Method for preparing protoplasts of clematis chelidonifolia leaves

Abstract

The application discloses a preparation method of Coptis teeta leaf protoplasts, which comprises the following steps: 1) cutting mature Coptis teeta leaves into filaments, and placing the filaments in an enzymolysis solution for enzymolysis treatment, wherein the mass-volume ratio of the Coptis teeta filaments to the enzymolysis solution is 1:8-12 g / mL, and the enzymolysis solution contains 2%-3% cellulase R-10 and 0.25%-1% isolated enzyme R-10; 2) filtering the mixed solution after the enzymolysis is completed by using a sterile filter screen to remove plant tissues and cell groups that are not completely enzymolyzed, taking the filtrate, centrifuging the filtrate at 800-1400 r / min for 2-5 min to remove supernatant, and obtaining a precipitate; and 3) resuspending the precipitate by using a CPW washing solution, collecting the cell precipitate after centrifugation, and obtaining the Coptis teeta leaf protoplasts. The Coptis teeta leaf protoplasts prepared by the method have high yield and high activity.

Description

Technical Field

[0001] This invention relates to the field of plant protoplast technology. More specifically, this invention relates to a method for preparing protoplasts from leaves of *Coptis chinensis*. Background Technology

[0002] Corydalis rhamnoides, belonging to the genus Corydalis in the family Papaveraceae, is mainly distributed in the rocky mountainous areas of Guangxi, Guizhou, and Yunnan provinces in China. It is a national second-class protected plant and a well-known traditional medicine of the Zhuang and Yao ethnic minorities. The whole plant is used medicinally, primarily for hepatitis, cirrhosis, and liver cancer, and has a broad market prospect. However, its scarce resources, particularly in the crevices of steep rocks in mountains, lead to a supply shortage. Therefore, there is an urgent need to utilize biotechnology to develop and protect existing resources and to innovate germplasm resources through genetic transformation.

[0003] Protoplasts are morphological and structural units that make up cells. Extracting protoplasts from plants and using them as materials for transgenic and mutant breeding is a novel method for cultivating new plant varieties. Because the cell walls of the extracted protoplasts are removed, each cell has the potential to develop into a plant, and they are more receptive to exogenous genetic material, allowing for rapid expression of exogenous DNA within the protoplast. By preparing protoplasts from *Coptis chinensis*, highly active protoplasts for transient expression analysis can be obtained; however, currently, technical information on obtaining *Coptis chinensis* protoplasts is very scarce.

[0004] There are two main methods for obtaining plant protoplasts: mechanical and enzymatic methods. Mechanical methods are complex and have a low yield, while enzymatic methods can rapidly obtain protoplasts in large quantities with minimal cell damage and are currently more widely used. Enzymatic methods often involve simultaneously adding pectinase and cellulase to decompose cells and remove the plant cell walls. However, plant tissues vary in cell wall thickness and vacuolar osmotic pressure due to differences in plant variety and development stage. Improper enzymatic methods can easily reduce the cell activity of protoplasts, lead to contamination by other microorganisms, and affect cell viability. Therefore, exploring methods for preparing protoplasts from *Coptis chinensis* has broad market prospects and is of great significance for the development, protection, and innovation of new germplasm resources of *Coptis chinensis*. Summary of the Invention

[0005] One objective of this invention is to provide a method for preparing protoplasts from Coptis chinensis leaves, wherein the protoplasts prepared by this method have high yield and high activity.

[0006] To achieve the objectives and other advantages of this invention, a method for preparing protoplasts from *Coptis chinensis* leaves is provided, comprising the following steps:

[0007] 1) Cut mature leaves of Coptis chinensis into filaments and place them in an enzymatic hydrolysate for enzymatic hydrolysis. The mass-to-volume ratio of Coptis chinensis filaments to enzymatic hydrolysate is 1:8-12 g / mL. The enzymatic hydrolysate contains 2%-3% cellulase R-10 and 0.25%-1% analyte R-10.

[0008] 2) Filter the enzymatically hydrolyzed mixture through a sterile filter to remove incompletely hydrolyzed plant tissues and cell clusters. Take the filtrate and centrifuge it at 800-1400 r / min for 2-5 min. Remove the supernatant to obtain the precipitate.

[0009] 3) The precipitate was resuspended with CPW washing solution, centrifuged, and the cell precipitate was collected to obtain the protoplasts of the leaves of Coptis chinensis.

[0010] Preferably, in the method for preparing the protoplasts of Coptis chinensis leaves, the mature leaves of Coptis chinensis are taken from Coptis chinensis seedlings that are 2 months old.

