mixture is cooled to room temperature, the CaCl2, the BSA and the β-mercaptoethanol are added, and then ultrapure water is added to make up to 15 mL to obtain the enzymatic solution.

[0137] After the enzymatic solution is prepared, the prepared enzymatic solution is filtered to remove bacteria by using a 0.45 μm filter membrane, and then the filtered enzymatic solution is poured into a 50 mL centrifuge tube for standby use.

[0138] 3. The method for preparing red radish protoplasts by using the enzymatic solution in claim 2, which comprises the following steps: (1) 30 cotyledon petioles with a length of about 3-5 mm are taken from red radish seedlings cultured for 4-6 days after 24 h of light shielding treatment. The cotyledon petioles are cut into thin filaments with a width of about 1 mm, and then the cut thin filaments are quickly placed in a 50 mL centrifuge tube containing 15 mL of the enzymatic solution. The centrifuge tube is wrapped with tin foil paper and placed in a dark place for vibration (28°C, 45 rpm) for enzymolysis for 4 h.

[0139] (2) A 70 μm cell filter screen is placed on a new 50 mL sterile centrifuge tube, about 1 mL of W5 solution is added to rotate and rinse the cell filter screen. The enzymolysis product obtained in step (1) is gently blown about 6-8 times by using a sterile 1 mL pipette tip with a cut end to sufficiently release the protoplasts, and then the enzymolysis product is transferred to the rinsed cell filter screen in batches for filtration. The unenzymolyzed tissue in the cell filter screen is gently pressed 3-5 times by using the piston of a 5 mL sterile syringe, and then 3 mL of W5 solution is slowly added to rinse the unenzymolyzed tissue, and the rinsing is repeated twice. The filtrate is collected, which is the filtered enzymatic solution.

[0140] (3) Centrifuge 100 g of the filtrated enzymatic hydrolysate obtained in step (2) for 7 min, discard the supernatant after centrifugation, resuspend the precipitate by slowly adding 3 mL of W5 solution along the wall of the centrifuge tube, filter the resuspended liquid through a 40 pm cell filter screen, continue to wash the residual tissue on the cell screen with 1 mL of W5 solution, repeat once again (i.e., wash the residual tissue on the cell screen with 1 mL of W5 solution), and collect the filtrate. Then centrifuge 100 g of the filtrate for 7 min, discard the supernatant, resuspend the precipitate with 3 mL of MMG solution, and obtain the red radish protoplasts.

[0141] (4) Use FDA (fluorescein diacetate) to detect the viability of red radish protoplasts. Take 100 μL of the protoplast suspension and place it in a 1.5 mL centrifuge tube wrapped in tin foil in advance, add 3 μL of 1 mg / mL FDA, and stain for 5 min in the dark. After staining, 50 μL is taken out and placed on a glass slide, and observed under an inverted fluorescence microscope (Thermo Fisher EVOS M3000) in a dark environment. Viable protoplasts will emit yellow-green fluorescence, and the number of yellow-green protoplasts is counted. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field is the protoplast viability value. The prepared protoplast viability value is 82.32%.

[0142] Count the protoplast viability: Protoplast viability = (number of yellow-green fluorescent protoplasts / total number of protoplasts) x 100%.

[0143] Count the protoplast concentration: Take the prepared protoplasts and add them to one end of the cover glass of a hemocytometer, wait for the liquid drop to fill the entire cover glass without air bubbles, and observe and count after standing for two minutes. The protoplast density prepared in this comparative example is about 17.54 x 10 5 individuals / mL.

[0144] Protoplast density (individuals / mL) = number of protoplasts in 25 squares x 10 4 x dilution factor.

[0145] Comparative Example 7 1. A composite enzyme, consisting of, in parts by mass, cellulase RS 0.5 parts, isolated enzyme R-10 0.5 parts, and hemicellulase 0.3 parts.

