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

Abstract

The invention provides enzymatic hydrolysate for preparing turnip protoplast and a preparation method and application of the turnip protoplast, and belongs to the technical field of protoplast preparation. According to the enzymatic hydrolysate for preparing the turnip protoplast and the preparation method of the turnip protoplast, provided by the invention, efficient degradation of turnip tissue cell walls can be realized, so that the turnip protoplast is released to the maximum extent, the yield of the protoplast is favorably improved, and meanwhile, the protoplast can keep relatively high activity. The result of the embodiment shows that the turnip protoplast can be efficiently prepared by taking the turnip stem cotyledons as the material by adopting the enzymatic hydrolysate, and the yield and activity of the obtained protoplast are relatively high.

Description

Technical Field

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

[0002] Turnip ( Brassica rapa var. rapa) is a biennial plant of the genus Brassica in the Brassicaceae family, also known as rutabaga, disk radish, round root, etc. Its main root is fleshy, thick and round at the upper part and slender at the lower part. Turnip is a traditional cultivated plant that can be used as medicine, food and feed. Its fleshy root tissue is dense, fresh, crispy and sweet, containing a large amount of vitamins, various minerals and amino acids, etc. It is warm in nature and has various effects such as detoxification, nourishing and increasing oxygen, enhancing immunity, anti-mutation, anti-radiation and anti-fatigue. Therefore, it has high application value. At present, the breeding of new turnip varieties mainly relies on traditional breeding methods such as natural selection and cross-breeding, which are time-consuming and have low efficiency. Utilizing existing biotechnology to accelerate the creation of germplasm resources can provide more research approaches for turnip breeding.

[0003] Plant protoplasts refer to plant cells without cell walls, which have certain reproductive ability and can develop into complete plants under appropriate culture conditions. Because protoplasts have no cell walls, they are relatively easy to overcome the barriers of distant hybridization incompatibility and uptake exogenous macromolecules such as organelles and proteins. They are widely used in subcellular localization, gene expression analysis, protein-protein interaction, gene editing, etc., and are considered ideal materials for developmental biology, cell biology and cytogenetics research. At present, there are very few relevant studies on the preparation of turnip protoplasts. Establishing and optimizing the preparation system of turnip protoplasts can provide a methodological basis for realizing the cell fusion of turnip protoplasts from different varieties and the efficient introduction and expression of foreign genes. Summary of the Invention

[0004] The purpose of the present invention is to provide an application of an enzymatic hydrolysate in the preparation of turnip protoplasts. The enzymatic hydrolysate can efficiently enzymolyze turnip plant tissues to achieve the preparation of turnip protoplasts, and the obtained protoplasts have a high yield and viability.

[0005] The purpose of the present invention is achieved by the following technical solutions: The present invention provides an application of an enzymatic hydrolysate in the preparation of turnip protoplasts. The enzymatic hydrolysate includes cellulase, macerozyme and pectinase; the mass ratio of cellulase, macerozyme and pectinase is (1.5 - 2.5):(0.8 - 1.0):(0.1 - 0.2).

[0006] The present invention provides an enzymolysis solution for preparing turnip protoplasts, which, based on the total volume of the enzymolysis solution, comprises: 1.5 - 2.5 wt.% cellulase, 0.8 - 1.0 wt.% macerozyme, 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.

[0007] The present invention provides a preparation method of the enzymolysis solution described in the above technical solution, comprising the following steps: Mix and heat cellulase, macerozyme, pectinase, mannitol, MES, and KCl to obtain a mixed enzymolysis solution; After cooling the mixed enzymolysis solution, mix it with CaCl₂, BSA, and β-mercaptoethanol and make up the volume to obtain the enzymolysis solution.

[0008] Preferably, the heating temperature is 54 - 56 °C; the heating time is 9 - 12 min.

[0009] The present invention provides a preparation method of turnip protoplasts, comprising the following steps: Use the enzymolysis solution described in the above technical solution or the enzymolysis solution prepared by the preparation method described in the above technical solution to enzymolyze turnip plant tissues to obtain turnip protoplasts.

[0010] Preferably, the enzymolysis temperature is 28 - 30 °C; the enzymolysis time is 3 - 4 h; the enzymolysis process is accompanied by oscillation; the rotation speed of the oscillation is 45 - 50 rpm.

[0011] Preferably, the plant tissues of turnip include the leaves and / or cotyledons of turnip; the preparation method of the cotyledons of turnip comprises: Cultivate turnip seeds through tissue culture for 4 - 6 d to obtain turnip seedlings; After subjecting the turnip seedlings to light avoidance treatment, take the cotyledons of turnip.

[0012] Preferably, during enzymolysis, the ratio of the number of cotyledon pieces to the volume of the enzymolysis solution is (20 - 30) pieces: 15 mL; the cotyledons are cut into filaments with a width of 0.5 - 1 mm for enzymolysis.

[0013] Preferably, after enzymolysis is completed, it further includes washing and resuspending the enzymolysis product; the solution for washing includes W5 solution; the solution for resuspending includes MMG solution.

[0014] 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.

[0015] Advantages of the present invention: The present invention provides an application of an enzymatic hydrolysate in the preparation of turnip protoplasts. The enzymatic hydrolysate includes cellulase, macerozyme, and pectinase; the mass ratio of cellulase, macerozyme, and pectinase is (1.5 - 2.5):(0.8 - 1.0):(0.1 - 0.2). In the enzymatic hydrolysate provided by the present invention, cellulase has the function of degrading cellulose in the plant cell wall, macerozyme is used in combination with cellulase to separate plant tissues into single cells, and the main function of pectinase is to degrade pectin in the plant cell wall to promote cell separation. Through the combined action of the three enzymes, the enzymatic hydrolysate can synergistically promote the lysis of the cell wall of turnip plant tissues, thereby maximizing the release of turnip protoplasts, which is beneficial to improving the yield of protoplasts and can also keep the protoplasts with high vitality at the same time.

[0016] Furthermore, the present invention also provides an enzymatic hydrolysate for preparing turnip protoplasts, including: 1.5 - 2.5 wt.% cellulase, 0.8 - 1.0 wt.% macerozyme, 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. On the basis of including cellulase, pectinase, and macerozyme, the enzymatic hydrolysate provided by the present invention also includes mannitol, MES, KCl, CaCl2, BSA, and β-mercaptoethanol. The corresponding components have the functions of stabilizing the osmotic pressure, maintaining the activity of protoplasts, and regulating the pH, etc., which is beneficial to the preparation of turnip protoplasts and is also beneficial to improving the yield and activity of turnip protoplasts, etc. The results of the examples of the present invention show that using the enzymatic hydrolysate with turnip cotyledons with petioles as the material, turnip protoplasts can be efficiently prepared, and the obtained protoplasts have a high yield and vitality. Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0018] Figure 1 Observation result diagram of turnip protoplasts prepared in Example 4; Figure 2 Observation result diagram of turnip protoplasts prepared in Comparative Example 3; Figure 3 Observation result diagram of the turnip protoplasts prepared in Comparative Example 4. Specific implementation manners

[0019] The present invention provides an application of an enzymatic hydrolysis solution in the preparation of turnip protoplasts. The enzymatic hydrolysis solution includes cellulase, macerozyme, and pectinase; the mass ratio of the cellulase, macerozyme, and pectinase is (1.5 - 2.5):(0.8 - 1.0):(0.1 - 0.2). In the enzymatic hydrolysis solution, the cellulase, macerozyme, and pectinase act on the turnip plant tissue comprehensively, and can synergistically play a role in promoting the lysis of the cell wall of the turnip plant tissue, thereby maximizing the release of turnip protoplasts, being beneficial to improving the protoplast yield, and at the same time enabling the protoplasts to maintain a relatively high activity. The enzymatic hydrolysis solution provided by the present invention can be used for preparing protoplasts of turnip plant tissues. The cotyledons of turnips have significant differences in the composition of cellulose, hemicellulose, pectin, etc. of their cell walls compared with other tissues such as turnip leaves, making it often difficult to prepare their protoplasts. In practice, the yield and viability of protoplast extraction from turnip cotyledons are often relatively low. For the petiolate cotyledons with relatively large cell differentiation potential, their protoplasts often have more significant advantages in subsequent applications. The enzymatic hydrolysis solution provided by the present invention can achieve the efficient extraction of protoplasts in the petiolate cotyledons of turnips during the preparation of turnip protoplasts, and improve the yield and viability of the protoplasts of the petiolate cotyledons.

