A method for separating and purifying waxberry protoplasts

Abstract

The application discloses a method for separating and purifying waxberry protoplasts. The method mainly comprises the following steps: culturing and obtaining sterile seedlings or cluster seedlings of the waxberry, and selecting tender new leaves at the top of the second to third leaves; cutting the leaves into strips and then placing the leaves in a permeation liquid for pretreatment; then placing the leaves in an enzymolysis liquid containing cellulase R-10, dissociation enzyme R-10 and pectinase Y-23, and performing enzymolysis under constant temperature, darkness and shaking conditions; and finally filtering through a cell sieve, centrifuging and resuspending to obtain the waxberry protoplasts. The yield of the protoplasts obtained by using the method is as high as 3.56x10 6 The application realizes efficient separation and purification of the waxberry protoplasts for the first time, and provides a key technical basis for molecular breeding and genetic improvement of the waxberry.

Description

Technical Field

[0001] This invention relates to the field of plant cell engineering, and specifically to a method for isolating and purifying myrica protoplasts. Background Technology

[0002] The Chinese bayberry (Morella rubra Lour.) is a specialty fruit tree of southern my country. Its brightly colored fruit, rich aroma, and sweet-sour taste make it a favorite among consumers both domestically and internationally, earning it the nickname "the agate of fruits." The fruit is rich in nutrients such as organic acids, flavonoids, vitamins, minerals, and dietary fiber. Furthermore, the entire Chinese bayberry plant is rich in various bioactive components, such as myricetin and its derivatives, and quercetin and its derivatives. These abundant nutrients and bioactive components give fresh Chinese bayberries certain antioxidant, anti-inflammatory, anti-tumor, and blood sugar-lowering effects. In recent years, the genetic breeding and gene improvement of fruit trees such as the Chinese bayberry have received increasing attention, with an urgent need to utilize modern molecular biology breeding methods to improve the quality of the people's "fruit basket." However, no researchers have yet reported on the preparation of Chinese bayberry protoplasts, leaving a gap in this technological system.

[0003] Protoplasts are living plant cells obtained after removing their cell walls. Because their plasma membranes are exposed, protoplasts readily accept exogenous genetic material delivery tools, such as plasmids and high-energy gold particles containing DNA, making them ideal materials for plant genetic transformation and molecular biology experiments. Furthermore, protoplasts possess a complete set of chromosomes, giving them totipotency. Under certain environmental conditions and physiological regulation, protoplasts can regenerate their cell walls, form callus clusters, further generate callus tissue, and ultimately form regenerated plants. Protoplasts can be used not only for transient transformation but also for scientific research such as plant somatic cell hybridization, protein-protein interactions, subcellular localization, promoter activity, and single-cell RNA sequencing. The establishment of this system will help fill the gap in genetic transformation methods in the molecular biology research of *Myrica rubra*, and promote the functional analysis and application research of genes related to stress resistance and quality improvement.

[0004] However, the technology for isolating and purifying *Myrica rubra* protoplasts remains lacking, hindering researchers from conducting in-depth studies of *Myrica rubra* using molecular biology methods. Isolating *Myrica rubra* protoplasts not only allows researchers to directly observe the subcellular structure of these protoplasts but also facilitates the homologous expression and verification of *Myrica rubra* genes, playing a crucial practical role in future breeding and genetic improvement of superior *Myrica rubra* varieties. Therefore, a method for extracting *Myrica rubra* protoplasts is urgently needed. Summary of the Invention

[0005] To address the lack of protoplast isolation and purification systems in existing technologies for *Myrica rubra*, this invention provides a method for isolating and purifying *Myrica rubra* protoplasts, offering a technical foundation for molecular breeding of *Myrica rubra*. This invention successfully isolates *Myrica rubra* protoplasts through the synergistic effect of a specific ratio between young *Myrica rubra* explants and enzymatic hydrolysate.

[0006] Based on the characteristics of the *Myrica rubra* species and the structure of its leaves, this invention pioneers a method for preparing protoplasts from *Myrica rubra* leaves. The waxy layer of *Myrica rubra* leaves is significantly thickened, making enzymatic hydrolysis of the cell walls of mature leaves difficult. Conventional methods using model plants or citrus protoplast isolation techniques often fail to successfully isolate complete protoplasts from *Myrica rubra* leaves. Therefore, it is necessary to develop a method specifically tailored to the characteristics of *Myrica rubra* leaves for the isolation and purification of its protoplasts.