[0011] Preferably, in the method for preparing the protoplasts of the leaves of *Coptis chinensis*, during enzymatic hydrolysis, the *Coptis chinensis* protoplast and the enzymatic hydrolysate are placed in the dark, at 24-26°C and 40 r / min on a shaker for 4-8 hours.

[0012] Preferably, in the method for preparing the protoplasts of *Coptis chinensis* leaves, the enzymatic hydrolysate is treated as follows before use: a mixed enzyme solution is prepared, consisting of 2%–3% cellulase R-10, 0.25%–1% analyte R-10, 20 mmol / L MME, 0.5 mol / L mannitol, 10 mmol / L CaCl2, and 20 mmol / L KCl. The mixed enzyme solution is activated in a 55°C water bath for 10 min, cooled to room temperature, and then 0.1% BSA is added to adjust the pH to 5.7. The solution is then filtered through a 0.45 μm microporous membrane for sterilization and stored at 4°C for later use.

[0013] Preferably, in the method for preparing protoplasts from *Coptis chinensis* leaves, the CPW washing solution comprises: 27.2 mg / L KH₂PO₄, 101 mg / L KNO₃, 1117.5 mg / L CaCl₂, 246 mg / L MgSO₄·7H₂O, 0.16 mg / L KI, 0.025 mg / L CuSO₄·5H₂O, 20 mmol / L MES, and 0.6–1.0 mol / L mannitol, with a pH of 5.8.

[0014] Preferably, in the method for preparing protoplasts from Coptis chinensis leaves, the centrifugation conditions in step 3) are the same as those in step 2), which are 1000-1200 r / min for 3 min.

[0015] Preferably, in the method for preparing protoplasts from Coptis chinensis leaves, the mass-to-volume ratio of Coptis chinensis filaments to enzymatic hydrolysate in step 1) is 1:10 g / mL, the enzymatic hydrolysate contains 3% cellulase R-10 and 0.75% dissociative enzyme R-10, and the enzymatic hydrolysis time is 4-7 h.

[0016] The mannitol concentration in the CPW washing solution in step 3) is 0.6 mol / L;

[0017] In steps 2) and 3), the centrifugation conditions are 1200 r / min for 3 min.

[0018] Preferably, in the method for preparing protoplasts from Coptis chinensis leaves, step 2) involves filtering the material sequentially using 200-mesh and 400-mesh sterile filters.

[0019] Preferably, in the method for preparing protoplasts from Coptis chinensis leaves, step 1) involves an enzymatic hydrolysis time of 7 hours.

[0020] Preferably, in the method for preparing protoplasts from Coptis chinensis leaves, in step 1), 2 μmol / L methyl jasmonate and 5 mg / L nano zinc oxide are added to the enzymatic hydrolysate before enzymatic hydrolysis, and the enzymatic hydrolysis time is 4 h.

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

[0022] (1) This invention, through research on the concentration of enzymes used in enzymatic hydrolysis, hydrolysis time, mannitol concentration, and centrifugation rate, has obtained a set of methods for efficiently obtaining protoplasts from Coptis chinensis leaves. This provides important technical support for the resource development and germplasm innovation of Coptis chinensis. Furthermore, this method yields approximately 20.77 × 10⁻¹¹ protoplasts with an activity level as high as 98.76% per gram of fresh Coptis chinensis leaf tissue. 4 indivual;

[0023] (2) By adding an appropriate concentration of methyl jasmonate and nano zinc oxide to the enzymatic hydrolysate, the present invention shortens the enzymatic hydrolysis time and further increases the number and activity of protoplasts. This may be because methyl jasmonate has an inducing and protective effect on protoplast cells, and nano zinc oxide has antibacterial and oxidative protective effects. The addition of the two reduces the loss of protoplasts and increases the yield and activity of protoplasts.

[0024] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0025] Figure 1 The protoplasts of *Coptis chinensis* leaves were obtained by the method for preparing the protoplasts of *Coptis chinensis* leaves in Experiment 1 of the present invention. Detailed Implementation

[0026] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings, so that those skilled in the art can implement it based on the description.

[0027] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0028] It should be noted that, unless otherwise specified, the experimental methods described in the following embodiments are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified. MES is sodium methyl ester sulfonate, BSA is bovine serum albumin, and the cell-protoplast washing solution is composed of: 27.2 mg / L KH2PO4, 101 mg / L KNO3, 1117.5 mg / L CaCl2, 246 mg / L MgSO4·7H2O, 0.16 mg / L KI, and 0.025 mg / L CuSO4·5H2O. The CPW washing solution used in this invention is prepared by adding 20 mmol / L MES and 0.6–1.0 mol / L mannitol to the cell-protoplast washing solution.

