Method for establishing rhodiola rosea protoplast extraction and purification system

Abstract

This invention belongs to the field of biotechnology, specifically relating to a stable and efficient method for extracting and purifying Rhodiola rosea protoplasts. Using Rhodiola rosea callus tissue as material, this invention establishes a highly efficient separation and purification system for Rhodiola rosea callus protoplasts based on enzymatic hydrolysis, determining the optimal enzyme combination, mannitol concentration, hydrolysis time, and centrifugation speed. Experimental verification shows that the protoplast yield using the Rhodiola rosea protoplast extraction and purification method described in this invention reaches 5.57 × 10⁻⁶. 6 The Rhodiola rosea protoplast extraction and purification system provided by this invention yields a large quantity of Rhodiola rosea protoplasts with minimal damage, few impurities, and high viability, providing experimental materials and technical support for gene function analysis, secondary metabolism regulation, and cell engineering research.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for establishing a system for the extraction and purification of Rhodiola rosea protoplasts. Background Technology

[0002] Rhodiola rosea ( Rhodiola crenulata (Hook. f. & Thomson) H. Ohba is a perennial herb belonging to the genus Rhodiola in the family Crassulaceae. As an important medicinal material in traditional Tibetan medicine, its roots and rhizomes are widely used for anti-hypoxia, anti-fatigue, and immune-boosting purposes. Rhodioloside, in particular, is considered a core efficacy marker due to its clear neuroprotective, cardiovascular regulatory, and anti-aging activities. However, due to slow growth in the wild, over-harvesting, and harsh habitat, Rhodiola grandiflora has been listed in the "National Key Protected Wild Plants List" and is endangered, leading to difficulties in the supply of raw materials for industrial production.

[0003] Plant protoplasts are naked, living cells enclosed by a plasma membrane after the cell wall has been removed. Due to the removal of the cell wall, protoplasts can efficiently take up exogenous nucleic acids and effector molecules, making them an ideal in vitro system for studying transient gene expression, protein-protein interaction verification, and secondary metabolic regulation. As a rare medicinal plant, *Rhodiola rosea* protoplasts can serve as excellent material for studying the regulation of rhodioloside synthesis. However, enzymatic hydrolysis conditions for *Rhodiola rosea* callus have not yet been reported. Therefore, to protect wild resources and meet the increasing demand for rhodioloside, establishing a stable and efficient protoplast preparation system for *Rhodiola rosea* is an urgent problem to be solved. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a stable and efficient method for establishing a Rhodiola rosea protoplast extraction and purification system.

[0005] The technical solution adopted by this invention to solve its technical problem is: a method for establishing a Rhodiola rosea protoplast extraction and purification system, comprising the following steps: A. Select Rhodiola rosea callus tissue and culture it in a culture medium for a period of time under dark conditions to induce chloroplast degradation and obtain light green or light yellow Rhodiola rosea callus tissue. B. Crush the loose, light green or light yellow Rhodiola rosea callus obtained in step A, cut the callus into small pieces, and put them into a container. C. Add the enzymatic hydrolysate to the container and carry out enzymatic hydrolysis under dark conditions to obtain the enzymatic hydrolysate mixture; D. The enzymatic hydrolysis mixture obtained after step C is filtered to remove residue and obtain protoplast enrichment solution. E. Wash the protoplast enrichment solution with cell washing solution; F. After purifying and centrifuging the washed protoplast enrichment solution, Rhodiola rosea protoplasts can be obtained by resuspending it in cell washing solution.

[0006] Furthermore, in step A, the culture medium formulation is as follows: MS powder 4.47 g / L + 6-BA 0.6-1.2 mg / L + NAA 0.2-0.3 mg / L + 2,4-D 0.1-0.2 mg / L + IAA 0.1-0.2 mg / L + agar 8-10 g / L + sucrose 25-30 g / L; pH value is 5.7-5.9.

[0007] Furthermore, in step A, the Rhodiola rosea callus is cultured in a culture medium at an ambient temperature of 25 ± 2℃; the pale green or pale yellow Rhodiola rosea callus needs to be cultured in the dark for 10-14 days.