[0007] In this invention, the waxy layer of the leaves of sterile bayberry seedlings and the clump-forming seedlings obtained from propagation is relatively thin, allowing liquids from the external environment to penetrate into the tender leaf tissue without the need to specifically remove the lower epidermis. After shredding, adding enzyme solution, vacuuming, and light-protected enzymatic hydrolysis, bayberry leaf protoplasts can be isolated from the leaves of sterile bayberry seedlings.

[0008] The specific technical solution of the present invention is as follows: This invention provides a method for isolating and purifying myrica protoplasts, comprising the following steps: S100: Select the pits of naturally cracked bayberries, sterilize them through sowing and cultivation to obtain sterile seedlings or propagate clump seedlings, take the tender leaves at the top of the morphology, divide them and place them in the permeation solution for pretreatment. S200: Transfer the pretreated material to the enzymatic hydrolysis solution, vacuum the solution, and then enzymatically hydrolyze it in the dark. S300: The enzymatic hydrolysis product was filtered, centrifuged, and washed and resuspended with W5 solution to obtain bayberry protoplasts; The enzymatic hydrolysate in step S200 comprises: 0.35-0.6 mol / L mannitol, 3.4-3.6% (w / v) cellulase R-10, 1.8-2.2% (w / v) dissociative enzyme R-10, 0.6% (w / v) pectinase Y-23, 0.01 mol / L LMES, and 0.01 mol / L CaCl2. Preferably, the pH of the enzymatic hydrolysate is 5.5-6.0; more preferably, the pH of the enzymatic hydrolysate is 5.7.

[0009] In some embodiments, sterilized bayberry seeds are sown on MS medium to obtain sterile seedlings. The method of obtaining sterile seedlings includes: selecting bayberry seeds with high viability in the current year, removing the outer seed coat, and sterilizing the seeds with 75% ethanol solution and 1% sodium hypochlorite solution with available chlorine concentration; after the bayberry seeds successfully germinate and produce sterile seedlings, the sterile seedlings are subcultured and propagated into clump-forming bayberry seedlings; preferably, the 2nd to 3rd tender leaves at the upper morphology of the clump-forming bayberry seedlings are used after about 3 to 4 weeks of propagation.

[0010] In some embodiments, the tender leaves mentioned in step S100 are taken from the 1st to 4th leaves at the morphological apex of a sterile or clump-forming waxberry seedling; preferably, 2 to 3 leaves.

[0011] In some embodiments, the leaf segmentation in step S100 involves dividing the leaf tissue into thin strips of 0.5 to 1 mm using a sharp blade; the pretreatment involves placing the segmented tissue blocks in a permeation solution for 10 to 30 minutes; preferably, the treatment time is 15 to 25 minutes; more preferably, the treatment time is 20 minutes.

[0012] In some embodiments, in step S100, the composition of the permeate is: 0.35~0.6 mol / L mannitol, 0.015~0.045 mol / L CaCl2, 0.01~0.025 mol / L KCl, and 0.01~0.025 mol / L MES; preferably, the concentration of mannitol is 0.45 mol / L; preferably, the concentration of CaCl2 is 0.03 mol / L; preferably, the concentration of KCl is 0.01 mol / L; preferably, the concentration of MES is 0.01 mol / L; preferably, the pH value of the permeate is 5.5~6.0; more preferably, the pH value of the permeate is 5.7.

[0013] Furthermore, the permeate is prepared from pure water.

[0014] In some embodiments, when the thin strip described in step S100 is placed in the permeation chamber, care should be taken to store it away from light.

[0015] In some embodiments, the cellulase used in this invention is cellulase R-10 (Yakult), and preferably the dissociation enzyme used is dissociation enzyme R-10 (Yakult) or pectinase Y-23 (Coolaber). In some embodiments, the preparation method of the enzymatic hydrolysate of the present invention is as follows: first, CaCl2 and MES are prepared as stock solutions respectively. After the remaining components of the enzymatic hydrolysate are mixed and preheated in a water bath at 50~70℃ for 5~30 min, they are naturally cooled to room temperature. Then, the CaCl2 stock solution and MES stock solution are added and the volume is adjusted. Preferably, the water bath preheating temperature is 65℃.

