Enzyme hydrolysate for preparing turnip protoplasts and preparation method and application of turnip protoplasts

Abstract

The present invention provides an enzymatic hydrolyzate for preparing turnip protoplasts, a method for preparing turnip protoplasts, and applications thereof, belonging to the technical field of protoplast preparation. The enzymatic hydrolyzate for preparing turnip protoplasts and the method for preparing turnip protoplasts provided by the present invention can efficiently degrade turnip tissue cell walls, thereby maximizing the release of turnip protoplasts, thereby facilitating an increase in protoplast yield and maintaining high protoplast activity. The present invention demonstrates, through examples, that using the enzymatic hydrolyzate with turnip petiole cotyledons as a raw material, can efficiently prepare turnip protoplasts, resulting in a high yield and high activity of the resulting protoplasts.

Description

Technical Field

[0001] The invention belongs to the technical field of protoplast preparation, and in particular relates to an enzymatic hydrolysate for preparing turnip protoplasts and a preparation method and application of the turnip protoplasts. Background Art

[0002] Turnip ( Brassica rapa Rapa var. rapa is a biennial plant of the genus Brassica in the family Cruciferae. It is also known as turnip, dish, and round root. Its taproot is fleshy, thick and round at the top and slender at the bottom. Turnip is a traditional cultivated plant used as medicine, food, and feed. Its fleshy roots are dense, crisp, and sweet. They contain a large amount of vitamins, various minerals, and amino acids. It is warm in nature and has many benefits, including detoxification, nourishment, oxygenation, immunity, anti-mutation, anti-radiation, and fatigue resistance. Therefore, it has high application value. Currently, the breeding of new turnip varieties mainly relies on traditional breeding methods such as natural selection and hybridization, which are time-consuming and inefficient. Leveraging existing biotechnology to accelerate the creation of germplasm resources can provide more research avenues for the breeding of turnip.

[0003] Plant protoplasts are plant cells with their cell walls removed. They possess a certain reproductive capacity and can develop into complete plants under appropriate culture conditions. Because protoplasts lack cell walls, they are relatively easy to overcome the incompatibility barrier of distant hybridization and to take up exogenous macromolecules such as organelles and proteins. They are widely used in subcellular localization, gene expression analysis, protein-protein interactions, gene editing, and are considered ideal materials for developmental biology, cell biology, and cytogenetics research. Currently, there is very little research on the preparation of turnip protoplasts. Establishing and optimizing a preparation system for turnip protoplasts can provide a methodological basis for achieving cell fusion of turnip protoplasts from different varieties and the efficient introduction and expression of exogenous genes. Summary of the Invention

[0004] The object of the present invention is to provide an application of an enzymatic hydrolysis solution in preparing turnip protoplasts. The enzymatic hydrolysis solution can efficiently hydrolyze turnip plant tissues to achieve turnip protoplast preparation, and the prepared protoplasts have high yield and activity.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] The present invention provides an application of an enzymatic hydrolysis solution in preparing turnip protoplasts. The enzymatic hydrolysis solution comprises cellulase, macerate and pectinase; and the mass ratio of the cellulase, macerate and pectinase is (1.5-2.5): (0.8-1.0): (0.1-0.2).

[0007] The invention provides an enzymatic hydrolysis solution for preparing turnip protoplasts. The enzymatic hydrolysis solution comprises, based on the total volume of the enzymatic hydrolysis solution, 1.5-2.5 wt.% of cellulase, 0.8-1.0 wt.% of macerate, 0.1-0.2 wt.% of pectinase, 0.55-0.6 mol / L of mannitol, 19-21 mmol / L of MES, 19-21 mmol / L of KCl, 9-11 mmol / L of CaCl2, 0.09-0.11 wt.% of BSA and 0.04-0.06 mmol / L of beta-mercaptoethanol.

[0008] The present invention provides a method for preparing the enzymatic hydrolysate described in the above technical solution, comprising the following steps:

[0009] The cellulase, macerate, pectinase, mannitol, MES and KCl are mixed and heated to obtain a mixed enzymatic hydrolysate;

[0010] The mixed enzymatic hydrolysate is cooled and then mixed with CaCl2, BSA and β-mercaptoethanol and the volume is fixed to obtain an enzymatic hydrolysate.

[0011] Preferably, the heating temperature is 54-56° C.; and the heating time is 9-12 minutes.

[0012] The present invention provides a method for preparing turnip protoplasts, comprising the following steps:

[0013] The enzymatic hydrolysis solution described in the above technical solution or the enzymatic hydrolysis solution prepared by the preparation method described in the above technical solution is used to enzymatically hydrolyze the turnip plant tissue to obtain turnip protoplasts.

[0014] Preferably, the enzymatic hydrolysis temperature is 28-30° C.; the enzymatic hydrolysis time is 3-4 h; the enzymatic hydrolysis process is accompanied by oscillation; and the oscillation speed is 45-50 rpm.

[0015] Preferably, the plant tissue of turnip includes leaves and / or cotyledons of turnip; and the method for preparing the cotyledons of turnip includes:

[0016] The turnip seeds were tissue cultured for 4-6 days to obtain turnip seedlings;

[0017] After protecting the turnip seedlings from light, take the cotyledons of the turnip.

[0018] Preferably, during enzymatic hydrolysis, the volume ratio of the number of cotyledons to the enzymatic hydrolysis solution is (20-30) pieces: 15 mL; the cotyledons are cut into filaments with a width of 0.5-1 mm for enzymatic hydrolysis.

[0019] Preferably, after the enzymatic hydrolysis is completed, the enzymatic hydrolysis product is further washed and resuspended; the washing solution includes W5 solution; and the resuspending solution includes MMG solution.

[0020] The present invention provides the application of the turnip protoplasts obtained by the preparation method described in the above technical solution in turnip breeding and / or bioinformatics.

[0021] Beneficial effects of the present invention:

[0022] The present invention provides an enzymatic hydrolysis solution for use in preparing turnip protoplasts. The enzymatic hydrolysis solution comprises cellulase, macerate, and pectinase; the mass ratio of the cellulase, macerate, and pectinase is (1.5-2.5): (0.8-1.0): (0.1-0.2). In the enzymatic hydrolysis solution provided by the present invention, the cellulase has the function of degrading cellulose in plant cell walls, the macerate is used in combination with the cellulase to separate plant tissue into single cells, and the pectinase mainly functions to degrade pectin in plant cell walls and promote cell separation. Through the combined action of the three enzymes, the enzymatic hydrolysis solution can synergistically promote the lysis of turnip plant tissue cell walls, thereby maximizing the release of turnip protoplasts, which is beneficial for increasing the protoplast yield while also maintaining a high level of protoplast viability.

[0023] Furthermore, the present invention also provides an enzymatic hydrolysis solution for preparing turnip protoplasts, comprising: 1.5-2.5 wt.% cellulase, 0.8-1.0 wt.% macerate, 0.1-0.2 wt.% pectinase, 0.55-0.6 mol / L mannitol, 19-21 mmol / L MES, 19-21 mmol / L KCl, 9-11 mmol / L CaCl2, 0.09-0.11 wt.% BSA, and 0.04-0.06 mmol / L β-mercaptoethanol. The enzymatic hydrolysis solution provided by the present invention, in addition to comprising cellulase, pectinase, and macerate, further comprises mannitol, MES, KCl, CaCl2, BSA, and β-mercaptoethanol. The corresponding components have the functions of stabilizing osmotic pressure, maintaining protoplast activity, and regulating pH, thereby facilitating the preparation of turnip protoplasts and improving the yield and activity of turnip protoplasts. The results of the examples of the present invention show that the enzymatic hydrolysate can be used to efficiently prepare turnip protoplasts using turnip petiole cotyledons as materials, and the obtained protoplasts have high yield and activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1This is a diagram showing the observation results of the turnip protoplasts prepared in Example 4;

[0026] Figure 2 This is the observation result of the turnip protoplasts prepared in Comparative Example 3;

[0027] Figure 3 This is the observation result of the turnip protoplasts prepared in Comparative Example 4. DETAILED DESCRIPTION

[0028] The present invention provides an enzymatic hydrolysis solution for preparing protoplasts from turnips. The enzymatic hydrolysis solution comprises cellulase, macerate, and pectinase; the mass ratio of the cellulase, macerate, and pectinase is (1.5-2.5):(0.8-1.0):(0.1-0.2). In the enzymatic hydrolysis solution, the cellulase, macerate, and pectinase act synergistically on turnip plant tissue, promoting cell wall lysis in turnip plant tissue, thereby maximizing the release of turnip protoplasts, thereby increasing protoplast yield and maintaining high protoplast activity. The enzymatic hydrolysis solution provided by the present invention can be used to prepare protoplasts from turnip plant tissue. The cotyledons of turnips often present significant challenges in preparing protoplasts due to their significantly different cell wall composition, including cellulose, hemicellulose, and pectin, compared to other tissues, such as turnip leaves. In practice, the yield and activity of protoplasts extracted from cotyledons are often low. However, the protoplasts from petiolated cotyledons, which have greater cell differentiation potential, often offer significant advantages in subsequent applications. The enzymatic hydrolysis solution provided by the invention can realize the efficient extraction of protoplasts from petiole cotyledons of turnip during the preparation of turnip protoplasts, thereby improving the yield and activity of the petiole cotyledon protoplasts.