[0008] Furthermore, in step B, the callus tissue is cut into small pieces with dimensions of 1-2 mm.

[0009] Further, in step C, the enzymatic hydrolysis process is as follows: First, prepare an enzymatic hydrolysate containing 1.0-2.0% cellulase, 0.5-1.0% cleavage enzyme, 0.4-0.7 mol / L mannitol, 2-5 mmol / L MES, and 5-10 mmol / L CaCl2, and bring it to a final volume using a sucrose-free CPW solution with a pH of 5.6-5.8; heat the conical flask containing the hydrolysate in a 50-60°C water bath for 5-15 minutes, cool it to room temperature, and then filter it using a 0.22-0.45 μm microporous membrane; next, add 1 g of chopped callus tissue to the hydrolysate at a ratio of 1 g of chopped callus tissue per 10-15 mL of hydrolysate, and then place the flask on a horizontal shaker and shake it at 60-80 r / min for 0.5-2 hours.

[0010] Furthermore, in step D, the enzymatic hydrolysis mixture is filtered using a filter screen with a pore size of 45 μm or 800 mesh.

[0011] Furthermore, in steps E and F, the cell washing solution is a W5 solution, and the buffer solution consists of 1.5-2.5 mmol / L MES, 140-160 mmol / L NaCl, 120-130 mmol / L CaCl2, and 4-8 mmol / L KCl; the pH value of the cell washing solution is 5.7-5.9.

[0012] Furthermore, in step E, the process of washing the protoplast enrichment solution with cell washing solution is as follows: First, slowly add cell washing solution to the protoplast enrichment solution and place it in a refrigerator at room temperature or 4°C for more than 20 minutes. The upper liquid is the protoplast enrichment layer solution.

[0013] Furthermore, in step F, the purification centrifugation includes the following steps: a. Transfer 10-20 mL of the protoplast enrichment solution from the upper layer to a 50 mL centrifuge tube, add 10-20 mL of cell washing solution, mix well, centrifuge at 600-900 r / min for 4-8 min, and discard the supernatant. b. Resuspend the precipitate in 10-25 mL of cell washing buffer, mix well, centrifuge again under the same conditions, and discard the supernatant. c. Resuspend the precipitate in 10-25 mL of cell washing buffer, mix well, centrifuge at 600-900 r / min for 2-4 min, and discard the supernatant. d. Add 0.5-1 mL of cell washing buffer to resuspend the protoplasts after precipitation.

[0014] Preferably, in step A, the culture medium formulation is: MS powder 4.47 g / L + 6-BA 1.0 mg / L + NAA 0.3 mg / L + 2,4-D 0.2 mg / L + IAA 0.2 mg / L + agar 8 g / L + sucrose 30 g / L; pH value is 5.7-5.9.

[0015] Preferably, in step A, the ambient temperature for culturing the Rhodiola rosea callus on the culture medium is 25±2℃; the light green or light yellow Rhodiola rosea callus needs to be cultured in the dark for 14 days.

[0016] Preferably, in step B, the callus tissue is cut into small pieces with dimensions of 1 mm each.

[0017] Preferably, in step C, the enzymatic hydrolysis process is as follows: First, an enzymatic hydrolysate is prepared, comprising 2.0% cellulase, 1.0% cleavage enzyme, 0.6 mol / L mannitol, 5 mmol / L MES, and 10 mmol / L CaCl2, and diluted to volume with a sucrose-free CPW solution with a pH of 5.6-5.8; the conical flask containing the enzymatic hydrolysate is heated in a 55°C water bath for 10 min, cooled to room temperature, and then filtered through a 0.22 μm microporous membrane; next, callus tissue cut into small pieces is added to the enzymatic hydrolysate at a ratio of 1 g of callus tissue cut into small pieces per 10 mL of enzymatic hydrolysate, and then the mixture is placed on a horizontal shaker and shaken at 80 r / min for 1.0 h.