[0029] A method for preparing protoplasts from Coptis chinensis leaves includes the following steps:

[0030] 1) Cut mature leaves of Coptis chinensis into filaments (preferably into 0.5-1 mm wide filaments), place them in an enzymatic hydrolysate for enzymatic hydrolysis, and the mass-volume ratio of Coptis chinensis filaments to enzymatic hydrolysate is 1:8-12 g / mL. The enzymatic hydrolysate contains 2%-3% cellulase R-10 and 0.25%-1% dissociative enzyme R-10.

[0031] 2) Filter the enzymatically hydrolyzed mixture through a sterile filter to remove incompletely hydrolyzed plant tissues and cell clusters. Take the filtrate and centrifuge it at 800-1400 r / min for 2-5 min. Remove the supernatant to obtain the precipitate.

[0032] 3) The precipitate was resuspended with CPW washing solution, centrifuged, and the cell precipitate was collected to obtain the protoplasts of the leaves of Coptis chinensis.

[0033] The method for preparing protoplasts from Coptis chinensis leaves according to the present invention requires only two centrifugations after enzymatic hydrolysis to obtain high-yield and highly active protoplasts.

[0034] More preferably, in the method for preparing the protoplasts of Coptis chinensis leaves, the mature leaves of Coptis chinensis are taken from Coptis chinensis seedlings that are 2 months old.

[0035] More preferably, in the method for preparing protoplasts from *Coptis chinensis* leaves, during enzymatic hydrolysis, the *Coptis chinensis* protoplast and enzymatic hydrolysate are placed in the dark, at 24–26°C and 40 r / min on a shaker for 4–8 h, and more preferably for 6–8 h.

[0036] More preferably, in the method for preparing protoplasts from *Coptis chinensis* leaves, the enzymatic hydrolysate is treated as follows before use: A mixed enzyme solution is prepared, comprising: 2%–3% cellulase R-10, 0.25%–1% cleavage enzyme R-10, 20 mmol / L MME, 0.5 mol / L mannitol, 10 mmol / L CaCl2, and 20 mmol / L KCl. The mixed enzyme solution is activated in a 55°C water bath for 10 min, cooled to room temperature, and then 0.1% BSA is added to adjust the pH to 5.7. The solution is then filtered through a 0.45 μm microporous membrane for sterilization and stored at 4°C for later use. In this invention, the cellulase R-10, cleavage enzyme R-10, BSA, and enzymatic hydrolysate are calculated as a mass-volume ratio, i.e., each 100 mL of enzymatic hydrolysate contains 2–3 g of cellulase R-10, 0.25–1 g of cleavage enzyme R-10, and 0.1 g of BSA.

[0037] More preferably, in the method for preparing protoplasts from *Coptis chinensis* leaves, the CPW washing solution comprises: 27.2 mg / L KH₂PO₄, 101 mg / L KNO₃, 1117.5 mg / L CaCl₂, 246 mg / L MgSO₄·7H₂O, 0.16 mg / L KI, 0.025 mg / L CuSO₄·5H₂O, 20 mmol / L MES, and 0.6–1.0 mol / L mannitol, with a pH of 5.8.

[0038] More preferably, in the method for preparing protoplasts from Coptis chinensis leaves, the centrifugation conditions in step 3) are the same as those in step 2), which are 1000-1200 r / min for 3 min.

[0039] More preferably, in the method for preparing protoplasts from Coptis chinensis leaves, in step 1), the mass-to-volume ratio of Coptis chinensis filaments to enzymatic hydrolysate is 1:10 g / mL, the enzymatic hydrolysate contains 3% cellulase R-10 and 0.75% dissociative enzyme R-10, and the enzymatic hydrolysis time is 4-7 h; more preferably, the enzymatic hydrolysis time is 7 h.

[0040] The mannitol concentration in the CPW washing solution in step 3) is 0.6 mol / L;

[0041] In steps 2) and 3), the centrifugation conditions are 1200 r / min for 3 min.

[0042] More preferably, in the method for preparing protoplasts from Coptis chinensis leaves, step 2) involves filtering the material sequentially using 200-mesh and 400-mesh sterile filters.

