A method for propagating fritillaria unibracteata by using protoplast culture

Abstract

The application relates to the technical field of medicinal material planting, in particular to a method for culturing and breeding Fritillaria unibractata by using protoplasts. The method comprises the following steps: taking inner layer tender scales of fresh Fritillaria unibractata bulbs; sequentially performing low-temperature pretreatment, disinfection treatment and antioxidation treatment on the Fritillaria bulb samples to obtain detoxification and antioxidation explants; sequentially performing induction inoculation, subculture purification, enzymatic hydrolysis treatment and purification treatment on the detoxification and antioxidation explants to obtain protoplast samples; sequentially performing initial culture, proliferation, adventitious bud differentiation, bulb induction, rooting culture, seedling raising, transplanting and maintenance on the protoplast samples to obtain Fritillaria unibractata mature bodies. The method combines a cheap antioxidant system, a multiproduct source polypeptide and a whole-process loss prevention strategy, thereby systematically improving the propagation rate of the protoplast culture of Fritillaria unibractata while controlling the cost.

Description

Technical Field

[0001] This application relates to the field of medicinal herb cultivation technology, and in particular to a method for propagating *Fritillaria purpurea* using protoplast culture. Background Technology

[0002] *Fritillaria thunbergii*, a perennial herbaceous plant belonging to the genus *Fritillaria* in the family Liliaceae, grows to a length of 15-23 cm. Its bulb consists of two scales. *Fritillaria thunbergii* is mainly distributed in northwestern Sichuan and southeastern Qinghai, growing in grasslands at altitudes of 3200-4500 m. It prefers cool, mild climates and is cold-hardy, thrives in moist conditions, dislikes high temperatures, and prefers shade. Propagation methods generally include seed propagation and underground bulb propagation. However, seed propagation requires a long time and has a low germination rate, making it difficult to meet the growing market demand. While underground bulb propagation allows for harvesting in a very short time, its low propagation coefficient still falls short of market demand.

[0003] Plant tissue culture technology can be used to obtain a large number of high-quality seedlings with consistent traits in a short period of time, which is expected to solve the problems of long growth cycle and low propagation coefficient of artificially cultivated fritillaria. The key to plant tissue culture technology lies in the preparation of protoplasts. Because the wild bulbs of Fritillaria thunbergii have a high bacterial load and a high risk of browning, young leaves and hypocotyls of sterile test-tube seedlings are generally used as protoplast donors. However, the propagation rate of fritillaria seedlings transformed from these protoplast donors is still low. Summary of the Invention

[0004] This application provides a method for propagating *Fritillaria purpurea* using protoplast culture to solve the following technical problem: how to improve the propagation rate of *Fritillaria purpurea* cultured using protoplasts.

[0005] In a first aspect, embodiments of this application provide a method for propagating *Fritillaria purpurea* using protoplast culture, the method comprising: Fresh, dark purple fritillary bulbs that are 2 to 3 years old, healthy, and free from pests and diseases are selected, and the inner tender scales are removed to obtain fritillary bulb samples. The fritillaria bulb samples were pretreated at low temperature to obtain explants; The explants were sequentially disinfected and subjected to antioxidant treatment to obtain detoxified and antioxidant explants. In the embryogenic callus induction medium, the detoxified antioxidant explants were sequentially induced, inoculated, and subcultured for purification to obtain embryogenic callus tissue. The embryonic callus was sequentially subjected to enzymatic hydrolysis and purification to obtain protoplast samples. The protoplast sample was initially cultured in protoplast culture medium to obtain microcall tissue; The micro-callus tissue was proliferated in a callus proliferation culture medium to obtain callus tissue; In a differentiation medium, the callus tissue was subjected to adventitious bud differentiation to obtain clustered buds; In a bulblet induction medium, the clustered buds were induced to form bulblets to obtain bulblets; The small bulbs were rooted in a rooting medium to obtain dark purple Fritillaria seedlings; The seedlings of the dark purple Fritillaria were successively hardened, transplanted and nurtured to obtain mature dark purple Fritillaria. The antioxidants used in the antioxidant treatment include polyvinylpyrrolidone and glutathione; The composite enzymatic hydrolysis solution used in the enzymatic hydrolysis treatment includes polyvinylpyrrolidone and glutathione. The embryogenic callus induction medium includes polyvinylpyrrolidone and glutathione; The protoplast culture medium includes polyvinylpyrrolidone, glutathione, and corn peptides; The callus proliferation culture medium includes corn peptides, glutathione, and astaxanthin; The differentiation culture medium includes glutathione and astaxanthin; The bulblet induction culture medium includes corn peptide, astaxanthin, and glutathione. The rooting medium includes glutathione and astaxanthin.

