Method for preparing stevia protoplast and application thereof

Abstract

The application discloses a preparation method of Stevia rebaudiana protoplast and application of the Stevia rebaudiana protoplast. The Stevia rebaudiana protoplast is prepared through the following steps: cutting leaves of Stevia rebaudiana tissue culture seedlings into strips, and placing the strips in a mannitol solution to perform pre-plasmolysis; draining the mannitol solution, and placing the strips in an enzyme solution; vacuumizing, and performing enzyme hydrolysis under the conditions of oscillation and light shielding; the enzyme solution comprises cellulase, dissociation enzyme, mannitol, MES, W5 buffer solution and glutathione; the W5 buffer solution is added with the glutathione; after the enzyme hydrolysis is completed, the enzyme solution is absorbed, the W5 buffer solution is added and shaken, a cell sieve is passed, the W5 buffer solution is eluted, and a filtrate is recovered; the filtrate is centrifuged, the supernatant is discarded, and the Stevia rebaudiana protoplast is obtained. The Stevia rebaudiana protoplast prepared by the method has high yield and high cell activity, is suitable for gene transient expression of Stevia rebaudiana, provides a stable single-cell test platform for basic research on Stevia rebaudiana molecular breeding, and has high scientific research and application values.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically to the field of plant cell engineering and crop molecular breeding technology. More specifically, this invention relates to a method for preparing stevia protoplasts and their application in transient transformation. Background Technology

[0002] After the cell wall is removed, protoplasts retain complete genetic information and totipotency, making them suitable for molecular experiments such as protein subcellular localization, transient expression of exogenous genes, cell fusion, and single-cell sequencing. Stable protoplast manipulation systems have been established in herbaceous model plants such as Arabidopsis thaliana and rice.

[0003] Stevia is a distinctive economic crop rich in steviol glycosides and various bioactive substances. my country ranks first globally in terms of cultivation, glycoside processing, and export. Existing research has covered areas such as stevia breeding, in vitro rapid propagation, and high-yield cultivation, but research bottlenecks remain in the supporting technologies for leaf protoplast isolation.

[0004] Currently, the demand for functional verification of genes related to stevia glycoside synthesis and stress tolerance continues to increase, making stable and efficient protoplast transformation platforms the core carriers for gene function analysis. The current mainstream method for preparing stevia protoplasts involves enzymatic hydrolysis using three compound enzymes (cellulase, dissociation enzyme, and pectinase). Young leaves are used as material, and the process involves enzymatic hydrolysis followed by sedimentation or density gradient centrifugation to remove cell debris. Because stevia mesophyll cells are rich in polyphenols and polysaccharides, they are highly susceptible to oxidation and browning during the enzymatic hydrolysis stage. This results in a low yield of intact protoplasts, numerous broken fragments, and low cell viability, failing to meet the needs of high-precision gene transformation and single-cell omics experiments, severely restricting stevia cell engineering and molecular breeding research. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide a method for preparing stevia protoplasts, which yields high-quality stevia protoplasts with high cell viability and high conversion efficiency for transient transformation.

[0006] The technical solutions for achieving the above-mentioned objectives include the following.

[0007] In a first aspect, the present invention provides a method for preparing stevia protoplasts, comprising the following steps:

[0008] S1. Cut the leaves of the stevia tissue culture seedlings into thin strips and place them in a mannitol solution for preplasmolysis.

[0009] S2. After the pre-plasmolysis of the leaves in step S1 is drained, the leaves are placed in the enzymatic hydrolysis solution, vacuumed, and enzymatically hydrolyzed in the dark under shaking conditions.

[0010] The enzymatic hydrolysate comprises 1 wt%–1.5 wt% cellulase, 0.25 wt%–0.35 wt% dissociative enzyme, 0.4 M–0.6 M mannitol, 1.8 mM–2.2 mM MES, 0.1 × W5 buffer, and 0.08 wt%–0.12 wt% glutathione.

[0011] S3. After the enzyme digestion in the dark is completed, aspirate the enzyme digest, add W5 buffer and shake, pass through a cell sieve, then elute with W5 buffer and recover the filtrate.