[0016] Furthermore, the water bath preheating time is 10-20 minutes, more preferably, the water bath preheating time is 10 minutes.

[0017] In some embodiments, the vacuuming conditions in step S200 are: vacuuming with a vacuum machine for 10-20 minutes, followed by slow venting for 3-10 minutes; preferably, the light-protected enzymatic hydrolysis conditions in step S200 are: a temperature of 25-30°C and a constant-temperature shaking speed of 40-80 r·min. -1 The enzymatic hydrolysis time is 2-12 h; more preferably, the temperature is 28℃; more preferably, the rotation speed of the constant temperature shaker is 50-70 r·min. -1 More preferably, the enzymatic hydrolysis time is 6-10 h.

[0018] In some embodiments, the filtration in step S300 uses a cell sieve with a pore size of 30-50 μm; preferably, the filtration in step S300 uses a cell sieve with a pore size of 40 μm; preferably, the centrifugation conditions in step S300 are: 20-25℃, rotation speed of 300-600 rpm, and centrifugation time of 2-5 min.

[0019] In some embodiments, the components of the W5 solution in step S300 are: 154 mmol / L NaCl, 125 mmol / L CaCl2, 5 mmol / L KCl, and 2 mmol / L MES. Preferably, the pH value of the W5 solution is 5.5 to 6.0; more preferably, the pH value of the W5 solution is 5.7.

[0020] Further, the method to terminate the enzymatic hydrolysis is to add an equal volume of W5 solution and filter it through a 40μm (325 mesh) cell sieve to remove impurities; preferably, washing a small amount of tissue block with W5 solution multiple times can promote the release of protoplasts; the purification method is centrifugation purification, with centrifugation purification 2-3 times; further, the parameters for the first centrifugation purification are 20℃, 500 rpm, 2-5 min, after which the supernatant is removed and the protoplasts are resuspended in W5; further, the parameters for the second and third centrifugation purification are 20℃, 300-400 rpm, 2-5 min, after which the protoplasts are resuspended in an appropriate amount of W5; In some embodiments, the bayberry variety is 'water chestnut'.

[0021] In some embodiments, the resuspended bayberry protoplasts are stored on ice; before observation under an optical microscope, the bayberry protoplasts are gently aspirated and mixed, and 40-60 μL of the protoplast suspension is dropped onto a glass slide and gently covered with a coverslip using tweezers; preferably, the yield of bayberry protoplasts is calculated using a hemocytometer. In some embodiments, the resuspension of protoplasts should be performed using a "de-sharpened syringe tip" and the resuspension of bayberry protoplasts should be done gently. In some embodiments, the protoplasts are protoplasts derived from myrica leaf mesophyll cells.

[0022] Compared with the prior art, the beneficial effects of this invention application are as follows: 1) For the first time, a relatively large number of myrica protoplasts were isolated from myrica leaves, with a protoplast yield of 3.56 × 10⁻⁶. 6 The concentration of protoplasts per mL solved the problem of difficult preparation of protoplasts from bayberry leaves.

[0023] 2) The separation and purification method in this invention yields high protoplasts with intact morphology, meeting the needs of subsequent transformation. Successfully obtaining bayberry protoplasts can also largely solve the problem of the lack of homology verification system in bayberry in the later stage, laying a technical foundation for the future molecular biology breeding of bayberry. Attached Figure Description

[0024] Appendix Figure 1 This is an optical microscope image of the myrica protoplasts extracted in Example 1 under a 40x microscope. Appendix Figure 2 This is an optical microscope image of the myrica protoplasts extracted in Example 2 under a 40x microscope. Appendix Figure 3 This is an optical microscope image of the myrica protoplasts extracted in Example 3 under a 40x microscope. Appendix Figure 4 This is an optical microscope image of the myrica protoplasts extracted in Example 3 under a 20x microscope. Appendix Figure 5 This is an optical microscope image of the myrica protoplasts extracted in Example 3 in a hemocytometer under a 10x microscope. Appendix Figure 6 This is an optical microscope image of the enzymatically digested protoplasts from non-young leaves of *Myrica rubra* extracted in Comparative Example 1, viewed under 40x magnification. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0026] The reagents involved in the examples are shown in Table 1. The table provides information on the manufacturers and catalog numbers of the reagents. The source of the reagents is for reference only and is not intended to limit their use.