[0006] Optionally, in the antioxidants used in the antioxidant treatment, the mass concentration of polyvinylpyrrolidone is 5 g / L to 10 g / L, and the mass concentration of glutathione is 0.2 g / L to 0.5 g / L; and / or In the embryogenic callus induction medium, the mass concentration of polyvinylpyrrolidone is 0.5 g / L to 1.0 g / L, and the mass concentration of glutathione is 0.1 g / L to 0.2 g / L; and / or In the protoplast culture medium, the mass concentration of the polyvinylpyrrolidone is 0.45 g / L to 0.55 g / L, the mass concentration of the glutathione is 0.05 g / L to 0.15 g / L, and the mass concentration of the corn peptide is 0.25 g / L to 0.35 g / L; and / or In the callus proliferation medium, the mass concentration of the corn peptide is 0.45 g / L to 0.55 g / L, the mass concentration of the glutathione is 0.05 g / L to 0.10 g / L, and the mass concentration of the astaxanthin is 0.01 g / L to 0.03 g / L; and / or In the differentiation medium, the mass concentration of glutathione is 0.095 g / L to 0.105 g / L, and the mass concentration of astaxanthin is 0.0095 g / L to 0.0105 g / L; and / or In the bulblet induction medium, the mass concentration of the corn peptide is 0.5 g / L to 1.0 g / L, the mass concentration of the astaxanthin is 0.02 g / L to 0.04 g / L, and the mass concentration of the glutathione is 0.045 g / L to 0.055 g / L; and / or In the rooting medium, the mass concentration of glutathione is 0.095 g / L to 0.105 g / L, and the mass concentration of astaxanthin is 0.0095 g / L to 0.0105 g / L.

[0007] Optionally, per 1 L, the embryogenic callus induction medium comprises: MS basal medium, 2,4-dichlorophenoxyacetic acid: 1.0 mg, α-naphthaleneacetic acid: 0.5 mg, vitamin C: 0.1 g, sucrose: 30 g, agar: 7 g, the polyvinylpyrrolidone: 0.5 g to 1.0 g, and the glutathione: 0.1 g to 0.2 g; and / or 1 L of the protoplast culture medium comprises: modified KM8P basal medium, 2,4-dichlorophenoxyacetic acid: 0.8 mg, 6-benzylaminopurine: 0.3 mg, mannitol: 0.45 mol, sucrose: 20 g, polyvinylpyrrolidone: 0.45 g to 0.55 g, glutathione: 0.05 g to 0.15 g, and corn peptide: 0.25 g to 0.35 g; and / or 1 L of the callus proliferation medium comprises: MS basal medium, 6-benzylaminopurine: 1.6 mg, α-naphthaleneacetic acid: 1.2 mg, vitamin C: 0.1 g, sucrose: 30 g, agar: 7 g, the corn peptide: 0.45 g to 0.55 g, the glutathione: 0.05 g to 0.10 g, the astaxanthin: 0.01 g to 0.03 g; and / or Based on 1L, the differentiation medium comprises: MS basal medium, 6-benzylaminopurine: 1.5mg, α-naphthaleneacetic acid: 0.2mg, sucrose: 30g, agar: 7g, the glutathione: 0.095g~0.105g, and the astaxanthin: 0.0095g~0.0105g; and / or 1 L of the bulblet induction medium comprises: MS basal medium, 6-benzylaminopurine: 0.5 mg, α-naphthaleneacetic acid: 1.0 mg, sucrose: 50 g, agar: 7 g, the corn peptide: 0.5 g to 1.0 g, the astaxanthin: 0.02 g to 0.04 g, and the glutathione: 0.045 g to 0.055 g; and / or 1L of the rooting medium comprises: 1 / 2 MS basal medium, indolebutyric acid: 0.5mg, sucrose: 20g, agar: 7g, glutathione: 0.095g~0.105g, and astaxanthin: 0.0095g~0.0105g.