[0043] More preferably, in the method for preparing protoplasts from Coptis chinensis leaves, in step 1), 2 μmol / L methyl jasmonate and 5 mg / L nano zinc oxide are added to the enzymatic hydrolysate before enzymatic hydrolysis, and the enzymatic hydrolysis time is 4 h.

[0044] The technical solution of the present invention may also include the following technical details to better achieve the technical effect: all the seedlings of Coptis chinensis in the present invention are obtained from the same source.

[0045] Example 1:

[0046] A method for preparing protoplasts from Coptis chinensis leaves includes the following steps:

[0047] 1) Cut mature leaves of Coptis chinensis into filaments and place them in an enzymatic hydrolysate for enzymatic hydrolysis. The mass-to-volume ratio of Coptis chinensis filaments to enzymatic hydrolysate is 1:8 g / mL. The enzymatic hydrolysate contains 2% cellulase R-10 and 0.25% analyte R-10.

[0048] 2) Filter the enzymatically hydrolyzed mixture through a sterile filter to remove incompletely hydrolyzed plant tissues and cell clusters. Take the filtrate, centrifuge it at 800 r / min for 2 min, remove the supernatant, and obtain the precipitate.

[0049] 3) The precipitate was resuspended with CPW washing solution, centrifuged, and the cell precipitate was collected to obtain the protoplasts of the leaves of Coptis chinensis.

[0050] Among them, mature leaves of Coptis chinensis were taken from Coptis chinensis seedlings that were 2 months old.

[0051] During enzymatic hydrolysis, the rocky stamens and the enzymatic hydrolysate were placed in the dark, at 24°C and 40 r / min on a shaker for 4 hours.

[0052] Before use, the enzymatic hydrolysate undergoes the following treatment: A mixed enzyme solution is prepared, consisting of 2% cellulase R-10, 0.25% analyte R-10, 20 mmol / L MES, 0.5 mol / L mannitol, 10 mmol / L CaCl2, and 20 mmol / L KCl. The mixed enzyme solution is activated in a 55℃ water bath for 10 min. After cooling to room temperature, 0.1% BSA is added to adjust the pH to 5.7. The solution is then filtered through a 0.45 μm microporous membrane for sterilization. After sterilization, it is stored at 4℃ for later use. Each 100 mL of enzymatic hydrolysate contains 2 g of cellulase R-10, 0.25 g of analyte R-10, and 0.1 g of BSA.

[0053] The CPW washing solution consists of: 27.2 mg / L KH2PO4, 101 mg / L KNO3, 1117.5 mg / L CaCl2, 246 mg / L MgSO4·7H2O, 0.16 mg / L KI, 0.025 mg / L CuSO4·5H2O, 20 mmol / L MES, and 0.6 mol / L mannitol. The pH of the CPW washing solution is 5.8.

[0054] The centrifugation conditions in step 3) are the same as those in step 2).

[0055] In step 2), the filtration process involves sequentially using 200-mesh and 400-mesh sterile filters.

[0056] Example 2:

[0057] A method for preparing protoplasts from Coptis chinensis leaves includes the following steps:

[0058] 1) Cut mature leaves of Coptis chinensis into filaments and place them in an enzymatic hydrolysate for enzymatic hydrolysis. The mass-to-volume ratio of Coptis chinensis filaments to enzymatic hydrolysate is 1:12 g / mL. The enzymatic hydrolysate contains 3% cellulase R-10 and 1% analyte R-10.

[0059] 2) Filter the enzymatically hydrolyzed mixture through a sterile filter to remove incompletely hydrolyzed plant tissues and cell clusters. Take the filtrate and centrifuge it at 1400 r / min for 5 min. Remove the supernatant to obtain the precipitate.

[0060] 3) The precipitate was resuspended with CPW washing solution, centrifuged, and the cell precipitate was collected to obtain the protoplasts of the leaves of Coptis chinensis.

[0061] Among them, mature leaves of Coptis chinensis were taken from Coptis chinensis seedlings that were 2 months old.

[0062] During enzymatic hydrolysis, the rocky stamens and the enzymatic hydrolysate were placed in the dark, at 26°C and 40 r / min on a shaker for 8 hours.

[0063] Before use, the enzymatic hydrolysate undergoes the following treatment: A mixed enzyme solution is prepared, consisting of 3% cellulase R-10, 1% analyte R-10, 20 mmol / L MES, 0.5 mol / L mannitol, 10 mmol / L CaCl2, and 20 mmol / L KCl. The mixed enzyme solution is activated in a 55℃ water bath for 10 min, cooled to room temperature, and then 0.1% BSA is added to adjust the pH to 5.7. The solution is then filtered through a 0.45 μm microporous membrane for sterilization. After sterilization, it is stored at 4℃ for later use. Each 100 mL of enzymatic hydrolysate contains 3 g of cellulase R-10, 1 g of analyte R-10, and 0.1 g of BSA.