[0020] The present invention provides an enzymatic hydrolysis solution for preparing turnip protoplasts, including, based on the total volume of the enzymatic hydrolysis solution: 1.5 - 2.5 wt.% cellulase, 0.8 - 1.0 wt.% macerozyme, 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.

[0021] In the present invention, based on the total volume of the enzymatic hydrolysis solution, the enzymatic hydrolysis solution includes 1.5 - 2.5 wt.% cellulase. As an optional implementation manner of the present invention, the mass percentage content 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 has no special limitation on the source of the cellulase, and conventional commercially available products in the art can be used. In the examples of the present invention, cellulase RS is taken as an example to illustrate the effect of the enzymatic hydrolysis solution. Adding cellulase to the enzymatic hydrolysis solution for preparing turnip protoplasts of the present invention has the effect of degrading cellulose in the cell wall of the turnip plant tissue.

[0022] In the present invention, based on the total volume of the enzymatic hydrolysate, the enzymatic hydrolysate comprises 0.8 - 1.0 wt.% macerozyme. As an alternative embodiment of the present invention, the mass percentage content of the macerozyme can be 0.8%, 0.9% or 1.0%. The present invention has no special limitation on the source of the macerozyme, and conventional commercially available products in the art can be used. In the examples of the present invention, macerozyme R-10 is taken as an example to illustrate the effect of the enzymatic hydrolysate. In the enzymatic hydrolysate for preparing turnip protoplasts of the present invention, the addition of macerozyme and cellulase promotes the separation of plant tissues into single cells.

[0023] In the present invention, based on the total volume of the enzymatic hydrolysate, the enzymatic hydrolysate comprises 0.1 - 0.2 wt.% pectinase. As an alternative embodiment of the present invention, the mass percentage content of the pectinase can be 0.1%, 0.15% or 0.2%. The present invention has no special limitation on the source of the pectinase, and conventional commercially available products in the art can be used. In the examples of the present invention, pectinase Y-23 is taken as an example to illustrate the effect of the enzymatic hydrolysate. In the enzymatic hydrolysate for preparing turnip protoplasts of the present invention, the addition of pectinase can degrade pectin in the cell wall of turnip plant tissues and promote cell separation.

[0024] In the present invention, based on the total volume of the enzymatic hydrolysate, the enzymatic hydrolysate comprises 0.55 - 0.6 mol / L mannitol. As an alternative embodiment of the present invention, the molar concentration of mannitol in the enzymatic hydrolysate can be 0.55, 0.56, 0.57, 0.58, 0.59 or 0.60 mol / L. The present invention has no special limitation on the source of the mannitol, and conventional commercially available products in the art can be used. In the present invention, the main function of adding mannitol to the enzymatic hydrolysate is to stabilize the osmotic pressure and maintain the activity of protoplasts.

[0025] In the present invention, based on the total volume of the enzymatic hydrolysate, the enzymatic hydrolysate comprises 19 - 21 mmol / L MES. As an alternative embodiment of the present invention, the molar concentration of MES in the enzymatic hydrolysate can be 19, 20 or 21 mmol / L. The present invention has no special limitation on the source of the MES, and conventional commercially available products in the art can be used. In the present invention, the addition of MES to the enzymatic hydrolysate is mainly to maintain the stability of the pH value of the system.

[0026] In the present invention, based on the total volume of the enzymatic hydrolysate, the enzymatic hydrolysate comprises 19 - 21 mmol / L KCl. As an alternative embodiment of the present invention, the molar concentration of KCl in the enzymatic hydrolysate can be 19, 20 or 21 mmol / L. The present invention has no special limitation on the source of the KCl, and conventional commercially available products in the art can be used. In the present invention, the addition of KCl to the enzymatic hydrolysate mainly functions to assist in regulating the osmotic pressure, maintain the osmotic pressure stability inside and outside the cell, help maintain the morphological integrity of the protoplast, and maintain the stability of the cell membrane.

[0027] In the present invention, based on the total volume of the enzyme digestion solution, the enzyme digestion solution comprises 9-11 mmol / L CaCl₂. As an optional embodiment of the present invention, the molar concentration of CaCl₂ in the enzyme digestion solution can be 9, 10 or 11 mmol / L. The present invention has no special limitation on the source of the CaCl₂, and conventional commercially available products in the art can be used. The main function of adding CaCl₂ to the enzyme digestion solution in the present invention is to stabilize the membrane structure of the protoplast and protect the protoplast from damage during the enzyme digestion process.

[0028] In the present invention, based on the total volume of the enzyme digestion solution, the enzyme digestion solution comprises 0.09-0.11 wt.% BSA. As an optional embodiment of the present invention, the mass percentage content of BSA in the enzyme digestion solution can be 0.09, 0.10 or 0.11 wt.%. The present invention has no special limitation on the source of the BSA, and conventional commercially available products in the art can be used. Adding BSA to the enzyme digestion solution in the present invention helps to maintain the stability and activity of the enzyme.

[0029] In the present invention, based on the total volume of the enzyme digestion solution, the enzyme digestion solution comprises 0.04-0.06 mmol / L β-mercaptoethanol. As an optional embodiment of the present invention, the molar concentration of β-mercaptoethanol in the enzyme digestion solution can be 0.04, 0.05 or 0.06 mmol / L. The present invention has no special limitation on the source of the β-mercaptoethanol, and conventional commercially available products in the art can be used. Adding β-mercaptoethanol to the enzyme digestion solution in the present invention can protect the protoplast from oxidative damage.

[0030] Three enzyme preparations, namely cellulase, macerozyme and pectinase, are added to the enzyme digestion solution provided by the present invention. The three enzymes can synergistically degrade the cell wall of the turnip tissue in the preparation of turnip protoplasts, thereby releasing the protoplasts to the greatest extent. At the same time, it is also beneficial to improve the activity of the protoplasts. Components such as mannitol, MES, KCl, CaCl₂, BSA and β-mercaptoethanol in the enzyme digestion solution have the effects of stabilizing the osmotic pressure, maintaining the activity of the protoplast and regulating the pH, etc., which is beneficial to the preparation of turnip protoplasts and is also beneficial to improving the yield and activity of turnip protoplasts, etc.

[0031] The present invention provides a preparation method of the enzyme digestion solution described in the above technical solution, comprising the following steps: Mix and heat cellulase, macerozyme, pectinase, mannitol, MES and KCl to obtain a mixed enzyme digestion solution; After cooling the mixed enzyme digestion solution, mix it with CaCl₂, BSA and β-mercaptoethanol and make up the volume to obtain the enzyme digestion solution.

[0032] The present invention mixes cellulase, macerozyme, pectinase, mannitol, MES acid-base buffer and KCl and heats them to obtain a mixed enzymatic hydrolysis solution. As an alternative embodiment of the present invention, the MES can be added in the form of 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 has no special limitation on the mixing method, and any conventional mixing method in the art can be used. After mixing, a mixed system is obtained. The present invention preferably heats the mixed system. As an alternative 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 min, or 9, 10, 11 or 12 min. The present invention has no special limitation on the heating method, and any conventional heating method in the art can be used. As an alternative embodiment of the present invention, the heating method can be water bath heating. The heating in the process of preparing the enzymatic hydrolysis solution in the present invention is mainly to promote the dissolution of the enzyme and improve the activity of the enzyme in the subsequent enzymatic hydrolysis process. After heating, a mixed enzymatic hydrolysis solution is obtained.

[0033] After obtaining the mixed enzymatic hydrolysis solution, the present invention cools the mixed enzymatic hydrolysis solution and then mixes it with CaCl2, BSA and β-mercaptoethanol and makes up the volume to obtain an enzymatic hydrolysis solution. The cooling in the present invention is preferably cooled to room temperature; the room temperature can be 20-25 °C, or 20, 21, 22, 23, 24 or 25 °C. After obtaining the enzymatic hydrolysis solution, the present invention preferably further includes filtering and sterilizing the enzymatic hydrolysis solution with a 0.45 μm filter membrane. The enzymatic hydrolysis solution sterilized by filtration in the present invention can be directly applied to the preparation of turnip protoplasts.