[0029] The invention provides an enzymatic hydrolysis solution for preparing turnip protoplasts. The enzymatic hydrolysis solution comprises, based on the total volume of the enzymatic hydrolysis solution, 1.5-2.5 wt.% of cellulase, 0.8-1.0 wt.% of macerate, 0.1-0.2 wt.% of pectinase, 0.55-0.6 mol / L of mannitol, 19-21 mmol / L of MES, 19-21 mmol / L of KCl, 9-11 mmol / L of CaCl2, 0.09-0.11 wt.% of BSA and 0.04-0.06 mmol / L of beta-mercaptoethanol.

[0030] In the present invention, the enzymolysis solution includes 1.5~2.5wt.% cellulase based on the total volume of the enzymolysis solution. As an optional embodiment of the present invention, the mass percentage of the cellulase can be 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4% or 2.5%. The present invention does not specifically limit the source of the cellulase, and conventional commercial products in the field can be used. In the examples of the present invention, cellulase RS is taken as an example to illustrate the effect of the enzymolysis solution. Adding cellulase to the enzymolysis solution for preparing turnip protoplasts of the present invention has the effect of degrading cellulose in the cell walls of turnip plant tissues.

[0031] In the present invention, the enzymatic hydrolysis solution includes 0.8~1.0wt.% macerate based on the total volume of the enzymatic hydrolysis solution. As an optional embodiment of the present invention, the mass percentage of the macerate can be 0.8%, 0.9% or 1.0%. The present invention does not specifically limit the source of the macerate, and conventional commercially available products in the field can be used. In the embodiment of the present invention, macerate R-10 is taken as an example to illustrate the effect of the enzymatic hydrolysis solution. The present invention adds macerate to the enzymatic hydrolysis solution for preparing turnip protoplasts to cooperate with cellulase to promote the separation of plant tissue into single cells.

[0032] In the present invention, the enzymolysis solution includes 0.1~0.2wt.% pectinase based on the total volume of the enzymolysis solution. As an optional embodiment of the present invention, the mass percentage of the pectinase can be 0.1%, 0.15% or 0.2%. The present invention does not specifically limit the source of the pectinase, and conventional commercial products in the field can be used. In the examples of the present invention, pectinase Y-23 is used as an example to illustrate the effect of the enzymolysis solution. Adding pectinase to the enzymolysis solution for preparing turnip protoplasts in the present invention can degrade pectin in the cell walls of turnip plant tissues and promote cell separation.

[0033] In the present invention, the enzymolysis solution includes 0.55 to 0.6 mol / L mannitol based on the total volume of the enzymolysis solution. As an optional embodiment of the present invention, the molar concentration of the mannitol in the enzymolysis solution can be 0.55, 0.56, 0.57, 0.58, 0.59 or 0.60 mol / L. The present invention is not particularly limited to the source of the mannitol, and conventional commercial products in this area can be used. In the present invention, the main effect of adding mannitol in the enzymolysis solution is to stabilize the osmotic pressure and maintain protoplast activity.

[0034] In the present invention, the enzymatic hydrolysis solution comprises 19 to 21 mmol / L MES, based on the total volume of the enzymatic hydrolysis solution. As an optional embodiment of the present invention, the molar concentration of MES in the enzymatic hydrolysis solution can be 19, 20, or 21 mmol / L. The present invention does not specifically limit the source of the MES; any commercially available product in the art can be used. In the present invention, the addition of MES to the enzymatic hydrolysis solution is primarily to maintain a stable pH value in the system.

[0035] In the present invention, the enzymolysis solution includes 19 to 21 mmol / L KCl based on the total volume of the enzymolysis solution. As an optional embodiment of the present invention, the molar concentration of KCl in the enzymolysis solution can be 19, 20 or 21 mmol / L. The present invention does not particularly limit the source of the KCl, and any conventional commercially available product in the art can be used. In the present invention, the addition of KCl to the enzymolysis solution mainly helps regulate osmotic pressure, maintains osmotic pressure stability inside and outside the cell, helps maintain the morphological integrity of the protoplasts, and maintains cell membrane stability.

[0036] In the present invention, the enzymolysis solution comprises 9 to 11 mmol / L CaCl2 based on the total volume of the enzymolysis solution. As an optional embodiment of the present invention, the molar concentration of CaCl2 in the enzymolysis solution can be 9, 10 or 11 mmol / L. The present invention is not particularly limited to the source of the CaCl2, and conventional commercial products in the art can be used. The main function of adding CaCl2 to the enzymolysis solution of the present invention is to stabilize the membrane structure of the protoplasts and protect the protoplasts from damage during the enzymolysis process.

[0037] In the present invention, the enzymatic hydrolysate includes 0.09-0.11 wt.% BSA, based on the total volume of the enzymatic hydrolysate. As an optional embodiment of the present invention, the mass percentage of BSA in the enzymatic hydrolysate may be 0.09, 0.10, or 0.11 wt.%. The present invention does not particularly limit the source of the BSA; any commercially available product in the art may be used. The addition of BSA to the enzymatic hydrolysate helps maintain enzyme stability and activity.

[0038] In the present invention, the enzymatic hydrolysis solution includes 0.04 to 0.06 mmol / L β-mercaptoethanol, based on the total volume of the enzymatic hydrolysis solution. As an optional embodiment of the present invention, the molar concentration of β-mercaptoethanol in the enzymatic hydrolysis solution can be 0.04, 0.05, or 0.06 mmol / L. The present invention does not particularly limit the source of the β-mercaptoethanol, and any commercially available product in the art can be used. The addition of β-mercaptoethanol to the enzymatic hydrolysis solution can protect protoplasts from oxidative damage.

[0039] The enzymatic hydrolysis solution provided by the present invention contains three enzyme preparations: cellulase, macerate, and pectinase. The three enzymes can synergistically degrade the cell walls of turnip tissue during the preparation of turnip protoplasts, thereby releasing the protoplasts to the greatest extent and improving the activity of the protoplasts. Mannitol, MES, KCl, CaCl2, BSA, and β-mercaptoethanol components in the enzymatic hydrolysis solution have the functions of stabilizing osmotic pressure, maintaining protoplast activity, and regulating pH, thereby facilitating the preparation of turnip protoplasts and improving the yield and activity of turnip protoplasts.

[0040] The present invention provides a method for preparing the enzymatic hydrolysate described in the above technical solution, comprising the following steps:

[0041] The cellulase, macerate, pectinase, mannitol, MES and KCl are mixed and heated to obtain a mixed enzymatic hydrolysate;

[0042] The mixed enzymatic hydrolysate is cooled and then mixed with CaCl2, BSA and β-mercaptoethanol and the volume is fixed to obtain an enzymatic hydrolysate.

[0043] In the present invention, cellulase, macerator, pectinase, mannitol, MES acid-base buffer, and KCl are mixed and heated to produce a mixed enzymatic hydrolysate. As an optional embodiment of the present invention, the MES can be added in the form of an MES acid-base buffer; the pH of the MES acid-base buffer can be 5.6-5.8, or 5.6, 5.7, or 5.8. The present invention does not specifically limit the mixing method; conventional mixing methods in the art can be employed. After mixing, a mixed system is obtained, and the present invention preferably heats the mixed system. As an optional embodiment of the present invention, the heating temperature can be 54-56°C, or 54, 55, or 56°C; the heating time can be 9-12 minutes, or 9, 10, 11, or 12 minutes. The present invention does not specifically limit the heating method; conventional heating methods in the art can be employed. As an optional embodiment of the present invention, the heating method can be water bath heating. Heating during the preparation of the enzymatic hydrolysate is primarily used to promote enzyme dissolution and enhance enzyme activity during subsequent enzymatic hydrolysis. After heating, a mixed enzymatic hydrolysate is obtained.

[0044] After obtaining the mixed enzymatic hydrolysate, the present invention cools the mixed enzymatic hydrolysate, mixes it with CaCl2, BSA, and β-mercaptoethanol, and then fixes the volume to obtain an enzymatic hydrolysate. The cooling method of the present invention is preferably to room temperature; the room temperature can be 20-25°C, or can be 20, 21, 22, 23, 24, or 25°C. After obtaining the enzymatic hydrolysate, the present invention preferably further includes filtering the enzymatic hydrolysate using a 0.45 μm filter membrane for sterilization. The sterilized filtered enzymatic hydrolysate of the present invention can be directly used in the preparation of turnip protoplasts.