[0008] Optionally, the embryonic callus tissue is subjected to enzymatic digestion and purification processes sequentially to obtain protoplast samples, including the following steps: Cellulase, dissociation enzyme, and mannitol were dissolved in CPW salt solution and then filtered and sterilized sequentially to obtain sterilized enzyme solution; Polyvinylpyrrolidone and glutathione were added to the sterilized enzyme solution to obtain a composite enzymatic hydrolysate. The embryonic callus was enzymatically hydrolyzed using the composite enzymatic hydrolysate to obtain an enzymatic hydrolysate mixture; The enzymatic hydrolysate was filtered to obtain the enzymatic hydrolysate filtrate. The enzymatic hydrolysis filtrate was centrifuged and washed to obtain the enzymatic hydrolysis wash. The enzymatically hydrolyzed washings were subjected to gradient centrifugation using sucrose to obtain protoplast samples; wherein the density of the protoplast samples was 1 × 10⁻⁶. 4 Cells / mL ~ 1×10 5 per mL.

[0009] Optionally, the composite enzymatic hydrolysate used in the enzymatic hydrolysis treatment includes polyvinylpyrrolidone, glutathione, cellulase, and cleavage enzyme; the mass ratio of the cellulase, the cleavage enzyme, the polyvinylpyrrolidone, and the glutathione is 150:50:(4.5~5.5):1.

[0010] Optionally, the enzymatic hydrolysis is carried out in the dark and under stirring conditions, the temperature of the enzymatic hydrolysis is 22℃~24℃, the time of the enzymatic hydrolysis is 4h~5h, and the rotation speed of the enzymatic hydrolysis is 30rpm~40rpm.

[0011] Optionally, the temperature of the low-temperature pretreatment is ≤4℃, and the time of the low-temperature pretreatment is 36h~54h.

[0012] Optionally, the explants are subjected to disinfection and antioxidant treatments sequentially to obtain detoxified and antioxidant explants, including the following steps: The explants were disinfected using carbendazim to obtain virus-free explants; The detoxified explants were subjected to antioxidant treatment in a sterile solution containing polyvinylpyrrolidone and glutathione to obtain detoxified antioxidant explants.

[0013] Optionally, the antioxidant treatment time is 10 min to 15 min.

[0014] Optionally, the initial culture time is 20-25 days; and / or The temperature for adventitious bud differentiation is 18℃~22℃, and the time for adventitious bud differentiation is 3 weeks~4 weeks; and / or The rooting culture temperature is 18℃~22℃, and the rooting culture time is 10d~15d.

[0015] The technical solutions provided in this application have the following advantages compared with the prior art: This application provides a method for culturing and propagating *Fritillaria purpurea* using protoplasts. This method comprehensively improves the protoplast propagation rate of *Fritillaria purpurea* by combining a low-cost antioxidant system, agricultural by-product-derived peptides, and a comprehensive damage prevention strategy: The method involves selecting the inner, tender scales of fresh *Fritillaria purpurea* bulbs, then pre-treating them at low temperature to inhibit the activity of polyphenol oxidase within the bulbs; subsequently, disinfection and antioxidant treatments are applied, utilizing the dual effects of polyvinylpyrrolidone (PVP) adsorbing polyphenols and glutathione reducing reactive oxygen species to prevent explant browning and reduce explant rejection rates. Then, a combined treatment process of induction inoculation, subculture purification, enzymatic hydrolysis, and purification is employed. PPVP and glutathione are added to the complex enzymatic hydrolysate to protect the exposed cell membranes formed during enzymatic hydrolysis, resulting in high-density protoplasts. Next, glutathione and corn peptides were added to the protoplast culture medium to provide nitrogen and carbon sources for the initial culture, as well as to enhance the antioxidant capacity of the initial culture and promote cell wall regeneration and division of the protoplasts. Then, a callus proliferation medium containing corn peptides, astaxanthin, and glutathione was used to promote microcallus proliferation and protect the cells of the microcallus, resulting in a large amount of callus tissue. Next, glutathione and trace amounts of astaxanthin were used to maintain the redox state of adventitious buds. Then, corn peptides and sucrose were used synergistically to promote dry matter accumulation, thereby accelerating bulb enlargement and forming stable small bulbs. Finally, through rooting and transplanting operations, glutathione and astaxanthin were added to the rooting medium to protect the root system of the small bulbs, which is beneficial for increasing the propagation rate of *Fritillaria purpurea*. Therefore, this method replaces expensive antioxidants with inexpensive polyvinylpyrrolidone and glutathione throughout the process, uses corn peptides to replace some nutrients and antioxidant components, and precisely delivers trace amounts of astaxanthin at the critical stage when cell division is active and most susceptible to oxidative damage, thereby reducing the cost of the antioxidant stage and enabling the mass reproduction of dark purple fritillaria. Attached Figure Description