[0034] The present invention provides a method for preparing turnip protoplasts, comprising the following steps: Using 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 to enzymatically hydrolyze turnip plant tissues to obtain turnip protoplasts.

[0035] As an alternative embodiment of the present invention, the turnip plant tissue includes the leaves and / or cotyledons of turnips. The enzyme solution provided by the present invention can be used to prepare protoplasts of turnip plant tissue, and can further prepare protoplasts of plant tissues such as turnip cotyledons that are difficult to extract protoplasts. The present invention has no special limitation on the method for obtaining the cotyledons of turnips, and any conventional method in the art can be used. As an alternative embodiment of the present invention, the cotyledons of turnips can be sterile cotyledons of turnips. As an alternative embodiment of the present invention, the method for preparing the cotyledons of turnips includes: culturing turnip seeds in tissue culture for 4-6 days to obtain turnip seedlings; after treating the turnip seedlings in the dark, taking the cotyledons of turnips. As an alternative embodiment of the present invention, the cotyledons can be cotyledons with petioles; the length of the petioles of the cotyledons with petioles can be 3-5 mm, or can also be 3, 4, or 5 mm.

[0036] In the present invention, the turnip seeds are preferably seeds with full seeds and intact epidermis. After obtaining the turnip seeds, the present invention preferably disinfects the turnip seeds before conducting tissue culture on the turnip seeds. The present invention does not specifically limit the disinfection method, and any conventional disinfection method in the art can be used. In the present invention, the disinfection method can be to first use alcohol for the first disinfection, and then use HgCl2 solution for the second disinfection. In the present invention, the alcohol is preferably alcohol with an ethanol volume fraction of 75%; the time for the first disinfection can be 15s. After the first disinfection is completed, the present invention preferably performs a second disinfection on the seeds that have completed the first disinfection. When the present invention performs the second disinfection, the mass concentration of the HgCl2 solution can be 0.2%. The time for the second disinfection of the present invention can be 5min. After the second disinfection is completed, the present invention preferably washes the obtained seeds. The present invention preferably uses sterile water to wash the seeds; the number of washings can be 5 times, and the time for each washing can be 1min. After the washing is completed, the present invention preferably absorbs the moisture on the surface of the seeds and then inoculates the seeds in the culture medium for tissue culture. The present invention does not specifically limit the inoculation method, and any conventional inoculation method in the art can be used. In the present invention, the culture medium uses 1 / 2MS culture medium as the basic culture medium, and also includes 1-1.5wt.% sucrose and 0.65-0.7wt.% agar. As an optional embodiment of the present invention, the sucrose can be 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5wt.%; the agar can 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 can be 5.75-5.85, or 5.75, 5.8 or 5.85. In the present invention, the temperature of the tissue culture can be 23-25°C, or 23, 24 or 25°C; the light intensity of the tissue culture can be 2500-3500Lx, or 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400 or 3500Lx; the photoperiod of the tissue culture can be 16h of light and 8h of darkness per day. In the present invention, the time of the tissue culture can be 4-6d, or 4, 5 or 6d. The present invention tissue culture is carried out for 4-6d to obtain turnip seedlings.

[0037] After obtaining the turnip seedlings, the present invention preferably performs 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 the turnip protoplasts.

[0038] The present invention uses the enzymatic hydrolysate to enzymatically hydrolyze the turnip plant tissue to obtain turnip protoplasts. As an alternative embodiment of the present invention, the present invention uses the enzymatic hydrolysate to enzymatically hydrolyze the cotyledons of turnips to obtain turnip protoplasts; the cotyledons can be petiolate cotyledons. When the present invention performs enzymatic hydrolysis, the ratio of the number of cotyledon pieces to the volume of the enzymatic hydrolysate can be (20 - 30) pieces: 15 mL, or can also be 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 cotyledons can be cut into filaments with a width of 0.5 - 1 mm for enzymatic hydrolysis, or can also be cut into filaments with a width of 0.5, 0.6, 0.7, 0.8, 0.9 or 1 mm for enzymatic hydrolysis.

[0039] As an alternative embodiment of the present invention, the temperature of the enzymatic hydrolysis is 28 - 30 °C, or can also be 28, 29 or 30 °C; the time of the enzymatic hydrolysis can be 3 - 4 h, or can also be 3, 3.5 or 4 h; the enzymatic hydrolysis process is accompanied by oscillation; the rotation speed of the oscillation is 45 - 50 rpm, or can also be 45, 46, 47, 48, 49 or 50 rpm; the enzymatic hydrolysis process is carried out in the dark. The enzymatic hydrolysis conditions of the present invention can ensure the optimal enzymatic hydrolysis conditions, and at the same time, can keep the protoplasts with higher activity. For example, if the enzymatic hydrolysis time is too long relative to the enzymatic hydrolysis time described in the present invention, it will cause the protoplasts without cell wall protection to be extremely easy to rupture and degrade, resulting in a decrease in the number and activity of protoplasts.

[0040] As an alternative embodiment of the present invention, during or after the enzymatic hydrolysis process, the enzymatic hydrolysis product can be gently pipetted with a sterile pipette tip with a cut end to fully release the protoplasts.

[0041] After the enzymatic hydrolysis is completed, the present invention obtains the protoplasts of Brassica rapa L. var. rapa. Alternatively, as an optional embodiment of the present invention, after obtaining the enzymatic hydrolysis product, the present invention preferably further includes washing and resuspending the enzymatic hydrolysis product to obtain protoplasts. After the enzymatic hydrolysis is completed, the present invention preferably filters the enzymatic hydrolysis product; the filtration is preferably carried out using a 70 µm cell strainer. Before using the cell strainer for filtration in the present invention, it is preferred to rinse the cell strainer by rotating with W5 solution. After the filtration is completed, the present invention preferably gently presses the unhydrolyzed tissue in the cell strainer with the piston of a sterile syringe, and then slowly adds W5 solution to wash the unhydrolyzed tissue; the number of presses can be 3 to 5 times, or 3, 4, or 5 times; the number of washes can be 2 to 3 times. As an optional embodiment of the present invention, the composition of the W5 solution includes: 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 pH of the solution is 5.7. After the filtration is completed, the present invention preferably collects the filtrate, that is, obtains the filtered enzymatic hydrolysis solution.

[0042] After obtaining the filtered enzymatic hydrolysis solution, the present invention preferably centrifuges the filtered enzymatic hydrolysis solution; the rotation speed of the centrifugation can be 100 g; the time of the centrifugation can be 7 min. After the centrifugation is completed, the present invention preferably discards the supernatant and takes the precipitate. The obtained precipitate is preferably resuspended with 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 washing is completed, the present invention preferably resuspends the washed protoplasts with MMG solution to obtain a solution containing Brassica rapa L. var. rapa protoplasts, that is, 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 pH of the solution is 5.7.

[0043] The method for preparing the Brassica rapa L. var. rapa protoplasts of the present invention is simple and efficient, shortens the enzymatic hydrolysis time, and can obtain a large number of Brassica rapa L. var. rapa protoplasts that maintain viability for a long time.

[0044] The present invention also provides the application of the Brassica rapa L. var. rapa protoplasts obtained by the above technical solution in Brassica rapa L. var. rapa breeding and / or bioinformatics. The application in bioinformatics includes any one or more of subcellular localization, gene expression analysis, protein-protein interaction, and gene editing.

[0045] In order to further illustrate the present invention, the technical solution provided by the present invention will be described in detail below with reference to the drawings and embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0046] Sources of raw materials used in the following protocols: Cellulase RS, Macerozyme R-10, and Pectolyase Y-23 were purchased from YAKULT PHARMACEUTICAL IND. CO., LTD.; MS medium was purchased from Phyto Tech LABS; MES, mannitol, KCl, CaCl2, BSA (bovine serum albumin), β-mercaptoethanol, agar, sucrose, etc. were purchased from Sangon Biotech (Sangon Biotech (Shanghai) Co., Ltd.); 0.45 μm filter membrane was purchased from Sartorius (Sartorius Stedim Biotech GmbH, Germany); disposable sterile cell strainer was purchased from Biosharp (Biosharp Life Sciences, Beijing Labgic Technology Co., Ltd.).