[0027] Table 1 All solutions involved in the examples need to be prepared and used immediately.

[0028] Terms and abbreviations used in this invention: MES: 2-(N-morpholino)ethanesulfonic acid. MS: Murashige and Skoog Medium (MS medium) 6BA: 6-Benzylaminopurine, (6-benzylaminopurine) IBA: Indole-3-butyric acid. Example 1 This embodiment provides a method for isolating and purifying myrica protoplasts, involving the following steps: (1) Select waxberry seeds harvested in the current year with high vigor. Choose waxberry seeds with naturally cracked seed coats and remove the hard outer seed coat with pliers, while avoiding mechanical damage to the waxberry kernel. After obtaining the seeds with the outer shell removed, disinfect the seed surface with 75% (v / v) solution for 30 seconds in a clean bench, and then wash with sterile water 2-3 times. After drying the waxberry seeds, disinfect them with a 1% sodium hypochlorite solution for 15 minutes, shaking the container moderately during the disinfection process. Then wash the seeds thoroughly with clean water 5-7 times. Inoculate the disinfected seeds into MS medium and treat them in the dark at an ambient temperature of 23℃ for about one week. Then, transfer the seeds to a light culture room at an ambient temperature of 23℃ for cultivation.

[0029] (2) After cultivating the seeds from step (1) for 3 to 6 weeks, sterile seedlings of bayberry are obtained. In a clean bench, the sterile seedlings of bayberry produced from the seeds are divided with a scalpel. The standard for division is to retain a 0.5 to 1 cm stem segment with leaves. Then, they are inoculated into a propagation medium and cultured for 3 to 6 weeks. They are placed in a light culture room with an ambient temperature of 23℃ until the plants survive and grow tender new leaves.

[0030] The culture medium formula is as follows: 2.2g MS powder, 10g sucrose, 100mg inositol, 8g agar, 300μL 1mol / L KOH stock solution, 1mL 6BA (stock solution concentration 1mg / L), and 200μL IBA (stock solution concentration 0.5mg / mL).

[0031] (3) Select tender leaves from the clump seedlings obtained in step (2), select the leaves at the top of the plant morphology of a single clump seedling, the leaves are light green, soft and not leathery, and select the 1st to 3rd leaves at the top of the morphology as explants for protoplast extraction.

[0032] Preferably, the second tender leaf at the top of the morphological apex of a clustered bayberry seedling is selected as the explant.

[0033] (4) Divide the tender leaves of the sterile bayberry seedlings obtained in step (3), cut the leaves perpendicular to the main vein with a sharp blade, divide the tissue into strips of 0.5-1 mm in size, and immediately place the leaf strips in a petri dish containing permeation solution. The permeate composition was: 0.45 mol / L mannitol, 0.03 mol / L CaCl2, 0.01 mol / L KCl, 0.01 mol / L LMES, with the balance being pure water, and the pH was adjusted to 5.7. Pre-treat tissue blocks with osmotic solution for 20-30 minutes, while moisturizing material simultaneously promotes plasmolysis of bayberry leaf mesophyll cells.

[0034] (5) Transfer the leaf strips treated in step (4) into the enzymatic hydrolysate; The enzymatic hydrolysate consisted of: cellulase R-10 3.4%, pectinase R-10 1.8%, pectinase Y-23 0.60%, mannitol 0.45 mol / L, 0.01 mol / L CaCl2, and 0.01 mol / L MES. The hydrolysate volume was 10 mL, with the remainder being pure water, and the pH was adjusted to 5.7. First, stock solutions of CaCl2 and MES were prepared at concentrations of 1 mol / L and 0.1 mol / L, respectively. After preheating the hydrolysate in a water bath at 65°C for 10 min, the hydrolysate was allowed to cool naturally to room temperature. Then, 100 μL of the CaCl2 stock solution and 1 mL of the MES stock solution were added, and finally, the volume was adjusted to 10 mL with pure water.

[0035] (6) Place the enzymatic hydrolysis system containing leaf strips in step (5) under light-proof conditions and vacuum it for 10-20 minutes. After vacuuming, gently open the gas valve and slowly release the gas for 5 minutes to ensure that the process of the enzymatic hydrolysate penetrating into the tissue is gradual.