[0064] The CPW washing solution consists of: 27.2 mg / L KH2PO4, 101 mg / L KNO3, 1117.5 mg / L CaCl2, 246 mg / L MgSO4·7H2O, 0.16 mg / L KI, 0.025 mg / L CuSO4·5H2O, 20 mmol / L MES, and 1.0 mol / L mannitol. The pH of the CPW washing solution is 5.8.

[0065] The centrifugation conditions in step 3) are the same as those in step 2).

[0066] In step 2), the filtration process involves sequentially using 200-mesh and 400-mesh sterile filters.

[0067] Example 3:

[0068] A method for preparing protoplasts from Coptis chinensis leaves includes the following steps:

[0069] 1) Cut mature leaves of Coptis chinensis into filaments and place them in an enzymatic hydrolysate for enzymatic hydrolysis. The mass-to-volume ratio of Coptis chinensis filaments to enzymatic hydrolysate is 1:10 g / mL. The enzymatic hydrolysate contains 2.5% cellulase R-10 and 0.75% analyte R-10.

[0070] 2) Filter the enzymatically hydrolyzed mixture through a sterile filter to remove incompletely hydrolyzed plant tissues and cell clusters. Take the filtrate and centrifuge it at 1000 r / min for 3 min. Remove the supernatant to obtain the precipitate.

[0071] 3) The precipitate was resuspended with CPW washing solution, centrifuged, and the cell precipitate was collected to obtain the protoplasts of the leaves of Coptis chinensis.

[0072] Among them, mature leaves of Coptis chinensis were taken from Coptis chinensis seedlings that were 2 months old.

[0073] During enzymatic hydrolysis, the rocky stamens and the enzymatic hydrolysate were placed in the dark, at 25°C and 40 r / min on a shaker for 6 hours.

[0074] Before use, the enzymatic hydrolysate undergoes the following treatment: A mixed enzyme solution is prepared, consisting of 2.5% cellulase R-10, 0.75% analyte R-10, 20 mmol / L MES, 0.5 mol / L mannitol, 10 mmol / L CaCl2, and 20 mmol / L KCl. The mixed enzyme solution is activated in a 55℃ water bath for 10 min. After cooling to room temperature, 0.1% BSA is added to adjust the pH to 5.7. The solution is then filtered through a 0.45 μm microporous membrane for sterilization. After sterilization, it is stored at 4℃ for later use. Each 100 mL of enzymatic hydrolysate contains 2.5 g cellulase R-10, 0.75 g analyte R-10, and 0.1 g BSA.

[0075] The CPW washing solution consists of: 27.2 mg / L KH2PO4, 101 mg / L KNO3, 1117.5 mg / L CaCl2, 246 mg / L MgSO4·7H2O, 0.16 mg / L KI, 0.025 mg / L CuSO4·5H2O, 20 mmol / L MES, and 0.8 mol / L mannitol. The pH of the CPW washing solution is 5.8.

[0076] The centrifugation conditions in step 3) are the same as those in step 2).

[0077] In step 2), the filtration process involves sequentially using 200-mesh and 400-mesh sterile filters.

[0078] Example 4:

[0079] A method for preparing protoplasts from Coptis chinensis leaves includes the following steps:

[0080] 1) Cut mature leaves of Coptis chinensis into filaments and place them in an enzymatic hydrolysate for enzymatic hydrolysis. The mass-to-volume ratio of Coptis chinensis filaments to enzymatic hydrolysate is 1:10 g / mL. The enzymatic hydrolysate contains 3% cellulase R-10 and 0.75% analyte R-10.

[0081] 2) Filter the enzymatically hydrolyzed mixture through a sterile filter to remove incompletely hydrolyzed plant tissues and cell clusters. Take the filtrate and centrifuge it at 1200 r / min for 3 min. Remove the supernatant to obtain the precipitate.

[0082] 3) The precipitate was resuspended with CPW washing solution, centrifuged, and the cell precipitate was collected to obtain the protoplasts of the leaves of Coptis chinensis.

[0083] Among them, mature leaves of Coptis chinensis were taken from Coptis chinensis seedlings that were 2 months old.