[0016] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0017] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 A flowchart illustrating a method for propagating *Fritillaria purpurea* using protoplast culture, provided in this application embodiment; Figure 2 A detailed flowchart of a method for culturing and propagating *Fritillaria purpurea* using protoplasts, provided in this application embodiment; Figure 3 This is a schematic diagram of the clustered buds in a method for propagating *Fritillaria purpurea* using protoplast culture provided in Embodiment 1 of this application; Figure 4 This is a nursery of transplanted *Fritillaria purpurea* seedlings in a method for propagating *Fritillaria purpurea* using protoplast culture provided in Embodiment 1 of this application. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0020] The range descriptions used in this application, such as numerical ranges and proportional ranges, include all possible sub-ranges and single numerical values ​​within that range. For example, the range descriptions of "1 to 6" or "1~6" cover all sub-ranges (such as 1 to 3, 2 to 5, etc.) and single numbers (such as 1, 2, 3, 4, 5, 6) between 1 and 6. Unless otherwise specified, the terms "comprising" and others used herein mean "including but not limited to"; relational terms such as "first" and "second" are used only to distinguish different entities or operations and do not imply an actual order or relationship; "and / or" indicates that multiple situations can exist individually or simultaneously; expressions such as "at least one," "multiple," and "at least one" refer to any combination of the corresponding objects, including combinations of single or multiple objects. The proportional relationships involved in this document, such as mass ratios and molar ratios, should be understood as the correspondence between the first and second terms of a proportional formula, according to the order of description. The raw materials, reagents, instruments, and equipment used herein can all be obtained by purchasing from the market or by existing methods.

[0021] Figure 1An exemplary flowchart of a method for culturing and propagating *Fritillaria purpurea* using protoplasts, provided in an embodiment of this application, is shown. like Figure 1 As shown in the embodiment of this application, a method for propagating *Fritillaria purpurea* using protoplast culture is provided, the method comprising: S1. Select healthy, disease-free, fresh dark purple fritillary bulbs that are 2 to 3 years old and free from pests and diseases, and remove the inner tender scales to obtain fritillary bulb samples. S2. The fritillaria bulb samples are subjected to low-temperature pretreatment to obtain explants; S3. The explants are subjected to disinfection and antioxidant treatment in sequence to obtain detoxified and antioxidant explants; S4. In the embryogenic callus induction medium, the detoxified antioxidant explants were sequentially induced, inoculated, and subcultured for purification to obtain embryogenic callus tissue; S5. The embryonic callus is subjected to enzymatic hydrolysis and purification treatment in sequence to obtain protoplast samples; S6. The protoplast sample is initially cultured in protoplast culture medium to obtain microcall tissue; S7. The micro-callus tissue is proliferated in a callus proliferation culture medium to obtain callus tissue; S8. In a differentiation medium, the callus tissue is subjected to adventitious bud differentiation to obtain clustered buds; S9. In a bulblet induction medium, the clustered buds are induced to form bulblets to obtain bulblets; S10. The small bulbs are rooted in a rooting medium to obtain dark purple Fritillaria seedlings; S11. The dark purple fritillary seedlings are successively hardened, transplanted and nurtured to obtain mature dark purple fritillary bodies; The antioxidants used in the antioxidant treatment include polyvinylpyrrolidone and glutathione; The composite enzymatic hydrolysis solution used in the enzymatic hydrolysis treatment includes polyvinylpyrrolidone and glutathione. The embryogenic callus induction medium includes polyvinylpyrrolidone and glutathione; The protoplast culture medium includes polyvinylpyrrolidone, glutathione, and corn peptides; The callus proliferation culture medium includes corn peptides, glutathione, and astaxanthin; The differentiation culture medium includes glutathione and astaxanthin; The bulblet induction culture medium includes corn peptide, astaxanthin, and glutathione. The rooting medium includes glutathione and astaxanthin.

[0022] It should be noted that the polyvinylpyrrolidone, glutathione, corn peptide and astaxanthin components used in this method need to be prepared into a stock solution first, and then filtered and sterilized through a 0.22μm filter membrane. These components are added after the culture medium is autoclaved and cooled to 50℃~60℃ to avoid the inactivation of these antioxidant components.

[0023] It should be noted that the embryogenic callus tissue must be selected from subcultured, stable, uniformly granulated, pale yellow tissue in the logarithmic growth phase; while aging, browning, and water-soaked embryogenic callus tissue will result in protoplasts with low viability and poor division ability. Therefore, the quality of the embryogenic callus tissue directly affects the success rate of *Fritillaria purpurea* culture.