[0036] (7) After vacuuming in step (6), wrap the enzymatic hydrolysis system with tin foil to block light, place it in a constant temperature shaker at 28℃, set the speed to 60 rpm, and the enzymatic hydrolysis time is 6~10h; In this embodiment, the enzymatic hydrolysis time is 6 hours.

[0037] (8) Observe the enzymatic hydrolysis system in step (7). If the liquid is a semi-transparent dark green, the enzymatic hydrolysis is successful and can be terminated. If the color of the enzymatic hydrolysis system is not a dark green, the time can be extended appropriately. The reaction is terminated by adding an equal volume of W5 solution; Further, the sample was filtered through a 40 μm (325 mesh) cell sieve to remove impurities, and the resulting filtrate contained protoplasts. The composition of solution W5 is: 154 mmol / L NaCl, 125 mmol / L CaCl2, 5 mmol / L KCl, 2 mmol / L MES, with the remainder being pure water, and pH=5.7. Example 1 used a gentle pressing motion on the tissue block to promote the release of the remaining protoplasts; Wash small amounts of tissue blocks repeatedly with W5 solution to facilitate the collection of remaining protoplasts.

[0038] (9) The filtrate obtained in step (8) is centrifuged for purification. The first centrifugation is set at 20°C, 500 rpm, and 2.5 min. After centrifugation, the supernatant is removed with an unremoved pipette tip, the precipitate of bayberry protoplasts is collected, and the W5 solution is taken with a pipette tip and the protoplasts are gently resuspended. The second centrifugation purification parameters are 20°C, 300~400 rpm, and 2.5 min. An appropriate amount of W5 is taken with a pipette tip and the protoplasts are resuspended. If you want to further reduce impurities, the centrifugation purification can be increased to a third time, with the centrifugation purification parameters being 20°C, 300~400 rpm, and 2.5 min.

[0039] (10) After collecting the protoplasts from step (9), they can be stored on ice. Before observation under an optical microscope, use a de-pointed pipette tip to slowly aspirate and mix the protoplasts to ensure they are fully and evenly suspended in the solution. Pipette 40-60 μL onto a glass slide, gently cover the slide with tweezers, and examine under an optical microscope. Figure 1 ).

[0040] Example 2 The concentrations of cellulase R-10 and analyte R-10 were optimized, with cellulase R-10 increasing from 3.4% to 3.6% and analyte R-10 increasing from 1.8% to 2.0%. The remaining implementation steps are the same as steps (1)-(10) in Example 1; The optimized enzymatic hydrolysate composition was as follows: cellulase R-10 3.6%, pectinase R-10 2.0%, pectinase Y-23 0.6%, mannitol 0.45 mol / L, 0.01 mol / L CaCl2, 0.01 mol / L MES, with a hydrolysis volume of 10 mL and the remainder being pure water, adjusted to pH 5.7. First, stock solutions of CaCl2 and MES were prepared at concentrations of 1 mol / L and 0.1 mol / L, respectively. After preheating the hydrolysate in a water bath at 65°C for 10 min, the hydrolysate was allowed to cool naturally to room temperature. Then, 100 μL of the CaCl2 stock solution and 1 mL of the MES stock solution were added, and finally, the volume was adjusted to 10 mL with pure water. Example 2: Optical microscopic observation results of the isolation and purification of Myrica rubra protoplasts are as follows: Figure 2 As shown.

[0041] Example 3 The concentration of the analyte R-10 was optimized, increasing it from 2.0% to 2.2%. The method of handling materials has been optimized, namely, no tissue blocks are pressed lightly; The remaining implementation steps are the same as steps (1)-(10) in Example 1; The optimized enzymatic hydrolysate composition is as follows: cellulase R-10 3.6%, pectinase R-10 2.2%, pectinase Y-23 0.6%, mannitol 0.45 mol / L, 0.01 mol / L CaCl2, 0.01 mol / L MES, with a hydrolysis volume of 10 mL and the remainder being pure water, adjusted to pH 5.7. First, stock solutions of CaCl2 and MES were prepared at concentrations of 1 mol / L and 0.1 mol / L, respectively. After preheating the hydrolysate in a water bath at 65°C for 10 min, the hydrolysate was allowed to cool naturally to room temperature. Then, 100 μL of the CaCl2 stock solution and 1 mL of the MES stock solution were added, and finally, the volume was adjusted to 10 mL with pure water.