[0018] Preferably, in step D, the enzymatic hydrolysis mixture is filtered using a filter screen with a pore size of 45 μm.

[0019] Preferably, in steps E and F, the cell washing solution is a W5 solution, and the buffer solution consists of 2.0 mmol / L MES, 154 mmol / L NaCl, 125 mmol / L CaCl2, and 5.0 mmol / L KCl; the pH value of the cell washing solution is 5.7-5.9.

[0020] Preferably, in step E, the process of washing the protoplast enrichment solution with cell washing solution is as follows: First, slowly add cell washing solution to the protoplast enrichment solution, place it in a refrigerator at 4°C for 30 minutes, and the upper liquid is the protoplast enrichment layer solution.

[0021] Preferably, in step F, the purification centrifugation step is as follows: a. Transfer 15 mL of the upper protoplast enrichment solution to a 50 mL centrifuge tube, add 15 mL of cell washing buffer, mix well, centrifuge at 700 r / min for 6 min, and discard the supernatant. b. Resuspend the precipitate in 20 mL of cell washing buffer, mix well, centrifuge again under the same conditions, and discard the supernatant. c. Resuspend the precipitate in 20 mL of cell washing buffer, mix well, centrifuge at 700 r / min for 3 min, and discard the supernatant; d. Add 0.5 mL of cell washing buffer to the precipitate and resuspend the protoplasts.

[0022] The advantages of this invention are: The Rhodiola rosea protoplast extraction and purification system and method described in this invention directly uses Rhodiola rosea callus tissue as material to prepare protoplasts, without the need for additional aseptic operations, and the whole process is simple and convenient.

[0023] This invention achieves the large-scale extraction of Rhodiola rosea protoplasts by regulating the components of the enzymatic hydrolysate.

[0024] This invention achieves stable maintenance of the osmotic pressure of Rhodiola rosea cells during the extraction process by setting different concentrations of mannitol in the cell washing solution.

[0025] The protoplast extraction and purification technique for Rhodiola rosea described in this invention yielded a protoplast yield of 5.57 × 10⁻⁶. 6 The number of protoplasts is 1 / g, and 80% of the protoplasts are intact, undamaged, and have few impurities. The quality and activity of the extracted protoplasts meet the requirements of the protoplast technology system.