[0047] Experimental tools used in the following protocols, such as forceps, dissecting blades, glass culture dishes, centrifuge tubes, pipette tips, etc. were sterilized at 121 °C for 25 min. The cell filtration sieve was a disposable sterile cell strainer, and the filter membrane was a 0.45 µm sterile microporous filter membrane; after wiping the laminar flow hood with 75% alcohol, it was irradiated with ultraviolet light for 30 min and ventilated for 10 min.

[0048] Prepare the MMG solution, with the composition: 0.4 mol / L mannitol, 15 mmol / L MgCl2 solution, 4 mmol / L MES, and the solution pH is 5.7.

[0049] Prepare the W5 solution, with the composition: 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.

[0050] Example 1 An enzyme solution for preparing turnip protoplasts, with the composition based on the total volume of the enzyme solution: 2.0% (mass concentration) Cellulase RS, 1.0% (mass concentration) Macerozyme R-10, 0.1% (mass concentration) Pectolyase Y-23, 0.55 mol / L mannitol at the final concentration, 20 mmol / L MES at the final concentration, MES was added in the form of a MES acid-base buffer with pH 5.7, 20 mmol / L KCl at the final concentration, 10 mmol / L CaCl2 at the final concentration, 0.1 wt.% BSA at the final concentration, and 0.05 mmol / L β-mercaptoethanol at the final concentration.

[0051] The preparation method of the enzymatic hydrolysate is as follows: According to the composition of the enzymatic hydrolysate, calculate the addition amounts of various substances. Then, mix cellulase RS, macerozyme R-10, pectinase Y-23, mannitol, MES acid-base buffer, and KCl, and perform a water bath at 55 °C for 10 min. After the water bath is completed, cool to room temperature, add CaCl2, BSA, and β-mercaptoethanol, and then add ultrapure water to make up to 15 mL to obtain the enzymatic hydrolysate.

[0052] 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 standby.

[0053] Examples 2-3 and Comparative Examples 1-2 Based on the total volume of the enzymatic hydrolysate, the compositions of the enzymatic hydrolysates for preparing turnip protoplasts in Examples 1-3 and Comparative Examples 1-2 are shown in Table 1 in detail. The preparation methods of the enzymatic hydrolysates in Examples 2-3 and Comparative Examples 1-2 are the same as those in Example 1.

[0054] Table 1 Compositions of the enzymatic hydrolysates in Examples 1-3

[0055] Example 4 A method for preparing turnip protoplasts, the steps are as follows: 1. Turnip tissue culture: Select about 100 turnip seeds with plump grains and intact epidermis, put them in a sterilized 50 mL centrifuge tube, and complete the steps of seed disinfection and inoculation in a laminar flow hood. Specifically as follows: First, disinfect with 75% ethanol aqueous solution for 15 s, then soak with 0.2% HgCl2 solution for 5 min, then wash with 45 mL of sterile water 5 times, each wash for about 1 min, then gently blot the water on the surface of the seeds with sterile filter paper, and place them on a 1 / 2MS + 1.5% sucrose + 0.7% agar (pH = 5.8 ± 0.05) medium for culture. The culture conditions are all: temperature 24 °C ± 1 °C, photoperiod 16 h light / 8 h dark, light intensity about 3000 Lx.

[0056] 2. Prepare the enzymatic hydrolysate (the enzymatic hydrolysate of Example 1) Based on the total volume of the enzymatic hydrolysate, the composition of the enzymatic hydrolysate is: cellulase RS with a mass concentration of 2.0%, macerozyme 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 is 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 wt.%, and β-mercaptoethanol with a final concentration of 0.05 mmol / L.

[0057] The preparation method of the 15 mL enzymatic hydrolysate is as follows: According to the composition of the enzymatic hydrolysate, calculate the addition amounts of various substances, then mix cellulase RS, macerozyme R-10, pectinase Y-23, mannitol, MES acid-base buffer, and KCl, and perform a water bath at 55 °C for 10 min. After the water bath is completed, cool to room temperature, add CaCl2, BSA, and β-mercaptoethanol, and then add ultrapure water to make up the volume to 15 mL to obtain the enzymatic hydrolysate. 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 standby.

[0058] 3. Dark-treat the turnip seedlings cultured for 4 - 6 days for 24 h, and then take a total of 30 petiolate cotyledons. The length of the petiole is about 3 - 5 mm. Cut the petiolate cotyledons into filaments with a width of 1 mm, and quickly put the cut leaves (with a width of 1 mm) into a 50 mL centrifuge tube containing 15 mL of enzymatic hydrolysate. Wrap the centrifuge tube with tinfoil and place it on a flat shaker, and perform enzymatic hydrolysis at 28 °C and 45 rpm in the dark for 3.5 h.

[0059] 4. Place a 70 µm cell filter sieve on a new 50 mL sterile centrifuge tube, and add about 1 mL of W5 solution to rotate and rinse the cell filter sieve. Gently pipette the enzymatic hydrolysis product obtained in step 3 about 6 - 8 times with a sterile 1 mL pipette tip with a cut end to fully release the protoplasts. Then transfer all the enzymatic hydrolysis products in batches to the rinsed cell sieve for filtration. Gently press the unhydrolyzed tissue in the cell filter sieve 3 - 5 times with the piston of a 5 mL sterile syringe, then slowly add 3 mL of W5 solution to rinse the unhydrolyzed tissue, and repeat the rinsing 2 more times. Collect the filtrate, which is the filtered enzymatic hydrolysate.

[0060] 5. Centrifuge the filtered enzymatic hydrolysate obtained in step 4 at 100 g 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. The resuspended liquid is then filtered through a 40 µm cell filter sieve. Continue to slowly add 1 mL of W5 solution to wash the residual tissue on the cell sieve, and repeat once (that is, slowly add 1 mL of W5 solution to wash the residual tissue on the cell sieve, the same below), and collect the filtrate. Then centrifuge the filtrate at 100 g for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain the turnip protoplast suspension.

[0061] 6. Use FDA (fluorescein diacetate) to detect the vitality 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 for 5 minutes in the dark. 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 vitality value. The vitality 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.

[0062] Statistical protoplast activity: protoplast activity = (number of protoplasts emitting yellow-green fluorescence / total number of protoplasts) × 100%.

[0063] Counting the protoplast concentration: Pipette the prepared protoplasts and drop them on one end of the cover glass of the blood cell counting plate. Wait for the droplet to fill the entire cover glass until no bubbles appear. After standing for two minutes, observe and count. The density of the protoplasts prepared in this example is about 34.22×10 5 Pieces / mL.

[0064] Protoplast density (pcs / mL) = number of protoplasts in 25 squares × 10 4 × dilution factor.

[0065] Example 5 A method for preparing turnip protoplasts, comprising the following steps: 1. The method of tissue culture of turnip is the same as that of Example 4.

[0066] 2. Preparation of enzymatic hydrolysis solution (the enzymatic hydrolysis solution of Example 2) Based on the total volume of the enzymatic hydrolysate, the composition of the enzymatic hydrolysate is: 1.5% mass concentration of cellulase RS, 1.0% mass concentration of macerate R-10, 0.1% mass concentration of pectinase Y-23, 0.55 mol / L final concentration of mannitol, 20 mmol / L final concentration of MES, MES added in the form of MES acid-base buffer with a pH of 5.7, 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.

[0067] The preparation method of 15 mL of enzymatic hydrolysate is the same as that in Example 4.

[0068] 3. Keep the turnip seedlings cultured for 4 - 6 days in the dark for 24 h. Then, take a total of 30 petiolate cotyledons, with the petiole length about 3 - 5 mm. Cut the petiolate cotyledons into filaments with a width of 1 mm, and quickly put the cut filaments (width 1 mm) of the leaves into a 50 mL centrifuge tube containing 15 mL of enzyme solution. Wrap the centrifuge tube with tin foil and place it on a flat shaker to oscillate in the dark. Under the conditions of 28 °C and 45 rpm, enzymolyze for 3.5 h.