[0042] Example 3: Optical microscopic observation results of the isolation and purification of Myrica rubra protoplasts are as follows: Figure 3 and Figure 4 As shown.

[0043] Aspirate 40-60 μL of protoplasts using a de-pointed pipette tip, preserve them on ice, and observe and count them under a light microscope on a 16×25 hematology counting plate. Figure 5 ); Protoplast yield (number / mL) = (average number of cells in a 5-square grid) × 104 × Dilution factor / Fresh weight of leaves; Based on the observed number of protoplasts in the five square cells, the average yield was calculated to be 3.56 × 10⁻⁶. 6 per mL.

[0044] Comparative Example 1 This comparative example provides a method for protoplast extraction. The core difference between this method and Examples 1-3 lies in the tenderness of the leaves from sterile bayberry seedlings. All other operational steps, reagent types and concentrations, and process parameters are completely consistent with Example 1. The specific results are as follows: Select the whole leaves (containing a large amount of non-young tissue) of the single-plant clump seedlings. The leaves are light green to dark green, most of them are hard and some are leathery. When cutting with a blade, you can feel obvious resistance.

[0045] Comparative Example 1: The results of optical microscopic observation of the isolation and purification of Myrica rubra protoplasts are attached. Figure 6 As shown, some cells exhibit deformed, elongated, or irregular spherical shapes, and there are many cell fragments, resulting in a relatively turbid background.

[0046] The above results show that the present invention selects young tissues as starting materials, which can effectively increase the yield of protoplasts and reduce the breakage rate. It solves the technical problems of low enzymatic hydrolysis efficiency and severe protoplast damage caused by thick cuticle and dense cell wall structure in non-young tissues, highlighting the superiority of the technical solution of the present invention.

[0047] The above embodiments and accompanying figures visually demonstrate the protoplasts isolated from young bayberry leaves using the method described in this invention (including permeation pretreatment, specific enzyme solution formulation, and purification steps). The accompanying microscopic images show that the isolated protoplasts are regularly spherical and evenly distributed, proving that this method can effectively remove cell walls while maintaining the integrity of the protoplast membrane structure.

[0048] This invention employs a specific separation and purification method, successfully isolating *Myrica rubra* protoplasts through the synergistic effect of a specific ratio of young *Myrica rubra* explants and enzymatic hydrolysate. This invention provides a method for the isolation and purification of *Myrica rubra* protoplasts, successfully achieving the dissociation of *Myrica rubra* protoplasts from explants and enabling the effective preparation of large quantities of relatively pure *Myrica rubra* mesophyll tissue protoplasts. The isolated protoplasts, after dilution, are sufficient for subsequent protoplast transient transformation, single-cell sequencing, and other procedures, providing a meaningful technical foundation for molecular biology research on *Myrica rubra*.

[0049] Those skilled in the art will understand that the steps, measures, and schemes in the various operations, methods, and processes discussed in this application can be alternated, modified, combined, or deleted; furthermore, other steps, measures, and schemes in the various operations, methods, and processes discussed in this application can also be alternated, modified, rearranged, decomposed, combined, or deleted; furthermore, the steps, measures, and schemes in the prior art that are similar to those disclosed in this application can also be alternated, modified, rearranged, decomposed, combined, or deleted. The technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification. The above-described embodiments are merely illustrative of several implementation methods of this disclosure, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of the patent for the embodiments of this disclosure. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the embodiments of this disclosure, and these all fall within the protection scope of the embodiments of this disclosure. Therefore, the protection scope of the embodiments of this disclosure should be determined by the appended claims. As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and detail can be made without departing from the spirit and scope of the present invention as defined in the appended claims.

[0050] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the present invention, such designs should fall within the protection scope of the present invention.