[0012] S4. Centrifuge the filtrate obtained in step S3, discard the supernatant, and obtain the stevia protoplasts;

[0013] In steps S2 and S3, 0.08 wt% to 0.12 wt% of glutathione is added to the W5 buffer solution.

[0014] In a second aspect, the present invention provides stevia protoplasts prepared by the above-described preparation method.

[0015] A third aspect of the present invention provides the application of the above-mentioned stevia protoplasts in transient transformation.

[0016] A fourth aspect of the present invention provides a method for transient transformation based on stevia protoplasts, comprising the following steps:

[0017] S1. The above-mentioned stevia protoplasts were resuspended in MMG solution, mixed with plasmids containing the target gene, and then PEG-CaCl2 solution was added, mixed well, and allowed to stand at room temperature in the dark; the PEG-CaCl2 solution included 40wt%~50wt% PEG-4000, 0.4 M~0.6 M mannitol and 0.08 M~0.12 M CaCl2;

[0018] S2. Add W5 buffer and mix well. Centrifuge and discard the supernatant.

[0019] S3. Add W5 buffer and mix well. Incubate in the dark, centrifuge, discard the supernatant, and you will get the transformed stevia protoplasts.

[0020] This invention uses young stevia leaves from tissue culture as experimental material. Enzymatic hydrolysis is performed using a solution containing a specific ratio of compound enzymes, a specific concentration of mannitol, and specific types and concentrations of antioxidants. The hydrolysis is supplemented by vacuum permeation, low-temperature, low-speed centrifugation, and washing with W5 buffer containing antioxidants. The resulting stevia protoplasts have a high yield (10.46 ± 0.62 × 10⁻⁶ protoplasts per gram of fresh leaf). 7 It has high cell viability (up to 95.88%±1.58%), is suitable for transient expression of stevia genes, and can be stably used for research such as protein subcellular localization, bimolecular fluorescence complementation, and single-cell sequencing.

[0021] The method for preparing and transiently transforming stevia leaf protoplasts established in this invention is simple to operate, time-saving, standardized, and highly reproducible. It can meet the needs of high-precision molecular experiments such as stevia gene function analysis and single-cell omics, providing a stable single-cell experimental platform for basic molecular breeding research of stevia. At the same time, it provides a reference for the construction of protoplast platforms for other economic crops in the Asteraceae family, and has high scientific research and application value. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the preparation process of stevia protoplasts in this invention. In A, bar = 3 cm, and in E, bar = 10 μm.

[0023] Figure 2 The activity test results of the stevia protoplasts prepared in Example 1 of this invention are shown, bar=10 μm.

[0024] Figure 3 The results show the effect of different concentrations of PEG-4000 on the transient conversion efficiency of protoplasts in Example 2 of this invention.

[0025] Figure 4 This is a subcellular localization map of proteins in Example 3 of the present invention, bar = 1 μm.

[0026] Figure 5 The image shows the bimolecular fluorescence complementary (BiFC) results in Example 3 of this invention, bar = 1 μm.

[0027] Figure 6 The results show the effects of different complex enzyme treatment groups on protoplast yield and activity in Example 4 of this invention.

[0028] Figure 7 The results show the effects of different concentrations of mannitol on protoplast yield and activity in Example 5 of this invention.

[0029] Figure 8 The results show the effects of different antioxidant treatment groups on protoplast yield and activity in Example 6 of this invention.

[0030] In the above figure, different letters indicate significant differences between treatments (P<0.05). Detailed Implementation