[0045] The present invention provides a method for preparing turnip protoplasts, comprising the following steps:

[0046] The enzymatic hydrolysis solution described in the above technical solution or the enzymatic hydrolysis solution prepared by the preparation method described in the above technical solution is used to enzymatically hydrolyze the turnip plant tissue to obtain turnip protoplasts.

[0047] As an optional embodiment of the present invention, the turnip plant tissue includes leaves and / or cotyledons of turnip. The enzymatic hydrolyzate provided by the present invention can be used to prepare protoplasts from turnip plant tissues, and can further be used to prepare protoplasts from plant tissues such as turnip cotyledons, which are difficult to extract protoplasts from. The present invention does not specifically limit the method for obtaining the cotyledons of turnip, and any conventional method in the art can be used to prepare them. As an optional embodiment of the present invention, the cotyledons of turnip can be sterile cotyledons of turnip. As an optional embodiment of the present invention, the method for preparing the cotyledons of turnip comprises: culturing turnip seed tissue for 4 to 6 days to obtain turnip seedlings; after protecting the turnip seedlings from light, taking the cotyledons of turnip. As an optional embodiment of the present invention, the cotyledons can be petiolated cotyledons; the length of the cotyledon petiole of the petiolated cotyledon can be 3 to 5 mm, or can be 3, 4 or 5 mm.

[0048] In the present invention, the turnip seeds are preferably seeds with plump grains and intact epidermis. After obtaining the turnip seeds, the present invention preferably disinfects them before conducting tissue culture. The present invention does not specify the disinfection method; conventional disinfection methods in the art can be used. The present invention can include a first disinfection with alcohol, followed by a second disinfection with a HgCl2 solution. In the present invention, the alcohol is preferably 75% ethanol by volume; the first disinfection can last for 15 seconds. After the first disinfection, the present invention preferably performs a second disinfection on the seeds that have undergone the first disinfection. The HgCl2 solution used in the second disinfection can have a mass concentration of 0.2%. The second disinfection can last for 5 minutes. After the second disinfection, the present invention preferably washes the seeds. The present invention preferably washes the seeds with sterile water; the washes can be performed five times, each lasting 1 minute. After washing, the present invention preferably removes moisture from the surface of the seeds and then inoculates the seeds into a culture medium for tissue culture. The present invention does not specify the inoculation method; conventional inoculation methods in the art can be used. In the present invention, the culture medium uses 1 / 2MS medium as the basic medium and also includes 1-1.5wt.% sucrose and 0.65-0.7wt.% agar. As an optional embodiment of the present invention, the sucrose content may be 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5wt.%; the agar content may be 0.65, 0.66, 0.67, 0.68, 0.69, or 0.7wt.%. As an optional embodiment of the present invention, the pH of the culture medium may be 5.75-5.85, or 5.75, 5.8, or 5.85. In the present invention, the tissue culture temperature can be 23-25°C, or 23, 24, or 25°C; the light intensity can be 2500-3500 Lx, or 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, or 3500 Lx; and the photoperiod can be 16 hours of light and 8 hours of darkness per day. In the present invention, the tissue culture duration can be 4-6 days, or 4, 5, or 6 days. In the present invention, turnip seedlings are obtained after 4-6 days of tissue culture.

[0049] After obtaining the turnip seedlings, the present invention preferably performs a light-shielding treatment on the turnip seedlings. The light-shielding treatment time of the present invention can be 24 to 48 hours, or can be 24, 25, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 hours. After the light-shielding treatment is completed, the present invention preferably takes the cotyledons of the turnip seedlings to prepare turnip protoplasts.

[0050] The present invention uses the enzymatic hydrolysis solution to enzymatically hydrolyze turnip plant tissue to obtain turnip protoplasts. As an optional embodiment of the present invention, the present invention uses the enzymatic hydrolysis solution to enzymatically hydrolyze turnip cotyledons to obtain turnip protoplasts; the cotyledons may be petiolated cotyledons. During the enzymatic hydrolysis, the volume ratio of the number of cotyledons to the enzymatic hydrolysis solution can be (20-30) cotyledons:15mL, or can be 20 cotyledons:15mL, 21 cotyledons:15mL, 22 cotyledons:15mL, 23 cotyledons:15mL, 24 cotyledons:15mL, 25 cotyledons:15mL, 26 cotyledons:15mL, 27 cotyledons:15mL, 28 cotyledons:15mL, 29 cotyledons:15mL, or 30 cotyledons:15mL. The cotyledons can be cut into filaments with a width of 0.5-1mm for enzymatic hydrolysis, or can be cut into filaments with a width of 0.5, 0.6, 0.7, 0.8, 0.9, or 1mm for enzymatic hydrolysis.

[0051] As an optional embodiment of the present invention, the temperature of the enzymolysis is 28~30°C, or 28, 29 or 30°C; the time of the enzymolysis can be 3~4h, or 3, 3.5 or 4h; the enzymolysis process is accompanied by oscillation; the rotation speed of the oscillation is 45~50rpm, or 45, 46, 47, 48, 49 or 50rpm; the enzymolysis process is carried out in the dark. The enzymolysis conditions of the present invention can ensure that the enzymolysis conditions are optimal, and at the same time, the protoplasts can maintain a higher activity. For example, if the enzymolysis time is too long relative to the enzymolysis time of the present invention, the protoplasts without cell wall protection will be easily broken and degraded, resulting in a decrease in the number and activity of the protoplasts.

[0052] As an optional embodiment of the present invention, during or after the enzymatic hydrolysis, the enzymatic hydrolysis product can be gently blown with a sterile pipette tip with a cut end to fully release the protoplasts.

[0053] After enzymatic hydrolysis is completed, the present invention obtains protoplasts of turnip. Alternatively, as an optional embodiment of the present invention, after obtaining the enzymatic hydrolysis product, the present invention preferably further comprises washing and resuspending the enzymatic hydrolysis product to obtain protoplasts. After enzymatic hydrolysis is completed, the present invention preferably filters the enzymatic hydrolysis product; the filtration is preferably performed using a 70µm cell filter. Before filtering using the cell filter, the present invention preferably uses W5 solution to rotate and rinse the cell filter. After filtration is completed, the present invention preferably uses the piston of a sterile syringe to gently press the undigested tissue in the cell filter, and then slowly adds W5 solution to rinse the undigested tissue; the number of presses can be 3-5 times, or 3, 4, or 5 times; the number of rinses can be 2-3 times. As an optional embodiment of the present invention, the W5 solution comprises: 2mmol / L MES, 154mmol / L NaCl solution, 125mmol / L CaCl2 solution, 5mmol / L KCl solution, and 5mmol / L glucose, with a solution pH of 5.7. After the filtration is completed, the present invention preferably collects the filtrate to obtain the filtered enzymatic hydrolyzate.

[0054] After obtaining the filtered enzymatic hydrolysate, the present invention preferably centrifuges the filtered enzymatic hydrolysate; the centrifugation speed can be 100g; and the centrifugation time can be 7 minutes. After the centrifugation is completed, the present invention preferably discards the supernatant and obtains the precipitate. The obtained precipitate is preferably resuspended in W5 solution. The present invention uses W5 solution to resuspend the precipitate mainly to wash the obtained protoplasts. As an optional embodiment of the present invention, the number of washes can be 2 to 3 times; after the washes are completed, the present invention preferably uses MMG solution to resuspend the washed protoplasts to obtain a solution containing turnip protoplasts, i.e., a protoplast suspension. As an optional embodiment of the present invention, the composition of the MMG solution includes: 0.4 mol / L mannitol, 15 mmol / L MgCl2 solution, 4 mmol / L MES, and the solution pH is 5.7.

[0055] The preparation method of the turnip protoplasts of the present invention is simple and efficient, shortens the enzymatic hydrolysis time, and can obtain a large number of turnip protoplasts that maintain activity for a long time.

[0056] The present invention also provides applications of the turnip protoplasts obtained by the preparation method described in the above technical solution in turnip breeding and / or bioinformatics. The bioinformatics applications include applications in any one or more of subcellular localization, gene expression analysis, protein-protein interaction, and gene editing.

[0057] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0058] The following raw materials were obtained from the following sources: Cellulase RS, macerozyme R-10, and pectolyase Y-23 were purchased from YAKULT PHARMACEUTICALIND. CO., LTD.; MS medium was purchased from Phyto Tech LABS; MES, mannitol, KCl, CaCl2, BSA (bovine serum albumin), β-mercaptoethanol, agar, and sucrose were purchased from Sangon (Shanghai Sangon Biotech Co., Ltd.); 0.45 μm filter membrane was purchased from Sartorius (Sartorius Stedim Biotech GmbH, Germany); and disposable sterile cell strainer was purchased from Biosharp (Biosharp Life Sciences, Beijing Labgic Technology Co., Ltd.).