[0026] The protoplasts obtained by this invention have dual application value: First, they can serve as a platform for germplasm innovation and genetic improvement, enabling polyploid breeding and the transfer of superior genes through protoplast fusion and plant regeneration technology, providing a scientific basis for the cultivation of new Rhodiola rosea varieties; Second, they can serve as a tool for the production and research of secondary metabolites, providing new pathways for the industrial production of active ingredients such as rhodioloside by constructing highly efficient transformation strains, or for the study of metabolic regulation mechanisms. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0028] Figure 1 This is a diagram of the Rhodiola rosea callus tissue and its treatment as described in Embodiment 1 of the present invention; Figure 2 This is a morphological diagram of the Rhodiola rosea protoplast described in Embodiment 1 of the present invention; Figure 3 This is the FDA fluorescence staining image of Rhodiola rosea protoplasts described in Example 1 of this invention; Figure 4 This is a graph showing the effect of different enzyme combinations described in Example 2 of the present invention on protoplast yield and viability; Figure 5 This is a graph showing the effect of different mannitol concentrations on protoplast yield and viability as described in Example 3 of this invention; Figure 6 These are FDA fluorescent staining images of different mannitol concentrations as described in Example 3 of this invention; Figure 7 This is a graph showing the effect of different enzymatic hydrolysis times on protoplast yield and viability as described in Example 4 of the present invention; Figure 8 These are FDA fluorescence staining images of different enzymatic hydrolysis times as described in Example 4 of this invention; Figure 9 This is a graph showing the effect of different centrifugation speeds on protoplast yield and viability as described in Example 5 of the present invention; Figure 10 These are FDA fluorescent staining images at different centrifugation speeds as described in Example 5 of this invention. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the preferred embodiments of this invention will be described in further detail below with reference to the examples. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0030] Example 1 The extraction and purification of Rhodiola rosea protoplasts includes the following steps: (1) Select callus tissue in good growth condition, inoculate it onto the culture medium, and culture for 14 days; the culture conditions are dark culture, temperature 25 ± 2℃; the culture medium formula is MS powder 4.47 g / L + 6-BA 1.0 mg / L + NAA 0.3 mg / L + 2,4-D 0.2 mg / L + IAA 0.2 mg / L + agar 8 g / L + sucrose 30 g / L; pH value is 5.7-5.9; (2) Preparation of enzymatic hydrolysate: 2.0% cellulase, 1.0% ionizing enzyme, 0.6 mol / L mannitol, 5 mmol / L MES, 10 mmol / L CaCl2, and make up to volume with sucrose-free CPW solution with pH 5.6-5.8; heat the conical flask containing the enzymatic hydrolysate in a 55°C water bath for 10 min, cool to room temperature, and filter with a 0.22 μm microporous membrane. Prepare and use immediately. (3) Preparation of cell washing solution W5: 2.0 mmol / L MES, 154 mmol / L NaCl, 125 mmol / L CaCl2, 5.0 mmol / L KCl; pH value is 5.7-5.9; sterilize at 121℃ for 15 min and store at 4℃. (4) Take out the callus after dark culture, remove the attached culture medium, weigh 2g of callus, crush it with a spatula or other items, cut it into small pieces with a blade with a length, width and height of 1mm, and put it into a 60mm culture dish; add 20mL of enzyme hydrolysate, shake well, seal with sealing film and wrap a layer of tin foil on the outside of the culture dish; place it on a horizontal shaker and shake at 80r / min at room temperature (25±2℃) for 1.0h.

[0031] (5) Filter the enzymatically digested mixture into a 50mL sterile centrifuge tube using a 45μm filter, then slowly add cell washing solution equal to the volume of the filtered mixture, place it in a 4℃ refrigerator for 30min to allow it to settle automatically, and the upper layer solution is the protoplast enrichment layer. (6) Take 15 mL of the protoplast enrichment solution from the upper layer into a 50 mL centrifuge tube, add 15 mL of cell washing buffer, mix well, centrifuge at 700 r / min for 6 min, and discard the supernatant; resuspend the precipitate with 20 mL of cell washing buffer, mix well, centrifuge again under the same conditions, and discard the supernatant; resuspend the precipitate with 20 mL of cell washing buffer, mix well, centrifuge at 700 r / min for 3 min, and discard the supernatant; resuspend the protoplasts in 0.5 mL of cell washing buffer.

[0032] (7) Protoplast yield was calculated using a hemocytometer. 10 μL of resuspended protoplast suspension was added dropwise to a 0.1 mm thick hemocytometer, and its morphology was observed and counted under a microscope. Each sample was counted three times to calculate the average. The formula for protoplast yield was: Protoplast yield (units / g) = (number of protoplasts per square in the large square of the hemocytometer within 0.1 mm of the hemocytometer) / (number of protoplasts per square in the large square of the hemocytometer within 0.1 mm of the hemocytometer). 3 (Number of protoplasts × 10) 4 × Total volume of protoplast suspension (mL) / Callus weight (g) (8) Protoplast viability was determined using fluorescein diacetate (FDA) staining; observation and counting were performed using an inverted fluorescence microscope. 5 μL of FDA stock solution was added to 100 μL of protoplast suspension, and the mixture was allowed to stand in the dark for 5 min. 20 μL of the stained protoplast suspension was observed and the FDA staining was recorded under an inverted fluorescence microscope, and images were acquired. The formula for calculating protoplast viability was: Protoplast viability (%) = Number of green fluorescent protoplasts / Total number of protoplasts × 100%. Figure 3 This is the FDA fluorescence staining image of Rhodiola rosea protoplasts described in Example 1 of this invention, by... Figure 3 The results showed that treating callus tissue with 2g of Rhodiola rosea in the dark, followed by enzymatic hydrolysis in 20mL of solution for 1.0h, resulted in a protoplast yield of 5.57×10⁻⁶. 6 Cells / g, with an average cell viability of 81.97%.