[0031] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0032] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0033] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0034] Stevia has a high content of polyphenols and secondary metabolites. Directly applying existing common protoplast preparation methods will result in problems such as insufficient enzymatic hydrolysis, severe browning, low protoplast yield, poor quality, low cell viability, and difficulty in transient transformation. Furthermore, the lack of a standardized transient transformation process makes it difficult to support molecular experiments such as gene function verification and protein-protein interaction. After extensive experimentation, the inventors of this invention discovered that using young stevia tissue-cultured leaves as experimental material, enzymatic hydrolysis is performed using a hydrolysate containing a specific ratio of compound enzymes, a specific concentration of mannitol, and specific types and concentrations of antioxidants. This is supplemented with vacuum permeation, low-temperature, low-speed centrifugation, and washing with W5 buffer containing antioxidants (i.e., adding antioxidants to both the hydrolysate and W5 buffer). The prototype glutathione, containing an active sulfhydryl group at its core, has several advantages. First, it competitively inhibits polyphenol oxidase, reducing quinone oxides and effectively alleviating browning caused by phenolic substances in leaves, preventing protoplast adhesion, aggregation, and loss, thus increasing protoplast yield. Second, it participates in the AsA-GSH cycle, scavenging reactive oxygen species generated by enzymatic stress, blocking lipid peroxidation of the plasma membrane, maintaining cell membrane integrity, and reducing cell rupture. Third, it protects the activity of cellulase and dissociative enzymes, ensuring sufficient cell wall dissociation. Based on these principles, it can effectively scavenge reactive oxygen species, inhibit polyphenol oxidative browning in stevia, and significantly reduce cell damage rate. Stevia protoplasts prepared using the method of this invention have a high yield (10.46 ± 0.62 × 10⁻⁶ protoplasts per gram of fresh leaves). 7 With high cell viability (up to 95.88%±1.58%), it is suitable for transient expression of Stevia genes and can be stably used for research such as protein subcellular localization, bimolecular fluorescence complementation, and single-cell sequencing. At the same time, it provides a reference for the construction of protoplast platforms for other economic crops in the Asteraceae family, and has high scientific research and application value.

[0035] Based on the prepared stevia protoplasts, the inventors of this invention further explored the transient transformation technology system. The transient transformation mediated by 40% PEG4000 achieved the highest efficiency (32.10%), significantly reducing protoplast fragmentation and aggregation. Furthermore, the feasibility of subcellular localization and BiFC analysis in stevia leaf protoplasts was verified.

[0036] In some embodiments of the present invention, a method for preparing stevia protoplasts is disclosed, comprising the following steps:

[0037] S1. Cut the leaves of the stevia tissue culture seedlings into thin strips and place them in a mannitol solution for preplasmolysis.

[0038] S2. After the pre-plasmolysis of the leaves in step S1 is drained, the leaves are placed in the enzymatic hydrolysis solution, vacuumed, and enzymatically hydrolyzed in the dark under shaking conditions.

[0039] The enzymatic hydrolysate comprises 1 wt%–1.5 wt% cellulase, 0.25 wt%–0.35 wt% dissociative enzyme, 0.4 M–0.6 M mannitol, 1.8 m M–2.2 m M MES, 0.1 × W5 buffer, and 0.08 wt%–0.12 wt% glutathione.

[0040] S3. After the enzyme digestion in the dark is completed, aspirate the enzyme digest, add W5 buffer and shake, pass through a cell sieve, then elute with W5 buffer and recover the filtrate.

[0041] S4. Centrifuge the filtrate obtained in step S3, discard the supernatant, and obtain the stevia protoplasts;

[0042] In S2 and S3, the W5 buffer solution contains 0.08 wt% to 0.12 wt% glutathione.

[0043] In one embodiment, in step S1, the stevia tissue culture seedlings are obtained by the following steps: after sterilizing the stevia seeds, they are inoculated into MS medium and cultured for 28 to 32 days. Then, stem segments are cut off and inserted into MS medium, and cultured for another 40 to 50 days.

[0044] In one embodiment, in step S1, the concentration of mannitol is 0.4 M to 0.6 M, and the pre-plasmolysis time is 12 min to 18 min.

[0045] In one embodiment, in step S1, the blades are 2 to 3 pairs of blades at the tip.

[0046] In one embodiment, in step S2, the enzymatic hydrolysate comprises 1.2 wt% to 1.5 wt% cellulase, 0.28 wt% to 0.32 wt% dissociative enzyme, 0.45 M to 0.55 M mannitol, 1.8 mM to 2.2 mM MES, 0.1×W5 buffer, and 0.09 wt% to 0.11 wt% glutathione.

[0047] In one embodiment, in step S2, the enzymatic hydrolysate comprises 1.5 wt% cellulase, 0.3 wt% dissociative enzyme, 0.5 M mannitol, 2.0 mM MES, 0.1×W5 buffer, and 0.1 wt% glutathione.