[0084] During enzymatic hydrolysis, the rocky stamens and the enzymatic hydrolysate were placed in the dark, at 25°C and 40 r / min on a shaker for 4 hours.

[0085] Before use, the enzymatic hydrolysate undergoes the following treatment: A mixed enzyme solution is prepared, consisting of 3% cellulase R-10, 0.75% analyte R-10, 20 mmol / L MES, 0.5 mol / L mannitol, 10 mmol / L CaCl2, and 20 mmol / L KCl. The mixed enzyme solution is activated in a 55℃ water bath for 10 min. After cooling to room temperature, 0.1% BSA is added to adjust the pH to 5.7. The solution is then filtered through a 0.45 μm microporous membrane for sterilization. After sterilization, it is stored at 4℃ for later use. Each 100 mL of enzymatic hydrolysate contains 3 g of cellulase R-10, 0.75 g of analyte R-10, and 0.1 g of BSA.

[0086] The CPW washing solution consists of: 27.2 mg / L KH2PO4, 101 mg / L KNO3, 1117.5 mg / L CaCl2, 246 mg / L MgSO4·7H2O, 0.16 mg / L KI, 0.025 mg / L CuSO4·5H2O, 20 mmol / L MES, and 0.6 mol / L mannitol. The pH of the CPW washing solution is 5.8.

[0087] The centrifugation conditions in step 3) are the same as those in step 2).

[0088] In step 2), the filtration process involves sequentially using 200-mesh and 400-mesh sterile filters.

[0089] Example 5:

[0090] Based on Example 4, the enzymatic hydrolysis time was modified to 7 hours. The rest of the process remained the same as in Example 4.

[0091] Example 6:

[0092] Based on Example 4, in the method for preparing protoplasts from *Coptis chinensis* leaves, in step 1), 2 μmol / L methyl jasmonate and 5 mg / L nano zinc oxide are added to the enzymatic hydrolysate before enzymatic hydrolysis, and the enzymatic hydrolysis time is 4 h. The rest is the same as in Example 4.

[0093] To further illustrate the technical effects of the method for preparing protoplasts from *Coptis chinensis* leaves according to the present invention, mature leaves of *Coptis chinensis* seedlings from the same source and aged 2 months were used for the following experiments, with each experiment repeated 3 times, to determine the yield and viability of *Coptis chinensis* protoplasts. Wherein:

[0094] 1. Methods for determining protoplast yield:

[0095] The protoplast yield was calculated using a 0.1mm XB-K-16 hemocytometer. The specific procedure was as follows: Gently wipe the protoplast with a cotton ball soaked in 75% alcohol, then rinse with distilled water and blot dry with absorbent paper. Place a coverslip over the counting chamber. Drip 10 μL of purified protoplast solution onto one edge of the coverslip, allowing it to seep into the counting chamber along the gap between the coverslip and the counting chamber until it is full. Place the counting chamber horizontally on the microscope stage and count the protoplasts in each of the 16 square cells within the central large square. Calculate the protoplast yield using the formula, expressing the yield as the number of protoplasts per gram of fresh weight.

[0096] The number of protoplasts in 1 mL of suspension = 1 large square of suspension (0.1 mm) 3 (Number of cells in) × 104 ;

[0097] Protoplast yield (cells / g) = number of protoplasts in 1 mL of suspension × total volume of protoplast suspension (mL) / total mass of material (g).

[0098] 2. Methods for determining protoplast viability:

[0099] The viability of protoplasts was determined using a 0.01% fluorescein diacetate (FDA) live staining method.

[0100] First, prepare a 5 mg / mL stock solution of FDA with acetone and store it at 4°C. The working concentration for detection is 0.01%, i.e., 25 μL of FDA per mL of protoplasts. After incubation for 5 min, count the number of protoplasts emitting yellow-green fluorescence and the total number of protoplasts under a fluorescence microscope. Take the average of three randomly selected fields of view. Protoplast viability is expressed as the proportion of yellow-green fluorescent protoplasts in a field of view to the total number of protoplasts in that field.

[0101] Protoplast viability = (number of protoplasts emitting green fluorescence / total number of protoplasts) × 100%.

[0102] Experiment 1: Investigating the effect of enzyme hydrolysate composition on the preparation method of protoplasts from Coptis chinensis leaves.

[0103] Twelve combinations of enzymatic hydrolysates with different concentrations of cellulase R-10 and ionase R-10 were prepared. The combinations of enzymatic hydrolysates used in the experiment are shown in Table 1.