[0146] 2. An enzymatic hydrolysate containing the complex enzyme of claim 1, consisting of 0.5 w / v% cellulase RS, 0.5 w / v% isolated enzyme R-10, 0.3 w / v% hemicellulase, mannitol with a final concentration of 0.55 mol / L, MES acid-base buffer with a pH of 5.7, a final concentration of 20 mmol / L of MES, a final concentration of 20 mmol / L of KCl, a final concentration of 10 mmol / L of CaCl2, a final concentration of 0.1% of BSA, and a final concentration of 0.05 mmol / L of β-mercaptoethanol, based on the total volume of the enzymatic hydrolysate.

[0147] The preparation method of the enzymatic hydrolysate is as follows: according to the composition of the enzymatic hydrolysate, the addition amount of each substance is calculated, then the cellulase RS, isolated enzyme R-10, hemicellulase, mannitol, MES acid-base buffer and KCl are mixed, and then water bathed at 55°C for 10 min. After the water bath is completed, it is cooled to room temperature, CaCl2, BSA and β-mercaptoethanol are added, and then ultrapure water is added to make up to 15 mL to obtain the enzymatic hydrolysate.

[0148] After the enzymatic hydrolysate is prepared, the prepared enzymatic hydrolysate is filtered with a 0.45 μm filter membrane to remove bacteria, and then poured into a 50 mL centrifuge tube for standby use.

[0149] 3. A method for preparing red radish protoplasts using the enzymatic hydrolysate of claim 2, the steps being as follows: (1) 30 cotyledon petiole pieces with a length of about 3-5 mm are taken from red radish seedlings cultured for 4-6 days after 24 h of light avoidance treatment. The cotyledon petiole pieces are cut into thin filaments with a width of about 1 mm, and the cut thin filaments are quickly placed in a 50 mL centrifuge tube containing 15 mL of the enzymatic hydrolysate, the centrifuge tube is wrapped with tin foil paper, and the centrifuge tube is placed in a dark place for light avoidance and shaking (28°C, 45 rpm) for 4 h.

[0150] (2) A 70 μm cell filter screen is placed on a new 50 mL sterile centrifuge tube, about 1 mL of W5 solution is added to rotate and rinse the cell filter screen. The enzymatic hydrolysate obtained in step (1) is gently blown about 6-8 times with a sterile 1 mL pipette tip with a cut end to fully release the protoplasts, and then the enzymatic hydrolysate is transferred to the rinsed cell filter screen in batches, the piston of a 5 mL sterile syringe is gently pressed on the unhydrolyzed tissue in the cell filter screen for 3-5 times, then 3 mL of W5 solution is slowly added to rinse the unhydrolyzed tissue, and the rinsing is repeated twice, and the filtrate is collected, which is the filtered enzymatic hydrolysate.

[0151] (3) Centrifuge 100 g of the filtrated enzymatic hydrolysate obtained in step (2) for 7 min, discard the supernatant after centrifugation, resuspend the precipitate by slowly adding 3 mL of W5 solution along the wall of the centrifuge tube, filter the resuspended liquid through a 40 pm cell filter screen, continue to wash the residual tissue on the cell screen with 1 mL of W5 solution, repeat once more (i.e., wash the residual tissue on the cell screen with 1 mL of W5 solution, and so on), and collect the filtrate. Then centrifuge 100 g of the filtrate for 7 min, discard the supernatant, resuspend the precipitate with 3 mL of MMG solution, and obtain the red radish protoplasts.

[0152] (4) Use FDA (fluorescein diacetate) to detect the viability of the red radish protoplasts. Take 100 μL of the protoplast suspension and place it in a 1.5 mL centrifuge tube wrapped with tin foil in advance, add 3 μL of 1 mg / mL FDA, and stain for 5 min in the dark. After staining, take out 50 μL and place it on a glass slide, and observe it under an inverted fluorescence microscope (Thermo Fisher EVOS M3000) in a dark environment. Viable protoplasts will emit yellow-green fluorescence, and the number of yellow-green protoplasts is counted. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field is the protoplast viability value. The prepared protoplast viability value is 80.36%.