[0024] It should be noted that the mannitol used in each culture medium in this method has an initial concentration of 0.45 mol / L. This concentration of mannitol can maintain the osmotic pressure of the fritillaria cells. The concentration of mannitol is gradually reduced as the fritillaria cell wall regenerates to avoid sudden changes in osmotic pressure that could lead to protoplast rupture or cell growth arrest.

[0025] It should be noted that if a high concentration of polyvinylpyrrolidone is used in the culture medium in this method, it may affect the osmotic pressure of the cells, while a high concentration of glutathione may interfere with redox signaling and affect cell proliferation.

[0026] It should be noted that the method for propagating *Fritillaria purpurea* using protoplast culture provided in this application embodiment systematically improves the propagation rate of *Fritillaria purpurea* protoplast culture while controlling costs through a combination of "inexpensive antioxidant system + agricultural by-product-derived peptides + comprehensive damage prevention strategy". The specific mechanism is as follows: I. Low-cost antioxidant system: Polyvinylpyrrolidone (PVP) + Glutathione (GSH) as an alternative to expensive solutions.

[0027] Traditional methods of controlling browning in dark purple Fritillaria tissue culture often rely on high concentrations of vitamin C, citric acid, or complex combinations of adsorbents, resulting in an extremely high rate of waste of Fritillaria tissue material due to browning. The core cost-reduction strategy of this method is: 1. PVP: PVP is an inexpensive polymer compound that can meet industrial-grade requirements. PVP inhibits the browning chain reaction from the source by physically adsorbing polyphenol substrates and blocking the contact between oxidases and substrates.

[0028] 2. GSH: GSH is a common biological reagent and is inexpensive; GSH contains active thiol groups (-SH), which directly reduce reactive oxygen species (ROS) and quinones, repairing oxidative damage.

[0029] 3. Mechanism for reducing costs: PVP and GSH form a dual defense of "adsorption + reduction" during the antioxidant treatment stage and the embryogenic callus induction stage, which significantly reduces the proportion of explants discarded due to browning and necrosis, and reduces the hidden costs of repeated material collection and repeated experiments.

[0030] Second, PVP+GSH is added directly during the enzymatic hydrolysis stage to reduce the protoplast damage rate.

[0031] During protoplast enzymatic hydrolysis, the cell wall is removed, and the cell membrane is directly exposed to the complex enzymatic hydrolysate, making it highly susceptible to rupture due to oxidative stress. This method involves adding PVP and GSH to the complex enzymatic hydrolysate used in the enzymatic hydrolysis treatment. 1. GSH can maintain the reducing environment of the enzymatic hydrolysis system and protect cell membrane lipids from oxidation; 2. PVP adsorbs polyphenols released from callus tissue, preventing polyphenols from oxidizing to form quinones and avoiding the poisoning of protoplasts by large amounts of quinones.

[0032] This method of adding PVP and GSH to the compound enzymatic hydrolysate avoids the problems of traditional methods, which require large amounts of starting material and multiple repeated enzymatic hydrolysates due to low protoplast survival rates. It achieves high-density protoplasts (1×10⁻⁶) with the minimum amount of enzyme and the shortest time (4-5 hours). 4 Cells / mL ~ 1×10 5 (pcs / mL).

[0033] III. Introduction of Corn Peptides: High-Value Utilization of Agricultural By-products.

[0034] Corn peptides are a byproduct of enzymatic hydrolysis of corn protein, belonging to the category of deep-processed agricultural products. Their cost is far lower than purified amino acids or expensive plant growth regulators. Replacing some plant growth regulators with corn peptides can provide some nutrition while preventing the oxidation of *Fritillaria purpurea*. 1. Adding corn peptides to protoplast culture medium can provide nitrogen and carbon sources for protoplasts with rigid walls regenerating, while also exerting antioxidant activity; 2. Adding corn peptides to the callus proliferation culture medium can promote the rapid proliferation of microcallus through the nutritional supplementation of corn peptides; 3. Adding corn peptides to the bulb induction medium can synergistically promote the accumulation of dry matter in bulbs and accelerate bulb enlargement, in conjunction with sucrose.

[0035] Therefore, corn peptides possess both nutritional and antioxidant functions, achieving a dual effect and reducing the amount of other expensive organic additives used in various culture media. Simultaneously, the hydrophobic amino acids (such as valine and leucine) and histidine abundant in corn peptides have metal chelating and free radical scavenging capabilities, which can replace some purified antioxidants.