[0048] In one embodiment, the W5 buffer contains 0.09 wt% to 0.11 wt% glutathione.

[0049] In one embodiment, in step S2, the preparation method of the enzymatic hydrolysate is as follows: add cellulase, ionizing enzyme, mannitol, and MES to 0.1×W5 buffer, bring the volume to a final volume with sterile water, heat in a water bath, cool to room temperature, add glutathione, filter through a microporous membrane, and store in the dark.

[0050] In one embodiment, in step S2, the oscillation speed is 40 rpm to 50 rpm.

[0051] In one embodiment, in step S2, the temperature of the light-protected enzymatic hydrolysis is 27°C to 29°C, and the time of the light-protected enzymatic hydrolysis is 3 h to 4 h.

[0052] In one embodiment, in step S3, the pore size of the cell sieve is 65 μm to 75 μm.

[0053] In one embodiment, in step S4, the centrifugation conditions are: 90 g to 110 g, 3 ℃ to 5 ℃.

[0054] Centrifuge for 4 to 6 minutes; the number of centrifugations is 2 to 3 times.

[0055] In other embodiments of the present invention, stevia protoplasts prepared by the above preparation method are disclosed.

[0056] In other embodiments of the present invention, the application of the above-mentioned stevia protoplast in transient transformation is disclosed.

[0057] In other embodiments of the present invention, a transient transformation method based on stevia protoplasts is disclosed, comprising the following steps:

[0058] S1. The above-mentioned stevia protoplasts were resuspended in MMG solution, mixed with plasmids containing the target gene, and then PEG-CaCl2 solution was added, mixed well, and allowed to stand at room temperature in the dark; the PEG-CaCl2 solution included 40wt%~50wt% PEG-4000, 0.4 M~0.6 M mannitol and 0.08 M~0.12 M CaCl2;

[0059] S2. Add W5 buffer and mix well. Centrifuge and discard the supernatant.

[0060] S3. Add W5 buffer and mix well. Incubate in the dark, centrifuge, discard the supernatant, and you will get the transformed stevia protoplasts.

[0061] In one embodiment, in step S1, the time for the light-protected resting period is 12 min to 18 min.

[0062] In one embodiment, in step S1, the PEG-CaCl2 solution comprises 40wt%~45wt% PEG-4000, 0.45 M~0.55 M mannitol, and 0.09 M~0.11 M CaCl2.

[0063] In one embodiment, in step S3, the temperature of the dark culture is 24℃~26℃, and the dark culture time is 22 h~26 h.

[0064] In one embodiment, in steps S2 and S3, the centrifugation conditions are: 90 g to 110 g, centrifugation at 3 ℃ to 5 ℃ for 4 min to 6 min.

[0065] In the following embodiments of the present invention, the Stevia seed Sr1, the subcellular localization vector, and the BiFC vector were all obtained from the Luo Ming research group at the South China Botanical Garden, Chinese Academy of Sciences. Cellulase (Cellulase R-10) and macerozyme (Macerozyme R-10) were purchased from Yakult, and all other reagents were purchased from Sigma.

[0066] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0067] Example 1: Method for preparing stevia protoplasts

[0068] This embodiment provides a method for preparing stevia protoplasts. Please refer to [link / reference needed]. Figure 1 Specifically, it includes the following steps:

[0069] 1. Preparation of Stevia tissue culture seedlings

[0070] (1) Seed disinfection

[0071] Select healthy Stevia Sr1 seeds, remove the pappus at the top of the seeds, rinse with sterile water, then transfer to 75% alcohol and shake for 1 min. Rinse twice with sterile water, then transfer to 2% sodium hypochlorite and shake for 10 min. Rinse three times with sterile water, 5 min each time. Finally, place the seeds on sterile filter paper for later use.

[0072] (2) Inoculation, germination and subculture

[0073] The sterilized seeds were inoculated into germination medium (MS medium containing 6.5 g / L agar + 0.5 mg / L 6-BA) and cultured at 25°C under 16 h light / 8 h dark (LD) conditions. After one month, once the seedlings had grown stem segments, the stem segments were cut off, the leaves were removed, and the seedlings were inoculated into subculture medium (MS medium containing 6.5 g / L agar + 0.5 mg / L IAA) and cultured for another 45 days under 25°C under 16 h light / 8 h dark (LD) conditions to obtain stevia tissue culture seedlings.