[0069] 4. Place a 70 µm cell strainer on a new 50 mL sterile centrifuge tube, and add about 1 mL of W5 solution to rotate and rinse the cell strainer. Gently pipette the enzymolysis product obtained in step 3 about 6 - 8 times with a sterile 1 mL pipette tip with a cut end to fully release the protoplasts. Then, transfer all the enzymolysis product in batches to the rinsed cell strainer for filtration. Gently press the unenzymolyzed tissue in the cell strainer 3 - 5 times with the piston of a 5 mL sterile syringe, and then slowly add 3 mL of W5 solution to rinse the unenzymolyzed tissue, and repeat the rinsing 2 more times. Collect the filtrate, which is the filtered enzymolysis solution.

[0070] 5. Centrifuge the filtered enzymolysis solution obtained in step 4 at 100 g 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. The resuspended liquid is filtered through a 40 µm cell strainer again. Continue to wash the residual tissue on the cell strainer with 1 mL of W5 solution and repeat once, and collect the filtrate. Then, centrifuge the filtrate at 100 g for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain the turnip protoplast suspension.

[0071] 6. Detect the viability of turnip protoplasts using FDA (fluorescein diacetate). Pipette 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 in the dark for 5 min. After staining, aspirate 50 μL and place it on 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 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. In this example, the protoplast viability value prepared is about 89.17%.

[0072] Statistical analysis of protoplast viability: Protoplast viability = (the number of protoplasts emitting yellow - green fluorescence / the total number of protoplasts) × 100%.

[0073] Statistical analysis of protoplast concentration: Pipette the prepared protoplasts and drop them at one end of the cover glass of a hemocytometer. Wait until the liquid droplet fills the entire cover glass without air bubbles, and observe and count after standing for two minutes. In this example, the density of the prepared protoplasts is about 31.76×105 cells / mL.

[0074] Protoplast density (cells / mL) = Number of protoplasts in 25 medium squares × 10 4 × Dilution factor.

[0075] Example 6 A method for preparing turnip protoplasts is as follows: 1. The method of turnip tissue culture is the same as that in Example 4.

[0076] 2. Prepare the enzyme solution (the enzyme solution in Example 3) Based on the total volume of the enzyme solution, the composition of the enzyme solution is: Cellulase RS with a mass concentration of 2.5%, Macerozyme R-10 with a mass concentration of 0.8%, 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 is 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.

[0077] The preparation method of 15 mL of the enzyme solution is the same as that in Example 4.

[0078] 3. Dark-treat the turnip seedlings cultured for 4 - 6 days for 24 h, and then take a total of 30 petiolate cotyledons, with the petiole length about 3 - 5 mm. Cut the petiolate cotyledons into filaments with a width of 1 mm, and quickly put the cut filaments (1 mm wide) into a 50 mL centrifuge tube containing 15 mL of the enzyme solution. Wrap the centrifuge tube with tin foil and place it on a flat shaker to oscillate in the dark at 28 °C and 45 rpm for 3.5 h for enzymatic hydrolysis.

[0079] 4. Place a 70 µm cell filter sieve on a new 50 mL sterile centrifuge tube, and add about 1 mL of W5 solution to rotate and rinse the cell filter sieve. Gently pipette the enzymatic hydrolysis product obtained in step 3 about 6 - 8 times with a sterile 1 mL pipette tip with a cut end to fully release the protoplasts, and then transfer all the enzymatic hydrolysis product in batches to the rinsed cell sieve for filtration. Gently press the unhydrolyzed tissue in the cell filter sieve 3 - 5 times with the piston of a 5 mL sterile syringe, then slowly add 3 mL of W5 solution to rinse the unhydrolyzed tissue, and repeat the rinsing 2 more times. Collect the filtrate, which is the filtered enzyme solution.

[0080] 5. Centrifuge 100 g of the filtered enzymatic hydrolysate obtained in step 4 for 7 min. After centrifugation, discard the supernatant. Slowly add 3 mL of W5 solution along the wall of the centrifuge tube to resuspend the precipitate. The resuspended liquid is then filtered through a 40-μm cell strainer. Continue to wash the residual tissue on the cell strainer with 1 mL of W5 solution and repeat once, and collect the filtrate. Then centrifuge the filtrate at 100 g for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain the turnip protoplast suspension.

[0081] 6. Detect the viability of turnip protoplasts using FDA (fluorescein diacetate). 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 in the dark for 5 min. After staining, aspirate 50 μL and place it on 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 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. In this example, the prepared protoplast viability value is approximately 85.40%.

[0082] Statistical protoplast viability: Protoplast viability = (number of protoplasts emitting yellow-green fluorescence / total number of protoplasts) × 100%.

[0083] Statistical protoplast concentration: Pipette the prepared protoplasts and add them to one end of the cover glass of a hemocytometer. Wait until the liquid droplet fills the entire cover glass without air bubbles, let it stand for two minutes, and then observe and count. In this example, the prepared protoplast density is approximately 30.74×10 5 cells / mL.

[0084] Protoplast density (cells / mL) = number of protoplasts in 25 medium squares × 10 4 × dilution factor.

[0085] Example 7 An enzymatic hydrolysate for preparing turnip protoplasts. Based on the total volume of the enzymatic hydrolysate, the composition of the enzymatic hydrolysate is as follows: cellulase RS with a mass concentration of 2.0%, macerozyme R-10 with a mass concentration of 1.0%, pectinase Y-23 with a mass concentration of 0.2%, mannitol with a final concentration of 0.55 mol / L, MES with a final concentration of 20 mmol / L, MES is added in the form of a 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 wt.%, and β-mercaptoethanol with a final concentration of 0.05 mmol / L.

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

[0087] Example 8 A method for preparing turnip protoplasts comprises the following steps: 1. The turnip tissue culture method is the same as that in Example 4.

[0088] 2. Prepare the enzyme solution, which is the enzyme solution in Example 7.

[0089] 3. Treat the turnip seedlings cultured for 4 - 6 days in the dark for 24 h. Then, take 30 cotyledons with petioles in total, and the length of the petioles is about 3 - 5 mm. Cut the cotyledons with petioles into filaments with a width of 1 mm, and quickly put the cut filaments (about 1 mm wide) into a 50 mL centrifuge tube containing 15 mL of the enzyme solution. Wrap the centrifuge tube with tin foil and place it on a flat shaker to oscillate in the dark. Under the conditions of 28 °C and 45 rpm, carry out enzyme digestion for 3.5 h.

[0090] 4. Place a 70 µm cell filter sieve on a new 50 mL sterile centrifuge tube, and add about 1 mL of W5 solution to rotate and rinse the cell filter sieve. Gently pipette the enzyme digestion product obtained in step 3 about 6 - 8 times with a sterile 1 mL pipette tip with a cut end to fully release the protoplasts. Then, transfer all the enzyme digestion products in batches to the rinsed cell sieve for filtration. Gently press the undigested tissue in the cell filter sieve 3 - 5 times with the piston of a 5 mL sterile syringe, and then slowly add 3 mL of W5 solution to rinse the undigested tissue, and repeat the rinsing 2 times. Collect the filtrate, which is the filtered enzyme solution.

[0091] 5. Centrifuge the filtered enzyme solution obtained in step 4 at 100 g 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. The resuspended liquid is filtered through a 40 µm cell filter sieve again. Continue to wash the residual tissue on the cell filter sieve with 1 mL of W5 solution, and repeat once, and collect the filtrate. Then, centrifuge the filtrate at 100 g for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain the turnip protoplast suspension.

[0092] 6. Detect the viability of turnip protoplasts using FDA (fluorescein diacetate). Pipette 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 in the dark for 5 min. After staining, aspirate 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 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. In this example, the protoplast viability value prepared is about 82.63%.

[0093] Statistical analysis of protoplast viability: Protoplast viability = (number of protoplasts emitting yellow-green fluorescence / total number of protoplasts) × 100%.