[0033] Example 2 Extraction and purification of Rhodiola rosea protoplasts using different enzyme combinations, including the following steps: Example 2 uses the same method as Example 1, except that different enzyme combinations (Tables 1-3) were used as hydrolysate formulations to enzymatically hydrolyze Rhodiola rosea callus. After initial screening of 24 formulations, 5 optimal formulations were selected for enzymatic hydrolysis of Rhodiola rosea callus, and their yield and activity were analyzed. Figure 4 This is a graph showing the effect of different enzyme combinations described in Example 2 of the present invention on protoplast yield and activity; (The graph is composed of...) Figure 4 It can be seen that, under the same mannitol concentration, enzymatic hydrolysis time, and centrifugation speed, the TM2 combination (2.0% cellulase + 1.0% dissociation enzyme) performed best, with an average yield of 6.63 × 10⁻⁶. 6 The number of cells / g was significantly higher than that of other combinations.

[0034] Table 1 Enzymatic hydrolysis combinations for FM group Table 1Enzymatic combination for the FM group

[0035] Table 2 Enzymatic digestion combinations for SM group Table 2Enzymatic combination for the SM group

[0036] Table 3 Enzymatic digestion combinations of TM group Table 3Enzymatic combination for the TM group

[0037] Example 3 Extraction and purification of Rhodiola rosea protoplasts with different mannitol concentrations, including the following steps: Example 3 follows the same method as Example 1, except that in Example 3, four different mannitol concentrations were used during enzymatic hydrolysis. Under the optimal enzyme combination and a fixed hydrolysis time of 1.0 h, four concentration gradients of 0.4, 0.5, 0.6, and 0.7 mol / L were established for protoplast preparation. Figure 5 This is a graph illustrating the effect of different mannitol concentrations on protoplast yield and viability, as described in Example 3 of this invention. Figure 5 It can be seen that as the mannitol concentration increases, the protoplast production continuously increases, reaching a peak at 0.6 mol / L with a production of 1.08 × 10⁻⁶. 7 With increasing mannitol concentration, protoplast production decreased and fragmentation increased. The 0.5 mol / L treatment group showed the highest viability, with an average viability exceeding 80%. The viability of the 0.6 mol / L treatment group was significantly higher than that of the 0.4 mol / L and 0.7 mol / L groups (P<0.01), but not significantly different from the 0.5 mol / L group (P>0.05). Notably, although the 0.5 mol / L group maintained high viability, its yield was significantly lower than that of the 0.6 mol / L group, indicating that while most cells could maintain activity under 0.5 mol / L osmotic pressure, cell release efficiency was low; therefore, the 0.6 mol / L treatment group showed the best effect. Figure 6 These are FDA fluorescent staining images of different mannitol concentrations as described in Example 3 of this invention; by Figure 6 It can be seen that the protoplasts in the 0.6 mol / L treatment group exhibited strong and uniform green fluorescence, with a staining positivity rate of approximately 80%, and the cells were plump, round, and regular in shape. Figure 6 C). In the 0.4 mol / L treatment group, although some protoplasts showed fluorescence, a large number of cell debris appeared in the background. Figure 6A). The positive rate of FDA staining in the 0.5 mol / L treatment group was approximately 77%, and the fluorescence intensity was similar to that of the 0.6 mol / L group. Although the impurities were less than those in the 0.4 mol / L group, some cells were damaged ( Figure 6 B). In the 0.7 mol / L treatment group, the vast majority of protoplasts had no fluorescence or only extremely weak fluorescence, and the cells were severely shrunk and showed irregular shapes ( Figure 6 D).