[0074] 2. Preparation of Stevia protoplasts

[0075] (1) Cut off the top three pairs of young leaves of the stevia tissue culture seedlings, cut them into strips of about 2 cm with a double-edged blade, and put them into 0.5 M mannitol solution for pre-plasmolysis for 15 min;

[0076] (2) After draining the pre-plasmolysis leaves, weigh 1 g of leaf filaments and place them in a triangular flask containing 10 mL of enzymatic hydrolysate. Vacuum at -700 mm Hg for 15 min and in the dark at 28℃ in a shaker at 45 rpm for 3.5 hours. The enzymatic hydrolysate includes 1.5% cellulase, 0.3% cleavage enzyme, 0.5 M mannitol, 2 mM MES (pH 5.7), and 0.1×W5 buffer. After adjusting the volume with sterile water, heat in a water bath at 55℃ for 10 min. After cooling to room temperature, add 0.1% reduced glutathione (prepared and used immediately) and filter through a 0.22 μm cellulose microporous membrane into a triangular flask. Store in the dark. The W5 buffer includes 2 mM pH 5.7 MES, 154 mM NaCl, 125 mM CaCl2, 5 mM KCl, and 0.1% reduced glutathione. The reduced glutathione should be prepared and used immediately.

[0077] (3) Place the Erlenmeyer flask on ice to stop the reaction; use a pipette to remove the enzyme digest (at this time, a small amount of free protoplasts will be found in the enzyme digest under a microscope), slowly add 10 mL of W5 buffer to the Erlenmeyer flask, shake manually for 10 s, and collect the liquid in the Erlenmeyer flask through a 70 μm cell sieve into a 50 mL round-bottom centrifuge tube. Wash the remaining leaf filaments with 20 mL of W5 buffer, and elute through the cell sieve again to collect the filtrate.

[0078] (4) Centrifuge the filtrate obtained from the previous step at 100 g and 4 ℃ for 5 min, discard the supernatant, slowly add 10 mL of W5 buffer (as above) to resuspend, centrifuge again at 100 g and 4 ℃ for 5 min, discard the supernatant, and you will get the stevia protoplasts.

[0079] (5) Use a pipette tip with the cut end to slowly resuspend the protoplasts in 2 mL of MMG solution (containing 0.5 M mannitol, 15 mM MgCl2 and 4 mM pH 5.7 MES), place on ice and use as soon as possible or place at 4 ℃ for no more than 24 h.

[0080] 3. Protoplast yield and activity

[0081] (1) Protoplast production

[0082] Use a pipette to draw 10 μL of the well-mixed protoplast suspension and inject it into the counting chamber of a hemocytometer. Observe the morphology of the protoplasts and count them using an optical microscope. Repeat 3 times.

[0083] Protoplast yield (N / g) = N' / 5 × 25 × 10 4 × Dilution factor / Mass of enzymatically digested sample (N = number of protoplasts, N' = number of protoplasts in 5 squares using the 5-point sampling method, counting according to the principle of "counting the top but not the bottom, counting the left but not the right").

[0084] The results showed that the yield of protoplasts was 10.46 ± 0.62 × 10⁻⁶. 7 g -1 FW.

[0085] (2) Viability of protoplasts

[0086] Take 100 μL of protoplast suspension, add FDA staining solution to make the final concentration 0.01% (FDA stock solution concentration is 10 mg / mL), let stand in the dark for 5 min and observe with a fluorescence microscope. Protoplasts that fluoresce are active. Repeat 3 times.

[0087] Protoplast viability (%) = (Number of protoplasts excited with yellow-green fluorescence under ultraviolet light / Total number of bright-field protoplasts) × 100.

[0088] Protoplast viability test results are as follows Figure 2 As shown, cells stained with fluorescent dyes are viable protoplasts with a viability of 95.88% ± 1.58%.