[0094] Statistical analysis of protoplast concentration: Pipette the prepared protoplasts and add them to one end of the coverslip of a hemocytometer. Wait until the liquid droplet fills the entire coverslip without air bubbles. After standing for two minutes, observe and count. In this example, the density of the prepared protoplasts is approximately 32.28×10 5 cells / mL.

[0095] Protoplast density (cells / mL) = number of protoplasts in 25 medium-sized squares × 10 4 × dilution factor.

[0096] Comparative Example 3 A method for preparing turnip protoplasts is as follows: 1. The method of turnip tissue culture is the same as in Example 4.

[0097] 2. Prepare the enzyme solution (the enzyme solution of Comparative Example 1).

[0098] 3. Treat the turnip seedlings cultured for 4 - 6 days in the dark for 24 hours. Then, take a total of 30 petiolate cotyledons, and the length of the petiole is about 3 - 5 mm. Cut the petiolate cotyledons into filaments with a width of 1 mm. Quickly put the cut filaments (width 1 mm) of the leaves into a 50 mL centrifuge tube containing 15 mL of the enzyme solution. Wrap the centrifuge tube with tin foil and place it on a flat shaker. Oscillate in the dark at 28°C and 45 rpm for 3.5 hours for enzymatic hydrolysis.

[0099] 4. Place a 70 µm cell filter sieve on a new 50 mL sterile centrifuge tube, and add about 1 mL of W5 solution to rotate and rinse the cell filter sieve. Gently pipette the enzymatic hydrolysis product obtained in step 3 about 6 - 8 times with a sterile 1 mL pipette tip with a cut end to fully release the protoplasts. Then, transfer all the enzymatic hydrolysis products in batches to the rinsed cell sieve for filtration. Gently press the unhydrolyzed tissue in the cell filter sieve 3 - 5 times with the piston of a 5 mL sterile syringe. Then, slowly add 3 mL of W5 solution to rinse the unhydrolyzed tissue, and repeat the rinsing 2 times. Collect the filtrate, which is the filtered enzymatic hydrolysis solution.

[0100] 5. Centrifuge the filtered enzymatic hydrolysis solution obtained in step 4 at 100 g for 7 minutes. 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. The resuspended liquid is then filtered through a 40 µm cell filter sieve. Continue to wash the residual tissue on the cell filter sieve with 1 mL of W5 solution and repeat once, and collect the filtrate. Then, centrifuge the filtrate at 100 g for 7 minutes, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain the turnip protoplast suspension.

[0101] 6. Detection of the viability of turnip protoplasts using FDA (fluorescein diacetate). Pipette 100 μL of the protoplast suspension into a 1.5 mL centrifuge tube wrapped with aluminum foil in advance, add 3 μL of 1 mg / mL FDA, and stain in the dark for 5 min. After staining, aspirate 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 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 protoplast viability value prepared in this comparative example is approximately 55.96%, as Figure 2 shown, Figure 2 the right figure in

[0102] is the FDA staining result, and the left figure is the protoplast image under the corresponding bright-field view.

[0103] Protoplast viability: Protoplast viability = (number of protoplasts emitting yellow-green fluorescence / total number of protoplasts) × 100%.

[0103] Statistical protoplast concentration: Pipette the prepared protoplasts and add them to one end of the cover glass of a hemocytometer. Wait until the liquid droplet fills the entire cover glass without air bubbles, and observe and count after standing for two minutes. The density of the protoplasts prepared in this comparative example is approximately 2.94×10 5 cells / mL.

[0104] Protoplast density (cells / mL) = number of protoplasts in 25 middle squares × 10 4 × dilution factor Comparative Example 4 A method for preparing turnip protoplasts, the steps are as follows: 1. The method of turnip tissue culture is the same as that in Example 4.

[0105] 2. Prepare the enzyme solution (the enzyme solution of Comparative Example 2).

[0106] 3. Dark-treat the turnip seedlings cultured for 4 - 6 days for 24 h, and then take a total of 30 petiolate cotyledons, and the length of the petiole is about 3 - 5 mm. Cut the petiolate cotyledons into filaments with a width of 1 mm, and quickly put the cut filaments (width 1 mm) of the leaves into a 50 mL centrifuge tube containing 15 mL of the enzyme solution. Wrap the centrifuge tube with aluminum foil and place it on a flat shaker, and shake in the dark at 28 °C and 45 rpm for 3.5 h for enzymatic hydrolysis.

[0107] 4. Place a 70-µm cell strainer on a new 50-mL sterile centrifuge tube, and add approximately 1 mL of W5 solution to rotate and rinse the cell strainer. Gently pipette the enzymatic digestion product obtained in step 3 about 6 - 8 times with a sterile 1-mL pipette tip with the end cut off to fully release the protoplasts. Then transfer all the enzymatic digestion product in batches to the rinsed cell strainer for filtration. Gently press the undigested tissue in the cell strainer 3 - 5 times with the piston of a 5-mL sterile syringe. After that, slowly add 3 mL of W5 solution to rinse the undigested tissue, and repeat the rinsing 2 more times. Collect the filtrate, which is the filtered enzymatic digestion solution.

[0108] 5. Centrifuge the filtered enzymatic digestion solution obtained in step 4 at 100 g 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 pellet. The resuspended liquid is then filtered through a 40-µm cell strainer. Continue to wash the residual tissue on the cell strainer with 1 mL of W5 solution and repeat once, and collect the filtrate. Then centrifuge the filtrate at 100 g for 7 min, discard the supernatant, and resuspend the pellet with 3 mL of MMG solution to obtain the turnip protoplast suspension.

[0109] 6. Detect the viability of turnip protoplasts using FDA (fluorescein diacetate). Pipette 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 in the dark for 5 min. After staining, pipette 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 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 protoplast viability value prepared in this comparative example is approximately 60.99%, as Figure 3 shown, Figure 3 the right figure in

[0110] is the FDA staining result, and the left figure is the image of protoplasts under the corresponding bright-field view.

[0111] Protoplast viability: Protoplast viability = (Number of protoplasts emitting yellow-green fluorescence / Total number of protoplasts) × 100%.

[0111] Statistical protoplast concentration: Pipette the prepared protoplasts and add them to one end of the cover glass of a hemocytometer. Wait until the liquid droplet fills the entire cover glass without air bubbles, and observe and count after standing for two minutes. The density of the protoplasts prepared in this comparative example is approximately 19.34×10 5 cells / mL.

[0112] Protoplast density (cells / mL) = Number of protoplasts in 25 middle squares × 10 4 × Dilution factor.

[0113] Comparative Example 5 A method for preparing turnip protoplasts comprises the following steps: 1. The method of turnip tissue culture is the same as that in Example 4.

[0114] 2. Prepare an enzyme digestion solution (the enzyme digestion solution in Example 1).

[0115] 3. Keep turnip seedlings cultured for 4 - 6 days in the dark for 24 h. Then, take 30 cotyledons with petioles in total, and the length of the petioles is about 3 - 5 mm. Cut the cotyledons with petioles into filaments with a width of 1 mm, and quickly put the cut filaments (width 1 mm) into a 50 mL centrifuge tube containing 15 mL of the enzyme digestion solution. Wrap the centrifuge tube with tin foil and place it on a flat shaker to oscillate in the dark. Under the conditions of 28 °C and 45 rpm, carry out enzyme digestion for 8 h.

[0116] 4. Place a 70 µm cell filter sieve on a new 50 mL sterile centrifuge tube, and add about 1 mL of W5 solution to rotate and rinse the cell filter sieve. Gently pipette the enzyme digestion product obtained in step 3 about 6 - 8 times with a sterile 1 mL pipette tip with a cut end to fully release the protoplasts. Then, transfer all the enzyme digestion products in batches to the rinsed cell sieve for filtration. Gently press the undigested tissue in the cell filter sieve 3 - 5 times with the piston of a 5 mL sterile syringe, and then slowly add 3 mL of W5 solution to rinse the undigested tissue, and repeat the rinsing 2 times. Collect the filtrate, which is the filtered enzyme digestion solution.