[0059] The experimental instruments used in the following protocols, such as tweezers, scalpel blades, glass culture dishes, centrifuge tubes, and pipette tips, were sterilized at 121°C for 25 min. The cell filter was a disposable sterile cell filter, and the filter membrane was a 0.45 µm sterile microporous filter. The clean bench was wiped with 75% alcohol, irradiated with ultraviolet light for 30 min, and ventilated for 10 min.

[0060] Prepare MMG solution with the following composition: 0.4 mol / L mannitol, 15 mmol / L MgCl2 solution, 4 mmol / L MES, and the pH value of the solution is 5.7.

[0061] Prepare W5 solution. The composition of W5 solution is: 2 mmol / L MES, 154 mmol / L NaCl solution, 125 mmol / L CaCl2 solution, 5 mmol / L KCl solution, 5 mmol / L glucose, and the solution pH is 5.7.

[0062] Example 1

[0063] The invention discloses an enzymatic hydrolysate for preparing turnip protoplasts. The enzymatic hydrolysate comprises, based on the total volume of the enzymatic hydrolysate, 2.0% cellulase RS, 1.0% machinase R-10, 0.1% pectinase Y-23, 0.55 mol / L mannitol, 20 mmol / L MES (MES acid-base buffer with a pH of 5.7), 20 mmol / L KCl, 10 mmol / L CaCl2, 0.1 wt.% BSA, and 0.05 mmol / L β-mercaptoethanol.

[0064] The preparation method of the enzymatic hydrolysate is as follows:

[0065] Based on the composition of the enzymatic hydrolysate, calculate the amount of each substance to be added. Then, mix Cellulase RS, Erosion Enzyme R-10, Pectinase Y-23, mannitol, MES acid-base buffer, and KCl, and incubate in a 55°C water bath for 10 minutes. After cooling to room temperature, add CaCl2, BSA, and β-mercaptoethanol, and then add ultrapure water to a final volume of 15 mL to obtain the enzymatic hydrolysate.

[0066] After obtaining the enzymatic hydrolysate, filter and sterilize the prepared enzymatic hydrolysate using a 0.45 μm filter membrane and pour it into a 50 mL centrifuge tube for later use.

[0067] Examples 2-3 and Comparative Examples 1-2

[0068] The compositions of the enzymatic hydrolysates for preparing turnip protoplasts in Examples 1-3 and Comparative Examples 1-2, based on the total volume of the enzymatic hydrolysates, are detailed in Table 1. The preparation methods of the enzymatic hydrolysates in Examples 2-3 and Comparative Examples 1-2 were the same as those in Example 1.

[0069] Table 1 Composition of the enzymatic hydrolysates of Examples 1 to 3

[0070]

[0071] Example 4

[0072] A method for preparing turnip protoplasts comprises the following steps:

[0073] 1. Turnip tissue culture: Select approximately 100 turnip seeds with plump, intact skins and place them in sterile 50mL centrifuge tubes. Sterilize and inoculate the seeds in a clean bench. The steps are as follows: sterilize with a 75% ethanol-water solution for 15 seconds, then soak in a 0.2% HgCl₂ solution for 5 minutes. Rinse with 45mL of sterile water five times, each wash lasting approximately 1 minute. Gently blot the seeds dry with sterile filter paper and culture on a 1 / 2 MS medium containing 1.5% sucrose and 0.7% agar (pH 5.8 ± 0.05). Culture conditions include a temperature of 24°C ± 1°C, a photoperiod of 16 hours light / 8 hours dark, and a light intensity of approximately 3000 lux.

[0074] 2. Preparation of enzymatic hydrolysis solution (the enzymatic hydrolysis solution of Example 1)

[0075] The composition of the enzymatic hydrolysis solution, based on the total volume of the enzymatic hydrolysis solution, is as follows: 2.0% cellulase RS by mass, 1.0% machinase R-10 by mass, 0.1% pectinase Y-23 by mass, 0.55 mol / L mannitol by mass, 20 mmol / L MES by mass, MES added in the form of MES acid-base buffer with a pH of 5.7, 20 mmol / L KCl by mass, 10 mmol / L CaCl2 by mass, 0.1 wt.% BSA by mass, and 0.05 mmol / L β-mercaptoethanol by mass.

[0076] To prepare 15 mL of enzymatic hydrolysate, calculate the amounts of each substance to be added based on the composition of the hydrolysate. Then, mix Cellulase RS, Measurase R-10, Pectinase Y-23, mannitol, MES acid-base buffer, and KCl. Incubate in a 55°C waterbath for 10 minutes. After cooling to room temperature, add CaCl2, BSA, and β-mercaptoethanol. Finally, add ultrapure water to a final volume of 15 mL to obtain the hydrolysate. After obtaining the hydrolysate, filter sterilize it using a 0.45 μm filter membrane and pour it into a 50 mL centrifuge tube for later use.

[0077] 3. Protect 4-6 day old turnip seedlings from light for 24 hours. Then, collect 30 petioled cotyledons (approximately 3-5 mm in length). Cut the petioles into 1 mm wide strips. Quickly place the 1 mm wide strips into a 50 mL centrifuge tube containing 15 mL of enzymatic hydrolysis solution. Wrap the tube in foil and place it on a plate shaker in the dark at 28°C, 45 rpm, and allow the enzymatic hydrolysis to proceed for 3.5 hours.

[0078] 4. Place a 70µm cell strainer on a new 50mL sterile centrifuge tube and add approximately 1mL of W5 solution to rinse the cell strainer. Gently pipette the enzymatic hydrolysate obtained in step 3 approximately 6-8 times with a sterile 1mL pipette tip to fully release the protoplasts. Then, transfer the enzymatic hydrolysate in batches to the rinsed cell strainer for filtration. Gently press the piston of a 5mL sterile syringe on the cell strainer 3-5 times to remove the undigested tissue. Then, slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinse twice and collect the filtrate to obtain the filtered enzymatic hydrolysate.

[0079] 5. Centrifuge the filtered enzymatic hydrolysate obtained in step 4 at 100g for 7 minutes. Discard the supernatant. Tilt the centrifuge tube and slowly add 3mL of W5 solution along the tube wall to resuspend the pellet. Filter the resuspended liquid through a 40µm cell strainer. Repeat this process by slowly adding 1mL of W5 solution to wash away any remaining tissue on the cell strainer. Repeat this process once more (i.e., slowly adding 1mL of W5 solution to wash away any remaining tissue on the cell strainer, similarly below). Collect the filtrate. Centrifuge the filtrate at 100g for 7 minutes, discard the supernatant, and resuspend the pellet in 3mL of MMG solution to obtain a protoplast suspension of turnip greens.

[0080] 6. Use FDA (fluorescein diacetate) to detect the activity of turnip protoplasts. Pipette 100μL of protoplast suspension, place it in a 1.5mL centrifuge tube wrapped in tin foil in advance, add 3μL of 1mg / mL FDA, and stain in the dark for 5 minutes. After staining, aspirate 50μL onto a glass slide and observe 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 will be counted. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field of view is the protoplast activity value. The activity value of the prepared protoplasts is about 88.09%, such as Figure 1 As shown, Figure 1 The middle right picture shows the FDA staining result, and the left picture shows the corresponding protoplast image under bright field vision. The scale bar in the picture is 100µm.

[0081] Statistical analysis of protoplast viability: protoplast viability = (number of protoplasts emitting yellow-green fluorescence / total number of protoplasts) × 100%.

[0082] Statistical protoplast concentration: Pipette the prepared protoplasts and drop them onto one end of the cover glass of the hemocytometer. Wait until the droplet fills the entire cover glass until no bubbles appear. Let it stand for two minutes and then observe and count. The density of the protoplasts prepared in this example is about 34.22×10 5 pieces / mL.

[0083] Protoplast density (cells / mL) = number of protoplasts in 25 squares × 10 4 × dilution factor.

[0084] Example 5

[0085] A method for preparing turnip protoplasts comprises the following steps:

[0086] 1. The method of tissue culture of turnip is the same as that in Example 4.

[0087] 2. Preparation of enzymatic hydrolysis solution (the enzymatic hydrolysis solution of Example 2)

[0088] The composition of the enzymatic hydrolysis solution, based on the total volume of the enzymatic hydrolysis solution, is as follows: cellulase RS with a mass concentration of 1.5%, machinase R-10 with a mass concentration of 1.0%, pectinase Y-23 with a mass concentration of 0.1%, mannitol with a final concentration of 0.55 mol / L, MES with a final concentration of 20 mmol / L, MES added in the form of MES acid-base buffer with a pH of 5.7, KCl with a final concentration of 20 mmol / L, CaCl2 with a final concentration of 10 mmol / L, BSA with a final concentration of 0.1%, and β-mercaptoethanol with a final concentration of 0.05 mmol / L.

[0089] The preparation method of 15 mL of enzymatic hydrolysate is the same as that in Example 4.