[0038] Example 4 Extraction and purification of Rhodiola protoplasts with different enzymolysis times, including the following steps: The method of Example 4 was the same as that of Example 1, except that in Example 4, when enzymolyzing, 4 different enzymolysis times were set, which were 0.5, 1.0, 1.5, and 2.0 h respectively; Figure 7 is the graph showing the influence of different enzymolysis times in Example 4 of the present invention on the protoplast yield and viability; from Figure 7 it can be seen that the yield of the 0.5 h treatment group was the lowest, only 2.95×10 6 cells / g. As the enzymolysis time gradually extended, it reached the peak at 1.5 h, which was 5.38×10 6 cells / g. After that, as the time extended, the yield decreased. The viability of the 0.5 h treatment group was the highest, reaching 68%. As the enzymolysis time extended, the viability showed a continuous downward trend and decreased sharply after 1.5 h; Figure 8 is the FDA fluorescence staining graph of different enzymolysis times in Example 4 of the present invention; from Figure 8 it can be seen that in the 0.5 h treatment group, the proportion of fluorescent positive cells was the highest, the fluorescence intensity was strong, the cell morphology was full, but there were many cell clumps that were not completely dissociated in the field of view, and the density of free protoplasts was low ( Figure 8 A), the positive rate of FDA staining in the 1.0 h treatment group was approximately 58%, the fluorescence intensity was good, the cell morphology was regular, and the density of free protoplasts increased significantly ( Figure 8 B), in the 1.5 h treatment group, the fluorescence intensity of some cells weakened, there were many cell debris in the field of view, and a few cells were weakly positive or unstained ( Figure 8 C), in the 2.0 h treatment group, the vast majority of cells had no fluorescence, the field of view was full of amorphous debris and membrane remnants, and only a very small number of cells retained weak fluorescence ( Figure 8 D); Example 5 Extraction and purification of Rhodiola protoplasts with different centrifugation speeds, including the following steps: The method of Example 5 was the same as that of Example 1, except that in Example 5, when purifying by centrifugation, different centrifugation speeds were selected, which were 300, 500, 700, and 900 r / min respectively; Figure 9This is a graph illustrating the effect of different centrifugation speeds on protoplast yield and viability, as described in Example 5 of this invention; Figure 9 As shown, cell viability significantly increased with increasing rotation speed, reaching a peak at 500 r / min with an average viability of over 80%, before gradually decreasing. The viability of the 500 r / min treatment group was slightly higher than that of the 700 r / min treatment group, but the difference was not significant (P>0.05). Figure 10 These are FDA fluorescent staining images at different centrifugation speeds as described in Example 5 of this invention; as shown... Figure 10 As shown, in the 300 r / min treatment group, the density of protoplasts in the field of view was low, and although some cells showed positive staining, the cell edges were slightly blurred. Figure 10 A). In the 500 r / min treatment group, the proportion of fluorescently positive cells was approximately 62%, with good cell morphology, but most cells showed weak positivity and low fluorescence intensity. Figure 10 B). In the 700 r / min treatment group, the proportion of fluorescent positive cells was higher, and compared with the 500 r / min treatment group, the fluorescence was brighter and more uniform, the cell morphology was fuller, and the background was cleaner. Figure 10 C). In the 900 r / min treatment group, although some cells still showed positive staining, local damage to the cell membrane surface and slight leakage of contents could be observed, and a few cells showed irregular morphology. Figure 10 D).

[0039] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for establishing a system for the extraction and purification of Rhodiola rosea protoplasts, characterized in that... Includes the following steps: A. Select Rhodiola rosea callus tissue and culture it in a culture medium for a period of time under dark conditions to induce chloroplast degradation and obtain light green or light yellow Rhodiola rosea callus tissue. B. Crush the loose, light green or light yellow Rhodiola rosea callus obtained in step A, cut the callus into small pieces, and put them into a container. C. Add the enzymatic hydrolysate to the container and carry out enzymatic hydrolysis under dark conditions to obtain the enzymatic hydrolysate mixture; D. The enzymatic hydrolysis mixture obtained after step C is filtered to remove residue and obtain protoplast enrichment solution. E. Wash the protoplast enrichment solution with cell washing solution; F. After purifying and centrifuging the washed protoplast enrichment solution, Rhodiola rosea protoplasts can be obtained by resuspending it in cell washing solution.