[0089] Example 2: Transient Transformation Method Based on Stevia Protoplasts

[0090] This embodiment provides a transient conversion method based on stevia protoplasts and compares the effects of different concentrations of PEG on the transient conversion efficiency. Specifically, it includes the following steps:

[0091] 1. Stevia protoplasts resuspended in MMG (prepared in Example 1) were placed on ice for 30 min. During this period, PEG-CaCl2 solutions of different concentrations were prepared, including different concentrations (w / v) of PEG-4000 (20%, 30%, 40%, 50%), 0.5 M mannitol, and 0.1 M CaCl2.

[0092] 2. Add 10 μL of endotoxin-free protoplasts with a concentration exceeding 1000 ng / μL to a 2 mL centrifuge tube; use a pipette tip with the tip cut off to slowly add 100 μL of protoplast suspension to the centrifuge tube and mix gently.

[0093] 3. Slowly add an equal volume of PEG-CaCl2 solution (110 μL) to the centrifuge tubes, mix gently, and let stand at room temperature in the dark for 15 min.

[0094] 4. Slowly add 1 mL of W5 buffer (ordinary W5 buffer) to the centrifuge tube and mix gently to stop the reaction. Centrifuge at 100g, 4 ℃ for 5 min, discard the supernatant, and then slowly add 1 mL of W5 buffer (ordinary W5 buffer) and mix gently. Incubate in the dark at 25℃ for 24 h.

[0095] 5. Centrifuge at 100 g for 5 min at 4 ℃, discard the supernatant, gently mix, and use for laser confocal observation.

[0096] The results are as follows Figure 3 As shown, the results indicate that under different PEG concentrations, the 40% PEG-4000 experimental group had the highest conversion efficiency, which was 32.10±3.77%. The average conversion efficiency of the 50% PEG-4000 experimental group was lower than that of the 40% PEG-4000 group. Although there was no significant difference, obvious protoplast fragmentation and aggregation were observed.

[0097] Example 3: Application of Stevia protoplasts

[0098] This embodiment utilizes stevia protoplasts to conduct subcellular localization experiments and bimolecular fluorescence complementation (BiFC) experiments, verifying its feasibility of application.

[0099] 1. Subcellular localization experiment

[0100] Using the transient transformation method based on stevia protoplasts (40% PEG-4000) described in Example 2, the pOX-GFP empty vector and the recombinant pOX-SrMYC2-GFP plasmid were transiently transformed into stevia protoplasts (prepared in Example 1), respectively. After 24 h of dark incubation, 2 μL of DAPI fluorescent dye (0.1 μg / mL) was added to the protoplast suspension, and the mixture was stained in the dark for 10 min. The eGFP and DAPI signals were then observed under a laser confocal fluorescence microscope.

[0101] The results are as follows Figure 4 As shown in the figure, the results indicate that the luminescence position of SrMYC2 under GFP excitation light is the same as that of DAPI staining, indicating that SrMYC2 is located in the cell nucleus.

[0102] 2. Bimolecular Fluorescence Complementary (BiFC) Experiment

[0103] The transient transformation method based on stevia protoplasts (40% PEG-4000) described in Example 2 was used to transform YFP... N -SrMYC2 and YFP C -SrJAZ plasmids (5 μL each) were co-transformed into stevia protoplasts (prepared in Example 1). After dark culture for 24 h, 2 μL of DAPI fluorescent dye (0.1 μg / mL) was added to the protoplast suspension, and YFP and DAPI signals were observed under a laser confocal fluorescence microscope.

[0104] The results are as follows Figure 5 As shown, the results indicate that the luminescence sites under YFP excitation light and the DAPI staining sites are co-localized, suggesting that SrMYC2 and SrJAZ interact in stevia protoplasts.

[0105] The results of this embodiment verify the feasibility of performing subcellular localization and BiFC analysis in stevia protoplasts.

[0106] Example 4: Comparison of the effects of different complex enzymes on the yield and activity of stevia protoplasts

[0107] This embodiment compares the effects of different complex enzyme concentrations in the enzymatic hydrolysate on the yield and activity of the final prepared protoplasts during the preparation of stevia protoplasts. Except for the different complex enzyme concentrations in the enzymatic hydrolysate in step 2 and the absence of added antioxidants (Table 1, N=4), all other steps are the same as in Example 1.