[0036] IV. Precise addition of trace amounts of astaxanthin: achieving highly effective protection with extremely low doses.

[0037] Astaxanthin is a potent antioxidant, but it is expensive. This method involves precise application at extremely low concentrations (during the callus proliferation, differentiation, and rooting stages), targeting only the critical phases when cell division is most active and susceptible to oxidative damage. 1. Callus proliferation stage: Astaxanthin + GSH + corn peptide work synergistically to protect rapidly dividing cells; 2. Differentiation and rooting stages: Trace amounts of astaxanthin work in conjunction with GSH to maintain redox homeostasis in adventitious buds and small bulbs.

[0038] This precise drip irrigation method avoids the cost burden of using high concentrations throughout the entire cycle, while ensuring that the reproduction rate does not decrease due to oxidative damage.

[0039] V. The high reproduction rate and closed-loop process of protoplast technology itself.

[0040] Protoplast culture is essentially a highly efficient propagation method: 1. Very little starting material: Only the inner layer of tender scales is needed, and a small-diameter bulb can produce a large number of protoplasts; 2. Extremely high reproduction coefficient: Each surviving protoplast can theoretically regenerate into a complete plant through cell division. After callus proliferation and adventitious bud differentiation, one protoplast can produce dozens to hundreds of clustered buds. 3. Closed-loop process: From protoplast → microcallus → callus proliferation → adventitious bud differentiation → bulblet induction → rooting, the entire process is completed in the culture medium, eliminating the need for the large number of bulbs that traditional propagation methods such as cuttings or division rely on.

[0041] In summary, the present application provides a method for propagating *Fritillaria purpurea* using protoplast culture. This method utilizes mechanisms such as PVP+GSH for continuous browning prevention, corn peptides to replace some expensive nutrients or antioxidants, precise micro-dose of astaxanthin, and a protoplast single-cell regeneration pathway. By using the inexpensive reagent PVP+GSH, it not only reduces material waste but also ensures protoplast integrity and improves explant survival. The use of the extremely low-cost agricultural byproduct corn peptides enhances cell division activity and callus proliferation efficiency. Furthermore, the use of a very small amount of astaxanthin reduces oxidative damage at critical stages, increasing differentiation rates. Finally, the protoplast single-cell regeneration pathway eliminates the need for significant bulb consumption, resulting in an exponential increase in the propagation coefficient. Therefore, this method does not rely on expensive new technologies or equipment. Instead, by optimizing the antioxidant combination (PVP, GSH, corn peptides, and astaxanthin) and controlling oxidative damage throughout the entire process, it achieves the highest material utilization and cell regeneration efficiency at the lowest reagent cost, thus realizing low-cost and high-efficiency propagation of *Fritillaria purpurea* protoplast culture.

[0042] In some optional embodiments, the antioxidant used in the antioxidant treatment has a mass concentration of 5 g / L to 10 g / L for polyvinylpyrrolidone and a mass concentration of 0.2 g / L to 0.5 g / L for glutathione; and / or In the embryogenic callus induction medium, the mass concentration of polyvinylpyrrolidone is 0.5 g / L to 1.0 g / L, and the mass concentration of glutathione is 0.1 g / L to 0.2 g / L; and / or In the protoplast culture medium, the mass concentration of the polyvinylpyrrolidone is 0.45 g / L to 0.55 g / L, the mass concentration of the glutathione is 0.05 g / L to 0.15 g / L, and the mass concentration of the corn peptide is 0.25 g / L to 0.35 g / L; and / or In the callus proliferation medium, the mass concentration of the corn peptide is 0.45 g / L to 0.55 g / L, the mass concentration of the glutathione is 0.05 g / L to 0.10 g / L, and the mass concentration of the astaxanthin is 0.01 g / L to 0.03 g / L; and / or In the differentiation medium, the mass concentration of glutathione is 0.095 g / L to 0.105 g / L, and the mass concentration of astaxanthin is 0.0095 g / L to 0.0105 g / L; and / or In the bulblet induction medium, the mass concentration of the corn peptide is 0.5 g / L to 1.0 g / L, the mass concentration of the astaxanthin is 0.02 g / L to 0.04 g / L, and the mass concentration of the glutathione is 0.045 g / L to 0.055 g / L; and / or In the rooting medium, the mass concentration of glutathione is 0.095 g / L to 0.105 g / L, and the mass concentration of astaxanthin is 0.0095 g / L to 0.0105 g / L.