[0117] 5. Centrifuge the filtered enzyme digestion solution obtained in step 4 at 100 g 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. The resuspended liquid is filtered through a 40 µm cell filter sieve, and continue to wash the residual tissue on the cell filter sieve with 1 mL of W5 solution, and repeat once, and collect the filtrate. Then, centrifuge the filtrate at 100 g for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain a turnip protoplast suspension.

[0118] 6. Detect the viability of turnip protoplasts using FDA (fluorescein diacetate). Pipette 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 in the dark for 5 min. After staining, aspirate 50 μL and place it on a glass slide, and observe it in the dark environment of an inverted fluorescence microscope (Thermo Fisher EVOS M3000). Viable protoplasts will emit yellow - green fluorescence, and 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 protoplast viability value prepared in this comparative example is about 71.36%.

[0119] Protoplast viability: Protoplast viability = (Number of protoplasts emitting yellow-green fluorescence / Total number of protoplasts) × 100%.

[0120] Statistical protoplast concentration: Pipette the prepared protoplasts and add them to one end of the cover glass of a hemocytometer. Wait until the liquid droplet fills the entire cover glass without air bubbles, let it stand for two minutes, and then observe and count. The density of the protoplasts prepared in this comparative example is approximately 22.52×10 5 cells / mL.

[0121] Protoplast density (cells / mL) = Number of protoplasts in 25 medium squares × 10 4 × Dilution factor.

[0122] Comparative Example 6 A method for preparing turnip protoplasts, the steps are as follows: 1. The turnip tissue culture method is the same as in Example 4.

[0123] 2. Prepare the enzyme solution Based on the total volume of the enzyme solution, the composition of the enzyme solution is: Cellulase RS with a mass concentration of 2.0%, Macerozyme R-10 with a mass concentration of 1.0%, Mannitol with a final concentration of 0.55 mol / L, MES with a final concentration of 20 mmol / L, MES is added in the form of a 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) and BSA (final concentration 0.1 wt.%), β-Mercaptoethanol (final concentration 0.05 mmol / L); The preparation method of the enzyme solution is the same as in Example 1.

[0124] 3. Dark-treat the turnip seedlings cultured for 4 - 6 days for 24 h, then take a total of 30 petiolate cotyledons, and the length of the petiole is about 3 - 5 mm. Cut the petiolate cotyledons into filaments with a width of 1 mm, quickly put the cut filaments (width about 1 mm) into a 50 mL centrifuge tube containing 15 mL of enzyme solution, wrap the centrifuge tube with tin foil and place it on a flat shaker to oscillate in the dark, and under the conditions of 28 °C and 45 rpm, enzymatically digest for 3.5 h.

[0125] 4. Place a 70 µm cell filter sieve on a new 50 mL sterile centrifuge tube, and add about 1 mL of W5 solution to rotate and rinse the cell filter sieve. Gently pipette the enzymatic digestion product obtained in step 3 about 6 - 8 times with a sterile 1 mL pipette tip with a cut end to fully release the protoplasts, then transfer all the enzymatic digestion products in batches to the rinsed cell sieve for filtration, gently press the undigested tissue in the cell filter sieve 3 - 5 times with the piston of a 5 mL sterile syringe, then slowly add 3 mL of W5 solution to rinse the undigested tissue, and repeat the rinsing 2 times, and collect the filtrate, which is the filtered enzyme solution.

[0126] 5. Centrifuge 100 g of the filtered and enzymolyzed solution obtained in step 4 for 7 min. After centrifugation, discard the supernatant. Slowly add 3 mL of W5 solution along the wall of the centrifuge tube to resuspend the precipitate. The resuspended liquid is then filtered through a 40-μm cell strainer. Continue to wash the residual tissue on the cell strainer with 1 mL of W5 solution and repeat once. Collect the filtrate. Then centrifuge the filtrate at 100 g for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain the turnip protoplast suspension.

[0127] 6. Detect the viability of turnip protoplasts using FDA (fluorescein diacetate). Pipette 100 μL of the protoplast suspension into a 1.5-mL centrifuge tube pre-wrapped with aluminum foil, add 3 μL of 1 mg / mL FDA, and stain in the dark for 5 min. After staining, aspirate 50 μL and place it on a glass slide. Observe under an inverted fluorescence microscope (Thermo Fisher EVOS M3000) in a dark environment. Viable protoplasts will emit yellow-green fluorescence. Count the number of yellow-green protoplasts. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field of view is the protoplast viability value. The prepared protoplast viability value is approximately 75.70%.

[0128] Protoplast viability: Protoplast viability = (number of protoplasts emitting yellow-green fluorescence / total number of protoplasts) × 100%.

[0129] Statistical protoplast concentration: Pipette the prepared protoplasts onto one end of the cover glass of a hemocytometer. Wait until the liquid droplet fills the entire cover glass without air bubbles. After standing for two minutes, observe and count. In this comparative example, the prepared protoplast density is approximately 23.36×10 5 cells / mL.

[0130] Protoplast density (cells / mL) = number of protoplasts in 25 medium squares × 10 4 × dilution factor.

[0131] Comparative Example 7 A method for preparing turnip protoplasts, the steps are as follows: 1. The turnip tissue culture method is the same as in Example 4.

[0132] 2. Prepare the enzymolysis solution Based on the total volume of the enzymatic hydrolysate, the composition and preparation method of the enzymatic hydrolysate are as follows: Macerozyme R-10 with a mass concentration of 1.0%, Pectolyase Y-23 with a mass concentration of 0.1%, mannitol (final concentration 0.55 mol / L), MES with a final concentration of 20 mmol / L, MES is added in the form of MES acid-base buffer with a pH of 5.7, KCl (final concentration 20 mmol / L), water bath at 55 °C for 10 min, after cooling to room temperature, add CaCl2 solution (final concentration 10 mmol / L) and BSA (final concentration 0.1 wt.%), β-mercaptoethanol (final concentration 0.05 mmol / L), make up the volume to 15 mL with ultrapure water, filter and sterilize the prepared enzymatic hydrolysate with a 0.45 μm filter membrane, and pour it into a 50 mL centrifuge tube for standby.

[0133] 3. Treat the turnip seedlings cultured for 4 - 6 days in the dark for 24 h, then take a total of 30 petiolate cotyledons, and the petiole length is about 3 - 5 mm. Cut the petiolate cotyledons into filaments with a width of 1 mm, quickly put the cut filaments (width about 1 mm) of the leaves into a 50 mL centrifuge tube containing 15 mL of enzymatic hydrolysate, wrap the centrifuge tube with tin foil and place it on a flat shaker to oscillate in the dark, and carry out enzymatic hydrolysis for 3.5 h at 28 °C and 45 rpm.

[0134] 4. Place a 70 µm cell strainer on a new 50 mL sterile centrifuge tube, and add about 1 mL of W5 solution to rotate and rinse the cell strainer. Gently pipette the enzymatic hydrolysis product obtained in step 3 about 6 - 8 times with a sterile 1 mL pipette tip with a cut end to fully release the protoplasts, then transfer all the enzymatic hydrolysis products in batches to the rinsed cell strainer for filtration, gently press the unhydrolyzed tissue in the cell strainer 3 - 5 times with the piston of a 5 mL sterile syringe, then slowly add 3 mL of W5 solution to rinse the unhydrolyzed tissue, and repeat the rinsing 2 more times, collect the filtrate, which is the filtered enzymatic hydrolysate.

[0135] 5. Centrifuge the filtered enzymatic hydrolysate obtained in step 4 at 100 g for 7 min, discard the supernatant after centrifugation, 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, continue to wash the residual tissue on the cell strainer with 1 mL of W5 solution, repeat 1 more time, and collect the filtrate. Then centrifuge the filtrate at 100 g for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain the turnip protoplast suspension.

[0136] 6. Detection of the viability of turnip protoplasts using FDA (fluorescein diacetate). 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 in the dark for 5 min. After staining, aspirate 50 μL and place it on 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 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 protoplast viability value prepared in this comparative example is approximately 69.46%.

[0137] Protoplast viability: Protoplast viability = (Number of protoplasts emitting yellow-green fluorescence / Total number of protoplasts) × 100%.