[0104] Except for the combination of enzyme hydrolysate, the experimental method of this invention is based on Example 4, with the enzymatic hydrolysis time set to 4h, the centrifugation conditions set to centrifugation at 1000r / min for 3min, and the mannitol concentration set to 0.6mol / L. The remaining methods and experimental parameters are the same as in Example 4, and the results are shown in Table 1.

[0105] Table 1. Effect of enzyme hydrolysate composition on the preparation method of protoplasts from Coptis chinensis leaves

[0106]

[0107]

[0108] Under the conditions of this experiment, the technical solution of group E11 is the optimal one. Figure 1 The protoplasts were obtained using the E11 technique. E11 refers to the enzymatic hydrolysate composed of 3% cellulase R-10 and 0.75% dissociative enzyme R-10.

[0109] Experiment 2: Study on the effect of mannitol concentration on the preparation method of protoplasts from Coptis chinensis leaves.

[0110] Using the mannitol concentration in the CPW washing solution as a variable, the mannitol concentrations were 0.2, 0.4, 0.6, 0.8, and 1.0 mol / L, respectively. Based on Example 4, the experimental method of this invention set the enzymatic hydrolysis time to 4 h, the centrifugation conditions to 1000 r / min for 3 min, and the composition of the enzymatic hydrolysate to 3% cellulase R-10 and 0.75% dissociative enzyme R-10. The remaining methods and experimental parameters were the same as in Example 4. The results are shown in Table 2.

[0111] Table 2. Effect of mannitol concentration on the preparation method of protoplasts from Coptis chinensis leaves

[0112] 0.2 0.68 100 0.4 4.9 94.34 0.6 15.4 96.39 0.8 13.2 100 1.0 10.4 100

[0113] Under the conditions of this experiment, the highest yield of protoplasts from *Coptis chinensis* leaves was achieved when the mannitol concentration in the CPW washing solution was 0.6 mol / L, with a high activity level of 96.39%.

[0114] Experiment 3: Investigating the effect of centrifugation rate on the preparation method of protoplasts from Coptis chinensis leaves.

[0115] Using centrifugation rate as a variable, centrifugation was carried out at rates of 800, 1000, 1200, and 1400 r / min for 3 min. Based on Example 4, the experimental method of this invention set the enzymatic hydrolysis time to 4 h, the enzymatic hydrolysate composition to be 3% cellulase R-10 and 0.75% analyte R-10, and the mannitol concentration to be 0.6 mol / L. The remaining methods and experimental parameters were the same as in Example 4. The results are shown in Table 3.

[0116] Table 3. Effect of centrifugation rate on the preparation method of protoplasts from Coptis chinensis leaves

[0117] 800 1.41 100 1000 3.08 98.55 1200 15.93 100 1400 7.12 99.18

[0118] Under the conditions of this experiment, the highest yield and activity of protoplasts from *Coptis chinensis* leaves were obtained by centrifuging at 1200 r / min for 3 min.

[0119] Experiment 4: Investigating the effect of enzymatic hydrolysis time on the preparation method of protoplasts from Coptis chinensis leaves.

[0120] With enzymatic hydrolysis time as the variable, the enzymatic hydrolysis times were 3, 4, 5, 6, 7, and 8 hours respectively. Based on Example 4, the experimental method of this invention set the composition of the enzymatic hydrolysate to 3% cellulase R-10 and 0.75% analyte R-10, the mannitol concentration to 0.6 mol / L, and the centrifugation conditions to 1200 r / min for 3 min. The remaining methods and experimental parameters were the same as in Example 4. The results are shown in Table 4.

[0121] Table 4. Effect of enzymatic hydrolysis time on the preparation method of protoplasts from Coptis chinensis leaves.

[0122]

[0123]

[0124] Under the conditions of this experiment, the protoplasts of Coptis chinensis leaves prepared by enzymatic hydrolysis for 7 hours were the best.

[0125] In summary, the present invention yielded the optimal technical solution for Example 5, wherein the enzymatic hydrolysis time is 7 hours, the hydrolysate composition is 3% cellulase R-10 and 0.75% analyte R-10, the mannitol concentration is 0.6 mol / L, and the centrifugation conditions are 1200 r / min for 3 minutes. Under this scheme, approximately 20.77 × 10⁻⁶ protoplasts with an activity as high as 98.76% can be obtained from 1 gram of fresh Coptis chinensis leaf tissue. 4 indivual.