[0153] Count the protoplast viability: Protoplast viability = (number of yellow-green fluorescent protoplasts / total number of protoplasts) x 100%.

[0154] Count the protoplast concentration: Take the prepared protoplasts and add them to one end of the cover glass of a hemocytometer, wait for the liquid drop to fill the entire cover glass without air bubbles, and observe and count after standing for two minutes. The protoplast density prepared in this comparative example is about 10.42 x 10 5 individuals / mL.

[0155] Protoplast density (individuals / mL) = 25 x 10 4 x dilution factor.

[0156] Comparative Example 8 1. A composite enzyme, consisting of, in parts by mass: cellulase RS 1.0 part, isolated enzyme R-10 1.0 part, and hemicellulase 0.3 part.

[0157] 2. The enzymatic hydrolysate containing the complex enzyme in 1, which is composed of 1.0 w / v% cellulase RS, 1.0 w / v% isolated enzyme R-10, 0.3 w / v% hemicellulase, mannitol with a final concentration of 0.55 mol / L, MES acid-base buffer with a pH of 5.7, a final concentration of 20 mmol / L of MES, a final concentration of 20 mmol / L of KCl, a final concentration of 10 mmol / L of CaCl2, a final concentration of 0.1% of BSA, and a final concentration of 0.05 mmol / L of β-mercaptoethanol, based on the total volume of the enzymatic hydrolysate.

[0158] The preparation method of the enzymatic hydrolysate is as follows: according to the composition of the enzymatic hydrolysate, the addition amount of each substance is calculated, then the cellulase RS, isolated enzyme R-10, hemicellulase, mannitol, MES acid-base buffer and KCl are mixed, and then water bathed at 55°C for 10 min. After the water bath is completed, it is cooled to room temperature, CaCl2, BSA and β-mercaptoethanol are added, and then ultrapure water is added to make up to 15 mL to obtain the enzymatic hydrolysate.

[0159] After the prepared enzymatic hydrolysate is obtained, the prepared enzymatic hydrolysate is filtered to remove bacteria using a 0.45 μm filter membrane, and then poured into a 50 mL centrifuge tube for standby use.

[0160] 3. The method for preparing red radish protoplasts using the enzymatic hydrolysate in 2, the steps are as follows: (1) 30 cotyledon petioles with a length of about 3-5 mm are taken from red radish seedlings cultured for 4-6 days after 24 h of light shielding treatment. The cotyledon petioles are cut into thin filaments with a width of 1 mm, and the cut thin filaments (about 1 mm wide) are quickly placed in a 50 mL centrifuge tube containing 15 mL of the enzymatic hydrolysate, the centrifuge tube is wrapped with tin foil paper and placed in a dark place for light shielding and shaking (28°C, 45 rpm) for 4 h.

[0161] (2) A 70 μm cell filter screen is placed on a new 50 mL sterile centrifuge tube, about 1 mL of W5 solution is added to rotate and rinse the cell filter screen. The cut end of a sterile 1 mL suction head is used to gently blow the enzymatic hydrolysate obtained in step (1) about 6-8 times to fully release the protoplasts, and then the enzymatic hydrolysate is transferred to the rinsed cell filter screen in batches, the piston of a 5 mL sterile syringe is used to gently press the unhydrolyzed tissue in the cell filter screen 3-5 times, then 3 mL of W5 solution is slowly added to rinse the unhydrolyzed tissue, and the rinsing is repeated twice, and the filtrate is collected, which is the filtered enzymatic hydrolysate.

[0162] (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.

[0163] (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%.

[0164] Protoplast viability: Protoplast viability = (Number of protoplasts emitting yellow-green fluorescence / Total number of protoplasts) × 100%.

[0165] 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.

[0166] Protoplast density (units / mL) = Number of protoplasts in 25 square cells × 10 4 × Dilution factor.

[0167] Comparative Example 9 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 3. The method for preparing carrot protoplasts using the two enzymatic hydrolysates is as follows: (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.

[0172] (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.

[0173] (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.