[0090] 3. Protect 4-6 day old turnip seedlings from light for 24 hours. Then, collect 30 petioled cotyledons (approximately 3-5 mm in length). Cut the petioles into 1 mm wide strips. Quickly place the 1 mm wide strips into a 50 mL centrifuge tube containing 15 mL of enzymatic hydrolysis solution. Wrap the tube in tinfoil and place it on a plate shaker in the dark at 28°C, 45 rpm, and incubate the enzymatic hydrolysis for 3.5 hours.

[0091] 4. Place a 70µm cell strainer on a new 50mL sterile centrifuge tube and add approximately 1mL of W5 solution to rinse the cell strainer. Gently pipette the enzymatic hydrolysate obtained in step 3 approximately 6-8 times with a sterile 1mL pipette tip to fully release the protoplasts. Then, transfer the enzymatic hydrolysate in batches to the rinsed cell strainer for filtration. Gently press the piston of a 5mL sterile syringe on the cell strainer 3-5 times to remove the undigested tissue. Then, slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinse twice and collect the filtrate to obtain the filtered enzymatic hydrolysate.

[0092] 5. Centrifuge the filtered enzymatic hydrolysate obtained in step 4 at 100g for 7 minutes. Discard the supernatant. Tilt the centrifuge tube and slowly add 3mL of W5 solution along the tube wall to resuspend the pellet. Filter the resuspended liquid through a 40µm cell strainer. Repeat this process with 1mL of W5 solution to wash any remaining tissue on the cell strainer. Collect the filtrate. Centrifuge the filtrate at 100g for 7 minutes, discard the supernatant, and resuspend the pellet in 3mL of MMG solution to obtain a protoplast suspension of turnip greens.

[0093] 6. Use FDA (fluorescein diacetate) to test the viability of turnip protoplasts. Pipette 100 μL of the protoplast suspension into a 1.5 mL centrifuge tube pre-wrapped with tin foil. Add 3 μL of 1 mg / mL FDA and stain for 5 minutes in the dark. After staining, aspirate 50 μL onto a glass slide and observe under an inverted fluorescence microscope (Thermo Fisher EVOS M3000) in the dark. 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 viability value of the protoplasts prepared in this example was approximately 89.17%.

[0094] Statistical analysis of protoplast viability: protoplast viability = (number of protoplasts emitting yellow-green fluorescence / total number of protoplasts) × 100%.

[0095] Statistical protoplast concentration: Pipette the prepared protoplasts and drop them onto one end of the cover glass of the hemocytometer. Wait until the droplet fills the entire cover glass until no bubbles appear. Let it stand for two minutes and then observe and count. The density of the protoplasts prepared in this example is about 31.76×10 5 pieces / mL.

[0096] Protoplast density (cells / mL) = number of protoplasts in 25 squares × 10 4 × dilution factor.

[0097] Example 6

[0098] A method for preparing turnip protoplasts comprises the following steps:

[0099] 1. The method of tissue culture of turnip is the same as that in Example 4.

[0100] 2. Preparation of enzymatic hydrolysis solution (the enzymatic hydrolysis solution of Example 3)

[0101] The composition of the enzymatic hydrolysis solution, based on the total volume of the enzymatic hydrolysis solution, is as follows: 2.5% mass concentration of cellulase RS, 0.8% mass concentration of machinase R-10, 0.1% mass concentration of pectinase Y-23, final concentration of mannitol of 0.55 mol / L, final concentration of MES of 20 mmol / L, MES added in the form of MES acid-base buffer with a pH of 5.7, final concentration of KCl of 20 mmol / L, final concentration of CaCl2 of 10 mmol / L, final concentration of BSA of 0.1%, and final concentration of 0.05 mmol / L of β-mercaptoethanol.

[0102] The preparation method of 15 mL of enzymatic hydrolysate is the same as that in Example 4.

[0103] 3. Protect 4-6 day old turnip seedlings from light for 24 hours. Then, collect 30 petioled cotyledons (approximately 3-5 mm in length). Cut the petioles into 1 mm wide strips. Quickly place the 1 mm wide strips into a 50 mL centrifuge tube containing 15 mL of enzymatic hydrolysis solution. Wrap the tube in foil and place it on a plate shaker in the dark at 28°C, 45 rpm, and allow the enzymatic hydrolysis to proceed for 3.5 hours.

[0104] 4. Place a 70µm cell strainer on a new 50mL sterile centrifuge tube and add approximately 1mL of W5 solution to rinse the cell strainer. Gently pipette the enzymatic hydrolysate obtained in step 3 approximately 6-8 times with a sterile 1mL pipette tip to fully release the protoplasts. Then, transfer the enzymatic hydrolysate in batches to the rinsed cell strainer for filtration. Gently press the piston of a 5mL sterile syringe on the cell strainer 3-5 times to remove the undigested tissue. Then, slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinse twice and collect the filtrate to obtain the filtered enzymatic hydrolysate.

[0105] 5. Centrifuge the filtered enzymatic hydrolysate obtained in step 4 at 100g for 7 minutes. Discard the supernatant. Tilt the centrifuge tube and slowly add 3mL of W5 solution along the tube wall to resuspend the pellet. Filter the resuspended liquid through a 40µm cell strainer. Repeat this process with 1mL of W5 solution to wash any remaining tissue on the cell strainer. Collect the filtrate. Centrifuge the filtrate at 100g for 7 minutes, discard the supernatant, and resuspend the pellet in 3mL of MMG solution to obtain a protoplast suspension of turnip greens.

[0106] 6. Use FDA (fluorescein diacetate) to test the viability of turnip protoplasts. Pipette 100 μL of the protoplast suspension into a 1.5 mL centrifuge tube pre-wrapped with tin foil. Add 3 μL of 1 mg / mL FDA and stain for 5 minutes in the dark. After staining, aspirate 50 μL onto a glass slide and observe under an inverted fluorescence microscope (Thermo Fisher EVOS M3000) in the dark. 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 viability value of the protoplasts prepared in this example was approximately 85.40%.

[0107] Statistical analysis of protoplast viability: protoplast viability = (number of protoplasts emitting yellow-green fluorescence / total number of protoplasts) × 100%.

[0108] Statistical protoplast concentration: Pipette the prepared protoplasts and drop them onto one end of the cover glass of the hemocytometer. Wait until the droplet fills the entire cover glass until no bubbles appear. Let it stand for two minutes and then observe and count. The density of the protoplasts prepared in this example is about 30.74×105 pieces / mL.

[0109] Protoplast density (cells / mL) = number of protoplasts in 25 squares × 10 4 × dilution factor.

[0110] Example 7

[0111] The invention discloses an enzymatic hydrolysate for preparing turnip protoplasts. The enzymatic hydrolysate comprises, based on the total volume of the enzymatic hydrolysate, 2.0% cellulase RS, 1.0% machinase R-10, 0.2% pectinase Y-23, 0.55 mol / L mannitol, 20 mmol / L MES (MES acid-base buffer with a pH of 5.7), 20 mmol / L KCl, 10 mmol / L CaCl2, 0.1 wt.% BSA, and 0.05 mmol / L β-mercaptoethanol.

[0112] The preparation method of the enzymatic hydrolysate is the same as that in Example 1.

[0113] Example 8

[0114] A method for preparing turnip protoplasts comprises the following steps:

[0115] 1. The tissue culture method of turnip is the same as that in Example 4.

[0116] 2. Prepare the enzymatic hydrolysate, which is the enzymatic hydrolysate of Example 7.

[0117] 3. Protect 4-6 day old turnip seedlings from light for 24 hours. Then, collect 30 petioled cotyledons (approximately 3-5 mm in length). Cut the petioles into 1 mm wide strips. Quickly place the 1 mm wide strips into a 50 mL centrifuge tube containing 15 mL of enzymatic hydrolysis solution. Wrap the tube in foil and place it on a plate shaker in the dark at 28°C, 45 rpm, and incubate the enzymatic hydrolysis for 3.5 hours.

[0118] 4. Place a 70µm cell strainer on a new 50mL sterile centrifuge tube and add approximately 1mL of W5 solution to rinse the cell strainer. Gently pipette the enzymatic hydrolysate obtained in step 3 approximately 6-8 times with a sterile 1mL pipette tip to fully release the protoplasts. Then, transfer the enzymatic hydrolysate in batches to the rinsed cell strainer for filtration. Gently press the piston of a 5mL sterile syringe on the cell strainer 3-5 times to remove the undigested tissue. Then, slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinse twice and collect the filtrate to obtain the filtered enzymatic hydrolysate.

[0119] 5. Centrifuge the filtered enzymatic hydrolysate obtained in step 4 at 100g for 7 minutes. Discard the supernatant. Tilt the centrifuge tube and slowly add 3mL of W5 solution along the tube wall to resuspend the pellet. Filter the resuspended liquid through a 40µm cell strainer. Repeat this process with 1mL of W5 solution to wash any remaining tissue on the cell strainer. Collect the filtrate. Centrifuge the filtrate at 100g for 7 minutes, discard the supernatant, and resuspend the pellet in 3mL of MMG solution to obtain a protoplast suspension of turnip greens.