2. The method for establishing the Rhodiola rosea protoplast extraction and purification system according to claim 1, characterized in that: In step A, the culture medium formulation is as follows: MS powder 4.47 g / L + 6-BA 0.6~1.2 mg / L + NAA 0.2~0.3 mg / L + 2,4-D 0.1~0.2 mg / L + IAA 0.1~0.2 mg / L + agar 8~10 g / L + sucrose 25~30 g / L; the pH of the culture medium is 5.7~5.9; the ambient temperature for culturing the Rhodiola rosea callus in the culture medium is 25 ± 2℃; the pale green or pale yellow Rhodiola rosea callus needs to be cultured in the dark for 10~14 days.

3. The method for establishing the Rhodiola rosea protoplast extraction and purification system according to claim 1, characterized in that: In step B, the callus tissue is cut into small pieces with dimensions of 1-2 mm.

4. The method for establishing the Rhodiola rosea protoplast extraction and purification system according to claim 1, characterized in that: In step C, the enzymatic hydrolysis process is as follows: First, an enzymatic hydrolysate is prepared, comprising 1.0-2.0% cellulase, 0.5-1.0% cleavage enzyme, 0.4-0.7 mol / L mannitol, 2-5 mmol / L MES, and 5-10 mmol / L CaCl2, and diluted to volume with a sucrose-free CPW solution with a pH of 5.6-5.8; the conical flask containing the enzymatic hydrolysate is heated in a water bath at 50-60°C for 5-15 minutes, cooled to room temperature, and then filtered through a 0.22-0.45 μm microporous membrane; next, callus tissue cut into small pieces is added to the enzymatic hydrolysate at a ratio of 1 g of callus tissue per 10-15 mL of enzymatic hydrolysate, and then the mixture is placed on a horizontal shaker and shaken at 60-80 r / min for 0.5-2 hours.

5. The method for establishing the Rhodiola rosea protoplast extraction and purification system according to claim 1, characterized in that: In step D, the enzymatic hydrolysis mixture is filtered using a filter screen with a pore size of 45 μm or 800 mesh.

6. The method for establishing the Rhodiola rosea protoplast extraction and purification system according to claim 1, characterized in that: In steps E and F, the cell washing solution is a W5 solution, and the buffer solution consists of 1.5~2.5 mmol / L MES, 140~160 mmol / L NaCl, 120~130 mmol / L CaCl2, and 4~8 mmol / L KCl; the pH value of the cell washing solution is 5.7~5.

9.

7. The method for establishing the Rhodiola rosea protoplast extraction and purification system according to claim 1, characterized in that: In step E, the process of washing the protoplast enrichment solution with cell washing solution is as follows: First, slowly add cell washing solution to the protoplast enrichment solution and place it in a refrigerator at room temperature or 4°C for more than 20 minutes. The upper liquid is the protoplast enrichment layer solution.

8. The method for establishing the Rhodiola rosea protoplast extraction and purification system according to claim 1, characterized in that: In step F, the purification centrifugation includes the following steps: a. Transfer 10-20 ml of the upper protoplast enrichment medium into a 50 ml centrifuge tube, add 10-20 ml of cell washing buffer, mix well, centrifuge at 600-900 r / min for 4-8 min, and discard the supernatant; b. Resuspend the precipitate in 10-25 ml of cell washing buffer, mix well, centrifuge again under the same conditions, and discard the supernatant. c. Resuspend the precipitate in 10-25 ml of cell washing buffer, mix well, centrifuge at 600-900 r / min for 2-4 min, and discard the supernatant; d. After precipitating, add 0.5~1ml of cell washing buffer to resuspend the protoplasts.

9. The application of Rhodiola rosea protoplasts prepared by any one of claims 1 to 8 in studies on transient gene expression, regulation of rhodioloside metabolism, protoplast fusion, or cell wall regeneration.

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