[0138] Statistical protoplast concentration: Pipette the prepared protoplasts and add them to one end of the cover glass of a hemocytometer. Wait until the liquid droplet fills the entire cover glass without air bubbles, let it stand for two minutes, and then observe and count. The protoplast density prepared in this comparative example is approximately 2.14×10 5 cells / mL.

[0139] Protoplast density (cells / mL) = Number of protoplasts in 25 middle squares × 10 4 × Dilution factor.

[0140] Comparative Example 8 A method for preparing turnip protoplasts, the steps are as follows: 1. The turnip tissue culture method is the same as in Example 4.

[0141] 2. Prepare the enzyme solution Based on the total volume of the enzyme solution, the composition and preparation method of the enzyme solution are as follows: Cellulase RS with a mass concentration of 2.0%, Pectinase Y-23 with a mass concentration of 0.1%, Mannitol (final concentration 0.55 mol / L), MES with a final concentration of 20 mmol / L, MES is added in the form of a MES acid-base buffer with a pH of 5.7, KCl (final concentration 20 mmol / L), water bath at 55 °C for 10 min, cool to room temperature, and then add CaCl2 solution (final concentration 10 mmol / L) and BSA (final concentration 0.1 wt.%), β-mercaptoethanol (final concentration 0.05 mmol / L), add ultrapure water to make up to 15 mL, filter and sterilize the prepared enzyme solution with a 0.45 μm filter membrane, and pour it into a 50 mL centrifuge tube for standby.

[0142] 3. Keep the turnip seedlings cultivated for 4 - 6 days in the dark for 24 h. Then, take a total of 30 petiolate cotyledons, with the petiole length about 3 - 5 mm. Cut the petiolate cotyledons into filaments with a width of 1 mm, and quickly put the cut filaments (about 1 mm wide) into a 50 mL centrifuge tube containing 15 mL of enzyme solution. Wrap the centrifuge tube with tin foil and place it on a flat shaker to oscillate in the dark. Digest at 28 °C and 45 rpm for 3.5 h.

[0143] 4. Place a 70 µm cell strainer on a new 50 mL sterile centrifuge tube, and add about 1 mL of W5 solution to rotate and rinse the cell strainer. Gently pipette the digestion product obtained in step 3 about 6 - 8 times with a sterile 1 mL pipette tip with a cut end to fully release the protoplasts. Then, transfer all the digestion product in batches to the rinsed cell strainer for filtration. Gently press the undigested tissue in the cell strainer 3 - 5 times with the piston of a 5 mL sterile syringe, and then slowly add 3 mL of W5 solution to rinse the undigested tissue, and repeat the rinsing 2 more times. Collect the filtrate, which is the filtered enzyme solution.

[0144] 5. Centrifuge the filtered enzyme solution obtained in step 3 at 100 g 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. The resuspended liquid is filtered through a 40 µm cell strainer again. Continue to wash the residual tissue on the cell strainer with 1 mL of W5 solution and repeat once, and collect the filtrate. Then, centrifuge the filtrate at 100 g for 7 min, discard the supernatant, and resuspend the precipitate with 3 mL of MMG solution to obtain the turnip protoplast suspension.

[0145] 6. Detect the viability of turnip protoplasts using FDA (fluorescein diacetate). Pipette 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 in the dark for 5 min. After staining, aspirate 50 μL and place it on 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 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 protoplast viability value prepared in this comparative example is about 78.20%.

[0146] Protoplast viability: Protoplast viability = (the number of protoplasts emitting yellow - green fluorescence / the total number of protoplasts) × 100%.

[0147] Statistical protoplast concentration: Pipette the prepared protoplasts and add them to one end of the cover glass of a hemocytometer. Wait until the liquid droplet fills 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 18.46×105 cells / mL.

[0148] Protoplast density (cells / mL) = Number of protoplasts in 25 medium squares × 10 4 × Dilution factor.

[0149] The yields and viabilities of turnip protoplasts in each example and comparative example are shown in Table 2.

[0150] Table 2 Yields and viabilities of turnip protoplasts in each example and comparative example

[0151] Note: The absence of the same lowercase letters indicates a significant difference at the P < 0.05 level.

[0152] As can be seen from Table 2, by comparing Example 4 with Comparative Examples 6 - 8, it can be obtained that when using an enzyme digestion solution containing three enzymes, namely cellulase, macerozyme, and pectinase, to prepare turnip protoplasts, the yield and viability of the obtained turnip protoplasts are significantly higher than those using any two of the enzymes. The effect of using cellulase, macerozyme, and pectinase simultaneously to prepare turnip protoplasts is the best. By comparing Examples 4 - 8 with Comparative Example 4, it can be obtained that the dosages of the three enzymes, cellulase, pectinase, and macerozyme, will have a significant impact on the preparation of turnip protoplasts. When the mass ratio of cellulase, macerozyme, and pectinase is (1.5 - 2.5):(0.8 - 1.0):(0.1 - 0.2), it is beneficial to improve the yield and viability of protoplasts. By comparing Example 4 with Comparative Example 5, it can be obtained that the enzyme digestion time significantly affects the yield and viability of turnip protoplasts. The enzyme digestion time should be reasonably controlled during the preparation of turnip protoplasts.

[0153] In summary, the enzyme digestion solution for preparing turnip protoplasts and the method for preparing turnip protoplasts provided by the present invention can significantly improve the yield and viability of turnip protoplasts.

[0154] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments without creative efforts based on these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of an enzymatic hydrolysate in the preparation of turnip protoplasts, wherein the enzymatic hydrolysate comprises cellulase, macerozyme and pectinase; the mass ratio of cellulase, macerozyme and pectinase is (1.5 - 2.5):(0.8 - 1.0):(0.1 - 0.2).

2. An enzymatic hydrolysis solution for preparing turnip protoplasts, characterized in that, Based on the total volume of the enzymatic hydrolysate, it comprises: 1.5 - 2.5 wt.% cellulase, 0.8 - 1.0 wt.% macerozyme, 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.

3. A method for preparing the enzymatic hydrolysate according to claim 2, characterized in that, It includes the following steps: Mix cellulase, macerozyme, pectinase, mannitol, MES and KCl and heat to obtain a mixed enzymatic hydrolysate; After cooling the mixed enzymatic hydrolysate, mix it with CaCl2, BSA and β-mercaptoethanol and make up the volume to obtain the enzymatic hydrolysate.

4. The preparation method according to claim 3, wherein, The heating temperature is 54 - 56 °C; the heating time is 9 - 12 min.

5. A method for preparing turnip protoplasts, characterized in that, It includes the following steps: Use the enzymatic hydrolysate prepared by the enzymatic hydrolysate described in claim 2 or the preparation method described in claim 3 or 4 to enzymatically hydrolyze turnip plant tissue to obtain turnip protoplasts.

6. The preparation method according to claim 5, characterized in that, The enzymatic hydrolysis temperature is 28 - 30 °C; the enzymatic hydrolysis time is 3 - 4 h; the enzymatic hydrolysis process is accompanied by oscillation; the rotation speed of the oscillation is 45 - 50 rpm.

7. The preparation method according to claim 5, characterized in that, The turnip plant tissue includes turnip leaves and / or cotyledons; The preparation method of the turnip cotyledons includes: Cultivate turnip seeds by tissue culture for 4 - 6 d to obtain turnip seedlings; After subjecting the turnip seedlings to light avoidance treatment, take the cotyledons of turnips.

8. The preparation method according to claim 7, wherein During enzymatic hydrolysis, the ratio of the number of cotyledon pieces to the volume of the enzymatic hydrolysate is (20 - 30) pieces: 15 mL; cut the cotyledons into filaments with a width of 0.5 - 1 mm for enzymatic hydrolysis.

9. The preparation method according to claim 5, characterized in that, After enzymatic hydrolysis is completed, it also includes washing and resuspending the enzymatic hydrolysis product; the solution for washing includes W5 solution; the solution for resuspending includes MMG solution.

10. Use of the turnip protoplasts obtained by the preparation method according to any one of claims 5 - 9 in turnip breeding and / or bioinformatics.

Images