[0120] 6. Use FDA (fluorescein diacetate) to test the viability of turnip protoplasts. Pipette 100 μL of the protoplast suspension into a 1.5 mL centrifuge tube pre-wrapped with tin foil. Add 3 μL of 1 mg / mL FDA and stain for 5 minutes in the dark. After staining, aspirate 50 μL onto a glass slide and observe under an inverted fluorescence microscope (Thermo Fisher EVOS M3000) in the dark. 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 viability value of the protoplasts prepared in this example was approximately 82.63%.

[0121] Statistical analysis of protoplast viability: protoplast viability = (number of protoplasts emitting yellow-green fluorescence / total number of protoplasts) × 100%.

[0122] Count the protoplast concentration: Pipette the prepared protoplasts and drop them onto one end of the cover glass of the hemocytometer. Wait until the drop fills the entire cover glass until no bubbles appear. Let it stand for two minutes and then observe and count. The density of the protoplasts prepared in this example is about 32.28×10 5 pieces / mL.

[0123] Protoplast density (cells / mL) = number of protoplasts in 25 squares × 10 4 × dilution factor.

[0124] Comparative Example 3

[0125] A method for preparing turnip protoplasts comprises the following steps:

[0126] 1. The method of tissue culture of turnip is the same as that in Example 4.

[0127] 2. Prepare enzymatic hydrolysis solution (the enzymatic hydrolysis solution of Comparative Example 1).

[0128] 3. Protect 4-6 day old turnip seedlings from light for 24 hours. Then, collect 30 petioled cotyledons (approximately 3-5 mm in length). Cut the petioles into 1 mm wide strips. Quickly place the 1 mm wide strips into a 50 mL centrifuge tube containing 15 mL of enzymatic hydrolysis solution. Wrap the tube in foil and place it on a plate shaker in the dark at 28°C, 45 rpm, and allow the enzymatic hydrolysis to proceed for 3.5 hours.

[0129] 4. Place a 70µm cell strainer on a new 50mL sterile centrifuge tube and add approximately 1mL of W5 solution to rinse the cell strainer. Gently pipette the enzymatic hydrolysate obtained in step 3 approximately 6-8 times with a sterile 1mL pipette tip to fully release the protoplasts. Then, transfer the enzymatic hydrolysate in batches to the rinsed cell strainer for filtration. Gently press the piston of a 5mL sterile syringe on the cell strainer 3-5 times to remove the undigested tissue. Then, slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinse twice and collect the filtrate to obtain the filtered enzymatic hydrolysate.

[0130] 5. Centrifuge the filtered enzymatic hydrolysate obtained in step 4 at 100g for 7 minutes. Discard the supernatant. Tilt the centrifuge tube and slowly add 3mL of W5 solution along the tube wall to resuspend the pellet. Filter the resuspended liquid through a 40µm cell strainer. Repeat this process with 1mL of W5 solution to wash any remaining tissue on the cell strainer. Collect the filtrate. Centrifuge the filtrate at 100g for 7 minutes, discard the supernatant, and resuspend the pellet in 3mL of MMG solution to obtain a protoplast suspension of turnip greens.

[0131] 6. Use FDA (fluorescein diacetate) to detect the activity of turnip protoplasts. Pipette 100μL of protoplast suspension, place it in a 1.5mL centrifuge tube wrapped in tin foil in advance, add 3μL of 1mg / mL FDA, and stain in the dark for 5 minutes. After staining, aspirate 50μL onto a glass slide and observe 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 of view is the protoplast activity value. The activity value of the protoplasts prepared in this comparative example is about 55.96%, such as Figure 2 As shown, Figure 2 The right picture is the FDA staining result, and the left picture is the corresponding protoplast image under bright field vision.

[0132] Protoplast viability: Protoplast viability = (number of protoplasts emitting yellow-green fluorescence / total number of protoplasts) × 100%.

[0133] Calculate the protoplast concentration: pipette the prepared protoplasts and drop them onto one end of the cover glass of the hemocytometer. Wait until the drop fills the entire cover glass until no bubbles appear. Let it stand for two minutes and then observe and count. The density of the protoplasts prepared in this comparative example is about 2.94×10 5 pieces / mL.

[0134] Protoplast density (cells / mL) = number of protoplasts in 25 squares × 10 4 × dilution factor

[0135] Comparative Example 4

[0136] A method for preparing turnip protoplasts comprises the following steps:

[0137] 1. The method of tissue culture of turnip is the same as that in Example 4.

[0138] 2. Prepare enzymatic hydrolysis solution (the enzymatic hydrolysis solution of Comparative Example 2).

[0139] 3. Protect 4-6 day old turnip seedlings from light for 24 hours. Then, collect 30 petioled cotyledons (approximately 3-5 mm in length). Cut the petioles into 1 mm wide strips. Quickly place the 1 mm wide strips into a 50 mL centrifuge tube containing 15 mL of enzymatic hydrolysis solution. Wrap the tube in foil and place it on a plate shaker in the dark at 28°C, 45 rpm, and allow the enzymatic hydrolysis to proceed for 3.5 hours.

[0140] 4. Place a 70µm cell strainer on a new 50mL sterile centrifuge tube and add approximately 1mL of W5 solution to rinse the cell strainer. Gently pipette the enzymatic hydrolysate obtained in step 3 approximately 6-8 times with a sterile 1mL pipette tip to fully release the protoplasts. Then, transfer the enzymatic hydrolysate in batches to the rinsed cell strainer for filtration. Gently press the piston of a 5mL sterile syringe on the cell strainer 3-5 times to remove the undigested tissue. Then, slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinse twice and collect the filtrate to obtain the filtered enzymatic hydrolysate.

[0141] 5. Centrifuge the filtered enzymatic hydrolysate obtained in step 4 at 100g for 7 minutes. Discard the supernatant. Tilt the centrifuge tube and slowly add 3mL of W5 solution along the tube wall to resuspend the pellet. Filter the resuspended liquid through a 40µm cell strainer. Repeat this process with 1mL of W5 solution to wash any remaining tissue on the cell strainer. Collect the filtrate. Centrifuge the filtrate at 100g for 7 minutes, discard the supernatant, and resuspend the pellet in 3mL of MMG solution to obtain a protoplast suspension of turnip greens.

[0142] 6. Use FDA (fluorescein diacetate) to detect the activity of turnip protoplasts. Pipette 100μL of protoplast suspension, place it in a 1.5mL centrifuge tube wrapped in tin foil in advance, add 3μL of 1mg / mL FDA, and stain in the dark for 5 minutes. After staining, aspirate 50μL onto a glass slide and observe 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 of view is the protoplast activity value. The protoplast activity value prepared in this comparative example is about 60.99%, such as Figure 3 As shown, Figure 3 The right picture is the FDA staining result, and the left picture is the corresponding protoplast image under bright field vision.

[0143] Protoplast viability: Protoplast viability = (number of protoplasts emitting yellow-green fluorescence / total number of protoplasts) × 100%.

[0144] Calculate the protoplast concentration: pipette the prepared protoplasts and drop them onto one end of the cover glass of the hemocytometer. Wait until the drop fills the entire cover glass until no bubbles appear. Let it stand for two minutes and then observe and count. The density of the protoplasts prepared in this comparative example is about 19.34×10 5 pieces / mL.

[0145] Protoplast density (cells / mL) = number of protoplasts in 25 squares × 10 4 × dilution factor.

[0146] Comparative Example 5

[0147] A method for preparing turnip protoplasts comprises the following steps:

[0148] 1. The method of tissue culture of turnip is the same as that in Example 4.

[0149] 2. Prepare enzymatic hydrolysis solution (the enzymatic hydrolysis solution of Example 1).

[0150] 3. Protect 4-6 day old turnip seedlings from light for 24 hours. Then, collect 30 petioled cotyledons (approximately 3-5 mm in length). Cut the petioles into 1 mm wide strips. Quickly place the 1 mm wide strips into a 50 mL centrifuge tube containing 15 mL of enzymatic hydrolysis solution. Wrap the tube in tinfoil and place it on a plate shaker in the dark at 28°C, 45 rpm, and incubate the enzymatic hydrolysis for 8 hours.

[0151] 4. Place a 70µm cell strainer on a new 50mL sterile centrifuge tube and add approximately 1mL of W5 solution to rinse the cell strainer. Gently pipette the enzymatic hydrolysate obtained in step 3 approximately 6-8 times with a sterile 1mL pipette tip to fully release the protoplasts. Then, transfer the enzymatic hydrolysate in batches to the rinsed cell strainer for filtration. Gently press the piston of a 5mL sterile syringe on the cell strainer 3-5 times to remove the undigested tissue. Then, slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinse twice and collect the filtrate to obtain the filtered enzymatic hydrolysate.