[0043] In these embodiments, among the antioxidants used in the antioxidant treatment, polyvinylpyrrolidone (PVP) at a concentration of 5 g / L to 10 g / L can strongly adsorb polyphenolic substrates released from the scale cuts through high concentrations of PPVP, thus blocking the initiation of browning. Additionally, glutathione at a concentration of 0.2 g / L to 0.5 g / L can reduce already formed quinones, repairing oxidative damage and achieving both explant detoxification and antioxidant effects. In the embryogenic callus induction medium, PPVP at a concentration of 0.5 g / L to 1.0 g / L can continuously adsorb phenols released during dedifferentiation. Furthermore, glutathione at a concentration of 0.1 g / L to 0.2 g / L can maintain a reducing environment and protect cell division activity. Through PPVP and glutathione, dedifferentiation of scale cells can be promoted to form embryogenic callus, rather than necrosis and browning, providing high-quality material for subsequent protoplast isolation.

[0044] In protoplast culture medium, polyvinylpyrrolidone (PVP) at a concentration of 0.45 g / L to 0.55 g / L can adsorb residual polyphenols and protect cell wall-less protoplasts. Additionally, glutathione at a concentration of 0.05 g / L to 0.15 g / L can maintain the reduced state of protoplast membrane lipids and prevent oxidative rupture. Furthermore, corn peptides at a concentration of 0.25 g / L to 0.35 g / L can provide both nitrogen and carbon nutrition and antioxidant support, promoting protoplast cell wall regeneration. Through the combined effects of PPVP, glutathione, and corn peptides, protoplast survival rate and cell wall regeneration efficiency are improved, ensuring the crucial transformation from single cell to microcallus.

[0045] In callus proliferation medium, corn peptides at a concentration of 0.45 g / L to 0.55 g / L can promote rapid division of microcallus tissue and chelate metal ions to prevent oxidation. In addition, glutathione at a concentration of 0.05 g / L to 0.10 g / L can maintain redox homeostasis of cells during the division phase of microcallus tissue. Furthermore, astaxanthin at a concentration of 0.01 g / L to 0.03 g / L can effectively scavenge ROS and protect cells during rapid proliferation. The corn peptides, glutathione, and astaxanthin in the callus proliferation medium can rapidly expand microcallus tissue into a large number of callus tissues, laying a quantitative foundation for subsequent differentiation.

[0046] In the differentiation medium, glutathione at a concentration of 0.095 g / L to 0.105 g / L can support the precise redox signals required for organ differentiation and promote differentiation; astaxanthin at a concentration of 0.0095 g / L to 0.0105 g / L can protect cells in the differentiation phase and prevent oxidative interference with hormone signals; glutathione and astaxanthin in the differentiation medium can promote the efficient differentiation of callus tissue into adventitious shoots and the formation of clustered shoots.

[0047] In the bulblet induction medium, corn peptides at a concentration of 0.5 g / L to 1.0 g / L can promote dry matter accumulation and bulb enlargement; astaxanthin at a concentration of 0.02 g / L to 0.04 g / L can protect enlarged cells and synergistically promote material accumulation with high sucrose concentration; glutathione at a concentration of 0.045 g / L to 0.055 g / L can maintain the reducing environment required for bulb development; corn peptides, astaxanthin, and glutathione in the bulblet induction medium can cause the base of the bud to swell into small bulbs with a diameter of 0.5 cm to 1 cm, thereby improving the transplant survival rate.

[0048] In the rooting medium, glutathione at a concentration of 0.095 g / L to 0.105 g / L can promote root cell elongation and root hair formation; astaxanthin at a concentration of 0.0095 g / L to 0.0105 g / L can protect young roots from oxidative stress; glutathione and astaxanthin in the rooting medium can induce white and robust roots with a rooting rate of >85% and shorten the seedling time.

[0049] In the antioxidants used for antioxidant treatment, the mass concentration of polyvinylpyrrolidone can be 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, or 10 g / L; and the mass concentration of glutathione can be 0.2 g / L, 0.25 g / L, 0.3 g / L, 0.35 g / L, 0.4 g / L, 0.45 g / L, or 0.5 g / L.

[0050] In the embryogenic callus induction medium, the mass concentration of the polyvinylpyrrolidone can be 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, or 1.0 g / L; and the mass concentration of the glutathione can be 0.1 g / L, 0.11 g / L, 0.12 g / L, 0.13 g / L, 0.14 g / L, 0.15 g / L, or 0.2 g / L.