[0108] Table 1

[0109]

[0110] The results are as follows Figure 6As shown, among the five complex enzyme treatment groups, the second group had the highest protoplast yield, which was 5.13 ± 0.61 × 10⁻⁶. 7 g -1 The FW group showed an activity of 89.98±4.94%; the group with the lowest protoplast yield and activity was group 5, with an average yield of only 1.67±0.74×10⁻⁶. 7 g -1 FW, with an activity of 69.56±7.01%.

[0111] Example 5: Comparison of the effects of different mannitol concentrations on the yield and activity of stevia protoplasts

[0112] This embodiment compares the effects of adding different concentrations of mannitol to the enzymatic hydrolysate during the preparation of stevia protoplasts on the yield and activity of the final protoplasts. Except for the different concentrations of mannitol in the enzymatic hydrolysate in step 2 (0.3 M, 0.4 M, 0.5 M, 0.6 M, N=4) and the absence of added antioxidants, all other steps are the same as in Example 1.

[0113] The results are as follows Figure 7 As shown, with increasing mannitol concentration in the enzymatic hydrolysate, the yield and activity of protoplasts both showed a trend of first increasing and then decreasing. The highest protoplast yield and activity were observed when the mannitol concentration was 0.5 M (5.17 ± 0.70 × 10⁻⁶). 7 g -1 FW, 90.26±3.80%.

[0114] Example 6: Comparison of the effects of different antioxidants on the yield and activity of stevia protoplasts

[0115] This embodiment compares the effects of using different antioxidants in the enzymatic hydrolysate during the preparation of stevia protoplasts on the yield and activity of the final protoplasts. Except for the different types and concentrations of antioxidants in the enzymatic hydrolysate in step 2 and W5 (Tables 2 and 3, N=4), all other steps are the same as in Example 1.

[0116] Table 2

[0117]

[0118] Table 3

[0119]

[0120] Stevia leaves are rich in polyphenols such as chlorogenic acid and isochlorogenic acid. During the enzymatic removal of cell walls, the broken cells release a large amount of phenolic substances, triggering biochemical reactions such as oxidative browning and ROS burst, which directly lead to protoplast breakage, inactivation and browning. Figure 8The results showed that, compared with the control group without added antioxidants (Group 1, yield was 4.88 ± 0.78 × 10⁻⁶), 7 g -1 The activity of stevia protoplasts was 87.03±3.61%. Adding DL-dithiothreitol (groups 2-4) to the enzymatic hydrolysate had almost no effect on increasing the yield of stevia protoplasts. However, adding L-ascorbic acid (groups 5-7) and reduced glutathione (groups 8-10) to the enzymatic hydrolysate significantly inhibited the oxidation of phenolic substances, significantly increased the yield and activity of stevia protoplasts, and reduced browning. The highest yield of stevia protoplasts (10.46±0.62×10⁻⁶) was observed when the concentration of reduced glutathione was 0.1% (group 9). 7 g -1 FW, activity can reach 95.88±1.58% ( Figure 8 (A) Compared with 0.1% reduced glutathione, the combination of reduced glutathione with DL-dithiothreitol or L-ascorbic acid in the enzymatic hydrolysate did not significantly increase the yield and activity of protoplasts. Figure 8 (B in the middle).

[0121] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0122] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing a Stevia rebaudiana protoplast, characterized by, Includes the following steps: S1. Cut the leaves of the stevia tissue culture seedlings into thin strips and place them in a mannitol solution for preplasmolysis. S2. After the pre-plasmolysis of the leaves in step S1 is drained, the leaves are placed in the enzymatic hydrolysis solution, vacuumed, and enzymatically hydrolyzed in the dark under shaking conditions. The enzymatic hydrolysate comprises 1 wt%–1.5 wt% cellulase, 0.25 wt%–0.35 wt% dissociative enzyme, 0.4 M–0.6 M mannitol, 1.8 mM–2.2 mM MES, 0.1×W5 buffer, and 0.08 wt%–0.12 wt% glutathione. S3. After the enzyme digestion in the dark is completed, aspirate the enzyme digest, add W5 buffer and shake, pass through a cell sieve, then elute with W5 buffer and recover the filtrate. S4. Centrifuge the filtrate obtained in step S3, discard the supernatant, and obtain the stevia protoplasts; In steps S2 and S3, 0.08 wt% to 0.12 wt% of glutathione is added to the W5 buffer solution.