[0152] 5. Centrifuge the filtered enzymatic hydrolysate obtained in step 4 at 100g for 7 minutes. Discard the supernatant. Tilt the centrifuge tube and slowly add 3mL of W5 solution along the tube wall to resuspend the pellet. Filter the resuspended liquid through a 40µm cell strainer. Repeat this process with 1mL of W5 solution to wash any remaining tissue on the cell strainer. Collect the filtrate. Centrifuge the filtrate at 100g for 7 minutes, discard the supernatant, and resuspend the pellet in 3mL of MMG solution to obtain a protoplast suspension of turnip greens.

[0153] 6. Use FDA (fluorescein diacetate) to test the viability of turnip protoplasts. Aspirate 100 μL of the protoplast suspension and place it in a 1.5 mL centrifuge tube pre-wrapped with tin foil. Add 3 μL of 1 mg / mL FDA and stain for 5 minutes in the dark. After staining, aspirate 50 μL onto a glass slide and observe under an inverted fluorescence microscope (Thermo Fisher EVOS M3000) in the dark. Viable protoplasts will emit a yellow-green fluorescence. Count the number of yellow-green protoplasts. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field of view is the protoplast viability value. The viability value of the protoplasts prepared in this comparative example was approximately 71.36%.

[0154] Protoplast viability: Protoplast viability = (number of protoplasts emitting yellow-green fluorescence / total number of protoplasts) × 100%.

[0155] Calculate the protoplast concentration: Pipette the prepared protoplasts and drop them onto one end of the cover glass of the hemocytometer. Wait until the drop fills the entire cover glass until no bubbles appear. Let it stand for two minutes and then observe and count. The density of the protoplasts prepared in this comparative example is about 22.52×10 5 pieces / mL.

[0156] Protoplast density (cells / mL) = number of protoplasts in 25 squares × 10 4 × dilution factor.

[0157] Comparative Example 6

[0158] A method for preparing turnip protoplasts comprises the following steps:

[0159] 1. The tissue culture method of turnip is the same as that in Example 4.

[0160] 2. Preparation of enzymatic hydrolysate

[0161] The composition of the enzymatic hydrolyzate, based on the total volume of the enzymatic hydrolyzate, is: 2.0% cellulase RS by mass concentration, 1.0% macerate R-10 by mass concentration, 0.55 mol / L mannitol, 20 mmol / L MES by mass concentration, MES added in the form of MES acid-base buffer with a pH of 5.7, KCl with a final concentration of 20 mmol / L, CaCl2 solution (final concentration 10 mmol / L), BSA (final concentration 0.1 wt.%), and β-mercaptoethanol (final concentration 0.05 mmol / L). The preparation method of the enzymatic hydrolyzate is the same as that in Example 1.

[0162] 3. Protect 4-6 day old turnip seedlings from light for 24 hours. Then, collect 30 petioled cotyledons (approximately 3-5 mm in length). Cut the petioles into 1 mm wide strips. Quickly place the 1 mm wide strips into a 50 mL centrifuge tube containing 15 mL of enzymatic hydrolysis solution. Wrap the tube in foil and place it on a plate shaker in the dark at 28°C, 45 rpm, and incubate the enzymatic hydrolysis for 3.5 hours.

[0163] 4. Place a 70µm cell strainer on a new 50mL sterile centrifuge tube and add approximately 1mL of W5 solution to rinse the cell strainer. Gently pipette the enzymatic hydrolysate obtained in step 3 approximately 6-8 times with a sterile 1mL pipette tip to fully release the protoplasts. Then, transfer the enzymatic hydrolysate in batches to the rinsed cell strainer for filtration. Gently press the piston of a 5mL sterile syringe on the cell strainer 3-5 times to remove the undigested tissue. Then, slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinse twice and collect the filtrate to obtain the filtered enzymatic hydrolysate.

[0164] 5. Centrifuge the filtered enzymatic hydrolysate obtained in step 4 at 100g for 7 minutes. Discard the supernatant. Tilt the centrifuge tube and slowly add 3mL of W5 solution along the tube wall to resuspend the pellet. Filter the resuspended liquid through a 40µm cell strainer. Repeat this process with 1mL of W5 solution to wash any remaining tissue on the cell strainer. Collect the filtrate. Centrifuge the filtrate at 100g for 7 minutes, discard the supernatant, and resuspend the pellet in 3mL of MMG solution to obtain a protoplast suspension of turnip greens.

[0165] 6. Use fluorescein diacetate (FDA) to test the viability of turnip protoplasts. Pipette 100 μL of the protoplast suspension into a 1.5 mL centrifuge tube pre-wrapped with tin foil. Add 3 μL of 1 mg / mL FDA and stain for 5 minutes in the dark. After staining, aspirate 50 μL onto a glass slide and observe under an inverted fluorescence microscope (Thermo Fisher EVOS M3000) in the dark. Viable protoplasts will emit a yellow-green fluorescence. Count the yellow-green protoplasts. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field of view is the protoplast viability value. The viability value of the prepared protoplasts is approximately 75.70%.

[0166] Protoplast viability: Protoplast viability = (number of protoplasts emitting yellow-green fluorescence / total number of protoplasts) × 100%.

[0167] Calculate the protoplast concentration: Pipette the prepared protoplasts and drop them onto one end of the cover glass of the hemocytometer. Wait until the drop fills the entire cover glass until no bubbles appear. Let it stand for two minutes and then observe and count. The density of the protoplasts prepared in this comparative example is about 23.36×10 5 pieces / mL.

[0168] Protoplast density (cells / mL) = number of protoplasts in 25 squares × 10 4 × dilution factor.

[0169] Comparative Example 7

[0170] A method for preparing turnip protoplasts comprises the following steps:

[0171] 1. The tissue culture method of turnip is the same as that in Example 4.

[0172] 2. Preparation of enzymatic hydrolysate

[0173] Based on the total volume of the enzymatic hydrolysate, the composition and preparation method of the enzymatic hydrolysate are as follows: 1.0% mass concentration of cleavage enzyme R-10, 0.1% mass concentration of pectinase Y-23, mannitol (final concentration 0.55 mol / L), MES final concentration 20 mmol / L, MES added in the form of MES acid-base buffer with a pH of 5.7, KCl (final concentration 20 mmol / L), incubated in a 55°C water bath for 10 min, cooled to room temperature, and then added with CaCl2 solution (final concentration 10 mmol / L) and BSA (final concentration 0.1 wt.%), and β-mercaptoethanol (final concentration 0.05 mmol / L), and the volume was made up to 15 mL with ultrapure water. The prepared enzymatic hydrolysate was filtered and sterilized using a 0.45 μm filter membrane and poured into a 50 mL centrifuge tube for later use.

[0174] 3. Protect 4-6 day old turnip seedlings from light for 24 hours. Then, collect 30 petioled cotyledons (approximately 3-5 mm in length). Cut the petioles into 1 mm wide strips. Quickly place the 1 mm wide strips into a 50 mL centrifuge tube containing 15 mL of enzymatic hydrolysis solution. Wrap the tube in foil and place it on a plate shaker in the dark at 28°C, 45 rpm, and incubate the enzymatic hydrolysis for 3.5 hours.

[0175] 4. Place a 70µm cell strainer on a new 50mL sterile centrifuge tube and add approximately 1mL of W5 solution to rinse the cell strainer. Gently pipette the enzymatic hydrolysate obtained in step 3 approximately 6-8 times with a sterile 1mL pipette tip to fully release the protoplasts. Then, transfer the enzymatic hydrolysate in batches to the rinsed cell strainer for filtration. Gently press the piston of a 5mL sterile syringe on the cell strainer 3-5 times to remove the undigested tissue. Then, slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinse twice and collect the filtrate to obtain the filtered enzymatic hydrolysate.

[0176] 5. Centrifuge the filtered enzymatic hydrolysate obtained in step 4 at 100g for 7 minutes. Discard the supernatant. Tilt the centrifuge tube and slowly add 3mL of W5 solution along the tube wall to resuspend the pellet. Filter the resuspended liquid through a 40µm cell strainer. Repeat this process with 1mL of W5 solution to wash any remaining tissue on the cell strainer. Collect the filtrate. Centrifuge the filtrate at 100g for 7 minutes, discard the supernatant, and resuspend the pellet in 3mL of MMG solution to obtain a protoplast suspension of turnip greens.

[0177] 6. Use FDA (fluorescein diacetate) to test the viability of turnip protoplasts. Aspirate 100 μL of the protoplast suspension and place it in a 1.5 mL centrifuge tube pre-wrapped with tin foil. Add 3 μL of 1 mg / mL FDA and stain for 5 minutes in the dark. After staining, aspirate 50 μL onto a glass slide and observe under an inverted fluorescence microscope (Thermo Fisher EVOS M3000) in the dark. Viable protoplasts will emit a yellow-green fluorescence. Count the number of yellow-green protoplasts. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field of view is the protoplast viability value. The viability value of the protoplasts prepared in this comparative example was approximately 69.46%.