[0174] (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%.

[0175] Protoplast viability: Protoplast viability = (Number of protoplasts emitting yellow-green fluorescence / Total number of protoplasts) × 100%.

[0176] 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.

[0177] Protoplast density (units / mL) = Number of protoplasts in 25 square cells × 10 4 × Dilution factor.

[0178] Comparative Example 10 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.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 3. The method for preparing carrot protoplasts using the two enzymatic hydrolysates is as follows: (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.

[0183] (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.

[0184] (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.

[0185] (4) Protoplast viability detection using FDA (fluorescein diacetate). 100 μL of protoplast suspension was placed in a 1.5 mL centrifuge tube pre-wrapped in aluminum foil. 3 μL of 1 mg / mL FDA was added, and 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%.

[0186] Protoplast viability: Protoplast viability = (Number of protoplasts emitting yellow-green fluorescence / Total number of protoplasts) × 100%.

[0187] 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.

[0188] Protoplast density (units / mL) = Number of protoplasts in 25 square cells × 10 4 × Dilution factor.

[0189] Comparative Example 11 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 3. The method for preparing carrot protoplasts using the two enzymatic hydrolysates is as follows: (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.

[0194] (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.

[0195] (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.

[0196] (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%.

[0197] Protoplast viability: Protoplast viability = (Number of protoplasts emitting yellow-green fluorescence / Total number of protoplasts) × 100%.

[0198] 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.

[0199] Protoplast density (units / mL) = Number of protoplasts in 25 square cells × 10 4 × Dilution factor.

[0200] Comparative Example 12 1. A complex enzyme, by mass parts, comprising: 1.0 part of cellulase RS, 0.5 part of analyte R-10 and 0.3 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, 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.

[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: (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.

[0205] (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.

[0206] (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.

[0207] (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%.

[0208] Protoplast viability: Protoplast viability = (Number of protoplasts emitting yellow-green fluorescence / Total number of protoplasts) × 100%.

[0209] 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.

[0210] Protoplast density (units / mL) = Number of protoplasts in 25 square cells × 10 4 × Dilution factor.

[0211] The yield and viability of carrot protoplasts in Examples 1-3 and Comparative Examples 1-12 are shown in Table 1.

[0212] Table 1. Yield and viability of carrot protoplasts in Examples 1-3 and Comparative Examples 1-12

[0213] Note: Different letters in the same column in the table indicate significant differences at the P < 0.05 level.

[0214] 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 a complex enzyme in the preparation of carrot protoplasts, wherein the complex enzyme comprises, by weight: Cellulase 1.0-1.5 parts, dissociation enzyme 0.3-0.5 parts and hemicellulase 0.3-0.5 parts.

2. The application according to claim 1, characterized in that, The cellulase includes cellulase RS; the analyte includes analyte R-10.

3. The application according to claim 1, characterized in that, By weight, the complex enzyme comprises: 1.0 part cellulase, 0.5 part dissociation enzyme, and 0.3 part hemicellulase; Alternatively, the complex enzyme comprises: 1.5 parts cellulase, 0.3 parts ionase, and 0.5 parts hemicellulase; Alternatively, the complex enzyme comprises: 1.0 part cellulase, 0.5 part dissociation enzyme, and 0.5 part hemicellulase.

4. An enzymatic hydrolysate for preparing carrot protoplasts, characterized in that, Based on the total volume of the enzymatic hydrolysate, it includes: 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.

5. The use of the enzymatic hydrolysate of claim 4 in any one or more of the following: preparing carrot protoplasts, improving protoplast activity, and improving protoplast yield.

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

7. The method according to claim 6, characterized in that, The enzymatic hydrolysis temperature is 28~30℃; the enzymatic hydrolysis time is 2.5~4h.

8. The method according to claim 6, characterized in that, The carrot plant tissue includes carrot seedling plant tissue; the carrot seedling plant tissue includes any one or more of the following: leaves, cotyledons, and petiolate cotyledons.

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

10. The method according to claim 9, 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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