Claims

1. A method for isolating and purifying myrica protoplasts, characterized in that, Includes the following steps: Step S100: Select the pits of naturally cracked bayberries, disinfect and sow them to obtain sterile seedlings or clump seedlings obtained through propagation, take the tender leaves at the top of the morphology, divide them and place them in a permeation solution for pretreatment. Step S200: Transfer the pretreated material to the enzymatic hydrolysate, and then enzymatically hydrolyze it in the dark after vacuuming. Step S300: Filter the enzymatic hydrolysis product, centrifuge, and wash and resuspend it with W5 solution to obtain bayberry protoplasts; In step S200, the enzymatic hydrolysate comprises: 0.35-0.6 mol / L mannitol, 3.4-3.6% (w / v) cellulase R-10, 1.8-2.2% (w / v) dissociative enzyme R-10, 0.6% (w / v) pectinase Y-23, 0.01 mol / L LMES, and 0.01 mol / L CaCl2. Preferably, the pH of the enzymatic hydrolysate is 5.5-6.0; more preferably, the pH of the enzymatic hydrolysate is 5.

7.

2. The method for isolating and purifying myrica protoplasts according to claim 1, characterized in that, In step S100, the tender leaves are taken from the 1st to 4th leaves at the morphological apex of sterile or clump-forming waxberry seedlings; preferably, 2 to 3 leaves.

3. The method for isolating and purifying myrica protoplasts according to claim 1, characterized in that, In step S100, the leaf segmentation involves using a sharp blade to divide the leaf tissue into thin strips of 0.5-1 mm; the pretreatment involves placing the segmented tissue blocks in a permeation solution for 10-30 minutes; preferably, the treatment time is 15-25 minutes; more preferably, the treatment time is 20 minutes.

4. The method for isolating and purifying myrica protoplasts according to claim 1, characterized in that, In step S100, the permeate consists of: 0.35~0.6 mol / L mannitol, 0.015~0.045 mol / L CaCl2, 0.01~0.025 mol / L KCl, and 0.01~0.025 mol / L MES; Preferably, the concentration of mannitol is 0.45 mol / L; preferably, the concentration of CaCl2 is 0.03 mol / L; preferably, the concentration of KCl is 0.01 mol / L; preferably, the concentration of MES is 0.01 mol / L; preferably, the pH value of the permeate is 5.5~6.0; more preferably, the pH value of the permeate is 5.

7.

5. The method for isolating and purifying myrica protoplasts according to claim 1, characterized in that, The preparation method of the enzymatic hydrolysate is as follows: first, CaCl2 and MES are prepared separately as stock solutions. After the remaining components of the enzymatic hydrolysate are mixed and preheated in a water bath at 50-70℃ for 5-30 minutes, they are naturally cooled to room temperature. Then, the CaCl2 stock solution and MES stock solution are added and the volume is adjusted. Preferably, the water bath preheating temperature is 65℃.

6. The method for isolating and purifying myrica protoplasts according to claim 5, characterized in that, The water bath preheating time is 10-20 minutes, more preferably 10 minutes.

7. The method for isolating and purifying myrica protoplasts according to claim 1, characterized in that, In step S200, the vacuuming conditions are as follows: vacuuming with a vacuum machine for 10-20 minutes, followed by slow venting for 3-10 minutes; preferably, the light-protected enzymatic hydrolysis conditions in step S200 are: a temperature of 25-30°C and a constant-temperature shaking incubator speed of 40-80 r·min. -1 The enzymatic hydrolysis time is 2-12 h; more preferably, the temperature is 28℃; more preferably, the rotation speed of the constant temperature shaker is 50-70 r·min. -1 More preferably, the enzymatic hydrolysis time is 6-10 h.

8. The method for isolating and purifying myrica protoplasts according to claim 1, characterized in that, In step S300, the filtration uses a cell sieve with a pore size of 30~50 μm; preferably, in step S300, the centrifugation conditions are: 20~25℃, rotation speed of 300~600 rpm, and centrifugation time of 2~5 min.

9. The method for isolating and purifying myrica protoplasts according to claim 1, characterized in that, In step S300, the composition of the W5 solution is: 154 mmol / L NaCl, 125 mmol / L CaCl2, 5 mmol / L KCl, and 2 mmol / L LMES. Preferably, the pH value of the W5 solution is 5.5~6.0; more preferably, the pH value of the W5 solution is 5.

7.

10. The method for isolating and purifying myrica protoplasts according to claim 1, characterized in that, The variety of bayberry mentioned is 'water chestnut'.

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