[0178] Protoplast viability: Protoplast viability = (number of protoplasts emitting yellow-green fluorescence / total number of protoplasts) × 100%.

[0179] Calculate the protoplast concentration: Pipette the prepared protoplasts and drop them onto one end of the cover glass of the hemocytometer. Wait until the drop fills the entire cover glass until no bubbles appear. Let it stand for two minutes and then observe and count. The density of the protoplasts prepared in this comparative example is about 2.14×105 pieces / mL.

[0180] Protoplast density (cells / mL) = number of protoplasts in 25 squares × 10 4 × dilution factor.

[0181] Comparative Example 8

[0182] A method for preparing turnip protoplasts comprises the following steps:

[0183] 1. The tissue culture method of turnip is the same as that in Example 4.

[0184] 2. Preparation of enzymatic hydrolysate

[0185] Based on the total volume of the enzymatic hydrolysate, the composition and preparation method of the enzymatic hydrolysate are as follows: 2.0% mass concentration of cellulase RS, 0.1% mass concentration of pectinase Y-23, mannitol (final concentration 0.55 mol / L), MES final concentration 20 mmol / L, MES added in the form of MES acid-base buffer with a pH of 5.7, KCl (final concentration 20 mmol / L), incubated in a 55°C water bath for 10 min, cooled to room temperature, added with CaCl2 solution (final concentration 10 mmol / L) and BSA (final concentration 0.1 wt.%), and β-mercaptoethanol (final concentration 0.05 mmol / L), and made up to 15 mL with ultrapure water. The prepared enzymatic hydrolysate was filtered and sterilized using a 0.45 μm filter membrane and poured into a 50 mL centrifuge tube for later use.

[0186] 3. Protect 4-6 day old turnip seedlings from light for 24 hours. Then, collect 30 petioled cotyledons (approximately 3-5 mm in length). Cut the petioles into 1 mm wide strips. Quickly place the 1 mm wide strips into a 50 mL centrifuge tube containing 15 mL of enzymatic hydrolysis solution. Wrap the tube in tinfoil and place it on a plate shaker in the dark. Enzymatic hydrolysis should continue at 28°C, 45 rpm, for 3.5 hours.

[0187] 4. Place a 70µm cell strainer on a new 50mL sterile centrifuge tube and add approximately 1mL of W5 solution to rinse the cell strainer. Gently pipette the enzymatic hydrolysate obtained in step 3 approximately 6-8 times with a sterile 1mL pipette tip to fully release the protoplasts. Then, transfer the enzymatic hydrolysate in batches to the rinsed cell strainer for filtration. Gently press the piston of a 5mL sterile syringe on the cell strainer 3-5 times to remove the undigested tissue. Then, slowly add 3mL of W5 solution to rinse the undigested tissue. Repeat the rinse twice and collect the filtrate to obtain the filtered enzymatic hydrolysate.

[0188] 5. Centrifuge the filtered enzymatic hydrolysate obtained in step 3 at 100g for 7 minutes. Discard the supernatant. Tilt the centrifuge tube and slowly add 3mL of W5 solution along the tube wall to resuspend the pellet. Filter the resuspended liquid through a 40µm cell strainer. Repeat this process with 1mL of W5 solution to wash any remaining tissue on the cell strainer. Collect the filtrate. Centrifuge the filtrate at 100g for 7 minutes, discard the supernatant, and resuspend the pellet in 3mL of MMG solution to obtain a protoplast suspension of turnip greens.

[0189] 6. Use fluorescein diacetate (FDA) to test the viability of turnip protoplasts. Aspirate 100 μL of the protoplast suspension and place it in a 1.5 mL centrifuge tube pre-wrapped with tin foil. Add 3 μL of 1 mg / mL FDA and stain for 5 minutes in the dark. After staining, aspirate 50 μL onto a glass slide and observe under an inverted fluorescence microscope (Thermo Fisher EVOS M3000) in the dark. Viable protoplasts will emit a yellow-green fluorescence. Count the yellow-green protoplasts. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field of view is the protoplast viability value. The viability value of the protoplasts prepared in this comparative example was approximately 78.20%.

[0190] Protoplast viability: Protoplast viability = (number of protoplasts emitting yellow-green fluorescence / total number of protoplasts) × 100%.

[0191] Calculate the protoplast concentration: pipette the prepared protoplasts and drop them onto one end of the cover glass of the hemocytometer. Wait until the drop fills the entire cover glass until no bubbles appear. Let it stand for two minutes and then observe and count. The density of the protoplasts prepared in this comparative example is about 18.46×10 5 pieces / mL.

[0192] Protoplast density (cells / mL) = number of protoplasts in 25 squares × 10 4 × dilution factor.

[0193] The yield and activity of the protoplasts of turnip in each embodiment and comparative example are shown in Table 2.

[0194] Table 2 The yield and activity of the protoplasts of turnip in each embodiment and comparative example

[0195]

[0196] Note: Numbers without the same lowercase letters indicate significant differences at the P < 0.05 level.

[0197] As shown in Table 2, a comparison between Example 4 and Comparative Examples 6-8 shows that the yield and activity of turnip protoplasts prepared using an enzymatic hydrolysis solution containing cellulase, macerate, and pectinase simultaneously was significantly higher than the yield and activity using any two of these enzymes. The best results were achieved using cellulase, macerate, and pectinase simultaneously. A comparison between Examples 4-8 and Comparative Example 4 shows that the dosage of cellulase, pectinase, and macerate significantly impacted turnip protoplast preparation. A mass ratio of cellulase, macerate, and pectinase of (1.5-2.5):(0.8-1.0):(0.1-0.2) significantly improved the yield and activity of turnip protoplasts. A comparison between Example 4 and Comparative Example 5 shows that the enzymatic hydrolysis time significantly affects the yield and activity of turnip protoplasts. The enzymatic hydrolysis time should be properly controlled during turnip protoplast preparation.

[0198] In summary, the enzymatic hydrolysate for preparing turnip protoplasts and the method for preparing turnip protoplasts provided by the present invention can significantly improve the yield and activity of turnip protoplasts.

[0199] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. An application of an enzymatic hydrolysate in preparing turnip protoplasts, the enzymatic hydrolysate comprising, based on the total volume of the enzymatic hydrolysate: 1.5-2.5wt.% cellulase, 0.8-1.0wt.% malonase, 0.1-0.2wt.% pectinase, 0.55-0.6mol / L mannitol, 19-21mmol / L MES, 19-21mmol / L KCl, 9-11mmol / L CaCl2, 0.09-0.11wt.% BSA, and 0.04-0.06mmol / L β1-mercaptoethanol; The enzymatic hydrolysis temperature is 28-30°C; the enzymatic hydrolysis time is 3-4 hours; the enzymatic hydrolysis process is accompanied by oscillation; the oscillation speed is 45-50 rpm; The cellulase is cellulase RS.

2. The application according to claim 1, characterized in that The preparation method of the enzymatic hydrolysate comprises the following steps: The cellulase, macerate, pectinase, mannitol, MES and KCl are mixed and heated to obtain a mixed enzymatic hydrolysate; The mixed enzymatic hydrolysate is cooled and then mixed with CaCl2, BSA and β1-mercaptoethanol, and the volume is fixed to obtain an enzymatic hydrolysate.

3. The preparation method according to claim 2, characterized in that: The heating temperature is 54-56° C.; the heating time is 9-12 minutes.

4. A method for preparing turnip protoplasts, characterized in that: The following steps are involved: The enzymatic hydrolysis solution of claim 1 is used to enzymatically hydrolyze turnip plant tissue to obtain turnip protoplasts; the enzymatic hydrolysis temperature is 28-30° C.; the enzymatic hydrolysis time is 3-4 hours; the enzymatic hydrolysis process is accompanied by oscillation; and the oscillation speed is 45-50 rpm.

5. The preparation method according to claim 4, characterized in that: The rape plant tissue includes leaves and / or cotyledons of rape; The preparation method of the cotyledons of turnip comprises: The turnip seeds were tissue cultured for 4-6 days to obtain turnip seedlings; After protecting the turnip seedlings from light, take the cotyledons of the turnip.

6. The preparation method according to claim 5, characterized in that: During the enzymatic hydrolysis, the volume ratio of the number of cotyledons to the enzymatic hydrolysis solution is (20-30) pieces: 15 mL; the cotyledons are cut into thin strips with a width of 0.5-1 mm for enzymatic hydrolysis.

7. The preparation method according to claim 4, characterized in that: After the enzymatic hydrolysis is completed, the enzymatic hydrolysis product is washed and resuspended; the washing solution includes W5 solution; the resuspending solution includes MMG solution.

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