[0126] Under these conditions, the present invention also conducted experiments on Example 6. The experimental materials were from the same source as those used in the above experiments. Each experiment was repeated three times to determine the protoplast yield and viability of *Coptis chinensis*. The results showed that the protoplast yield under the technical solution of Example 6 was 23.58 × 10⁻⁶. 4 The yield and activity of the prepared *Coptis chinensis* protoplasts were 100% (number per g). The yield and activity of *Coptis chinensis* protoplasts obtained under this method were higher than those obtained under the methods of Examples 4 and 5, specifically the yield and activity of *Coptis chinensis* protoplasts obtained after 4 hours and 7 hours of enzymatic hydrolysis in Example 4. This indicates that adding appropriate concentrations of methyl jasmonate and nano-zinc oxide to the enzymatic hydrolysate not only shortened the hydrolysis time but also further increased the quantity and activity of *Coptis chinensis* protoplasts.

[0127] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for preparing protoplasts of Coptis teeta leaves, characterized by, Includes the following steps: 1) Cut mature leaves of Coptis chinensis into filaments and place them in an enzymatic hydrolysate for enzymatic hydrolysis. The mass-to-volume ratio of Coptis chinensis filaments to enzymatic hydrolysate is 1:8~12 g / mL. The enzyme composition in the enzymatic hydrolysate is 2%~3% cellulase R-10 and 0.25%~1% analyte R-10. Before enzymatic hydrolysis, add 2 μmol / L methyl jasmonate and 5 mg / L nano zinc oxide to the enzymatic hydrolysate. The enzymatic hydrolysis time is 4 h. 2) Filter the enzymatically hydrolyzed mixture through a sterile filter to remove incompletely hydrolyzed plant tissues and cell clusters. Take the filtrate and centrifuge it at 1000~1400 r / min for 2~5 min. Remove the supernatant to obtain the precipitate. 3) Resuspend the precipitate with CPW washing solution containing 0.6~1.0 mol / L mannitol, and collect the cell precipitate after centrifugation to obtain the protoplasts of Coptis chinensis leaves.

2. The method for preparing protoplasts from *Coptis chinensis* leaves as described in claim 1, characterized in that, Mature leaves of Coptis chinensis were taken from seedlings of Coptis chinensis that were 2 months old.

3. The method for preparing protoplasts from *Coptis chinensis* leaves as described in claim 1 or 2, characterized in that, During enzymatic hydrolysis, the rocky stamens and the enzymatic hydrolysate were placed in the dark, at 24-26℃ and 40 r / min on a shaker for 4 hours.

4. The method for preparing protoplasts from *Coptis chinensis* leaves as described in claim 3, characterized in that, Before using the enzymatic hydrolysate, the following treatment should be performed: First, prepare a mixed enzyme solution with the following composition: 2%~3% cellulase R-10, 0.25%~1% analyte R-10, 20 mmol / L MES, 0.5 mol / L mannitol, 10 mmol / L CaCl2, and 20 mmol / L KCl. Activate the mixed enzyme solution in a 55℃ water bath for 10 min. After cooling to room temperature, add 0.1% BSA and adjust the pH to 5.

7. Sterilize by filtering through a 0.45 μm microporous membrane. After sterilization, store at 4℃ for later use.

5. The method for preparing protoplasts from *Coptis chinensis* leaves as described in claim 4, characterized in that, The composition of the CPW washing solution is: 27.2 mg / L KH2PO4, 101 mg / L KNO3, 1117.5 mg / L CaCl2, 246 mg / L MgSO4·7H2O, 0.16 mg / L KI, 0.025 mg / L CuSO4·5H2O, 20 mmol / L MES, and 0.6~1.0 mol / L mannitol. The pH of the CPW washing solution is 5.

8.

6. The method for preparing protoplasts from *Coptis chinensis* leaves as described in claim 5, characterized in that, The centrifugation conditions in step 3) are the same as those in step 2), which are 1000~1200 r / min for 3 min.

7. The method for preparing protoplasts from *Coptis chinensis* leaves as described in claim 6, characterized in that, In step 1), the mass-to-volume ratio of the fibrous material of *Rhizophora stylosa* to the enzymatic hydrolysate is 1:10 g / mL, and the enzyme composition in the enzymatic hydrolysate is 3% cellulase R-10 and 0.75% analyte R-10. The mannitol concentration in the CPW washing solution in step 3) is 0.6 mol / L; In steps 2) and 3), the centrifugation conditions are 1200 r / min for 3 min.

8. The method for preparing protoplasts from *Coptis chinensis* leaves as described in claim 1, characterized in that, In step 2), the filtration process involves sequentially using 200-mesh and 400-mesh sterile filters.

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