2. The method for preparing stevia protoplasts according to claim 1, characterized in that, In step S2, the enzymatic hydrolysate comprises 1.2 wt%~1.5 wt% cellulase, 0.28 wt%~0.32 wt% dissociative enzyme, 0.45 M~0.55 M mannitol, 1.8 mM~2.2 mM MES, 0.1×W5 buffer, and 0.09 wt%~0.11 wt% glutathione; Preferably, the enzymatic hydrolysate comprises 1.5 wt% cellulase, 0.3 wt% dissociative enzyme, 0.5 M mannitol, 2.0 mM MMES, 0.1 × W5 buffer and 0.1 wt% glutathione.

3. The method for preparing stevia protoplasts according to claim 1, characterized in that, In steps S2 and S3, 0.09 wt% to 0.11 wt% of glutathione is added to the W5 buffer solution.

4. The method for preparing stevia protoplasts according to claim 1, characterized in that, In step S2, the preparation method of the enzymatic hydrolysate is as follows: add cellulase, ionizing enzyme, mannitol, and MES to 0.1×W5 buffer, bring the volume to a final volume with sterile water, heat in a water bath, cool to room temperature, add glutathione, filter through a microporous membrane, and store in the dark.

5. The method for preparing stevia protoplasts according to claim 1, characterized in that, In step S1, the stevia tissue culture seedlings are obtained through the following steps: after sterilizing the stevia seeds, they are inoculated into MS medium and cultured for 28 to 32 days. Then, stem segments are cut off and inserted into MS medium, and cultured for another 40 to 50 days. And / or, the concentration of mannitol is 0.4 M to 0.6 M, and the pre-plasmolysis time is 12 min to 18 min; And / or, the blades are 2 to 3 pairs of blades at the tip.

6. The method for preparing stevia protoplasts according to claim 1, characterized in that, In step S2, the oscillation speed is 40 rpm to 50 rpm; And / or, in step S2, the temperature of the light-protected enzymatic hydrolysis is 27℃~29℃, and the time of the light-protected enzymatic hydrolysis is 3 h~4 h; And / or, in step S3, the pore size of the cell sieve is 65 μm to 75 μm; And / or, in step S4, the centrifugation conditions are: 90 g to 110 g, centrifugation at 3 ℃ to 5 ℃ for 4 min to 6 min; the number of centrifugations is 2 to 3 times.

7. Stevia protoplasts prepared by the preparation method according to any one of claims 1 to 6.

8. The application of the stevia protoplast as described in claim 7 in transient transformation.

9. A method for transient transformation based on stevia protoplasts, characterized in that, Includes the following steps: S1. The stevia protoplasts described in claim 7 are resuspended in MMG solution, mixed with plasmids containing the target gene, and then PEG-CaCl2 solution is added, mixed, and allowed to stand at room temperature in the dark; the PEG-CaCl2 solution comprises 40wt%~50wt% PEG-4000, 0.4 M~0.6 M mannitol, and 0.08 M~0.12 M CaCl2; S2. Add W5 buffer and mix well. Centrifuge and discard the supernatant. S3. Add W5 buffer and mix well. Incubate in the dark, centrifuge, discard the supernatant, and you will get the transformed stevia protoplasts.

10. The transient transformation method based on stevia protoplasts according to claim 9, characterized in that, In step S1, the PEG-CaCl2 solution comprises 40wt%~45wt% PEG-4000, 0.45 M~0.55 M mannitol, and 0.09 M~0.11 M CaCl2; And / or, in step S1, the time for light-protected standing is 12 min to 18 min; And / or, in step S3, the temperature of the dark culture is 24℃~26℃, and the time of the dark culture is 22 h~26 h; And / or, in steps S2 and S3, the centrifugation conditions are: 90 g to 110 g, centrifugation at 3 ℃ to 5 ℃ for 4 min to 6 min.

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