[0051] In protoplast culture medium, the mass concentration of the polyvinylpyrrolidone can be 0.45 g / L, 0.46 g / L, 0.47 g / L, 0.48 g / L, 0.49 g / L, 0.50 g / L, or 0.55 g / L; the mass concentration of the glutathione can be 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, 0.09 g / L, 0.10 g / L, or 0.15 g / L; and the mass concentration of the corn peptide can be 0.25 g / L, 0.26 g / L, 0.27 g / L, 0.28 g / L, 0.29 g / L, 0.30 g / L, or 0.35 g / L.

[0052] In the callus proliferation medium, the mass concentration of the corn peptide can be 0.45 g / L, 0.46 g / L, 0.47 g / L, 0.48 g / L, 0.49 g / L, 0.50 g / L, or 0.55 g / L; the mass concentration of the glutathione can be 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, 0.09 g / L, or 0.10 g / L; and the mass concentration of the astaxanthin can be 0.01 g / L, 0.015 g / L, 0.020 g / L, 0.025 g / L, or 0.03 g / L.

[0053] In the differentiation medium, the mass concentration of glutathione can be 0.095 g / L, 0.096 g / L, 0.097 g / L, 0.098 g / L, 0.099 g / L, 0.100 g / L, or 0.105 g / L; the mass concentration of astaxanthin can be 0.0095 g / L, 0.0096 g / L, 0.0097 g / L, 0.0098 g / L, 0.0099 g / L, 0.0100 g / L, or 0.0105 g / L.

[0054] In the bulblet induction medium, the mass concentration of the corn peptide can be 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, or 1.0 g / L; the mass concentration of the astaxanthin can be 0.02 g / L, 0.025 g / L, 0.03 g / L, 0.035 g / L, or 0.04 g / L; and the mass concentration of the glutathione can be 0.045 g / L, 0.046 g / L, 0.047 g / L, 0.048 g / L, 0.049 g / L, 0.050 g / L, or 0.055 g / L.

[0055] In the rooting medium, the mass concentration of glutathione can be 0.095 g / L, 0.096 g / L, 0.097 g / L, 0.098 g / L, 0.099 g / L, 0.100 g / L, or 0.105 g / L, and the mass concentration of astaxanthin can be 0.0095 g / L, 0.0096 g / L, 0.0097 g / L, 0.0098 g / L, 0.0099 g / L, 0.0100 g / L, or 0.0105 g / L.

[0056] It should be noted that if significant browning occurs during this process, the mass concentrations of polyvinylpyrrolidone and glutathione can be temporarily increased by 30% to 50%, while simultaneously using dark culture conditions and low-temperature treatment to control the spread of browning.

[0057] It should be noted that, since astaxanthin is a fat-soluble carotenoid, its solubility in aqueous culture medium is extremely low. Therefore, it is necessary to prepare astaxanthin stock solution first, and then dispense it according to the usage requirements of astaxanthin. The preparation process of astaxanthin stock solution is as follows: (1) Dissolve synthetic astaxanthin powder in anhydrous ethanol to prepare a stock solution of 10 mg / mL. After filtration and sterilization through a 0.22μm organic phase filter membrane, dispense it into brown ampoules and store it at -20℃ in the dark as astaxanthin stock solution; (2) When astaxanthin is needed, after autoclaving the remaining components of the culture medium and cooling it to 50℃~60℃, add the corresponding volume of astaxanthin stock solution according to the final concentration of the culture medium, mix it quickly, and dispense it.

[0058] In some alternative embodiments, the embryogenic callus induction medium, per 1 L, comprises: MS basal medium, 2,4-dichlorophenoxyacetic acid: 1.0 mg, α-naphthaleneacetic acid: 0.5 mg, vitamin C: 0.1 g, sucrose: 30 g, agar: 7 g, the polyvinylpyrrolidone: 0.5 g to 1.0 g, and the glutathione: 0.1 g to 0.2 g; and / or 1 L of the protoplast culture medium comprises: modified KM8P basal medium, 2,4-dichlorophenoxyacetic acid: 0.8 mg, 6-benzylaminopurine: 0.3 mg, mannitol: 0.45 mol, sucrose: 20 g, polyvinylpyrrolidone: 0.45 g to 0.55 g, glutathione: 0.05 g to 0.15 g, and corn peptide: 0.25 g to 0.35 g; and / or 1 L of the callus proliferation medium comprises: MS basal medium, 6-benzylaminopurine: 1.6 mg, α-naphthaleneacetic acid: 1.2 mg, vitamin C: 0.1 g, sucrose: 30 g, agar: 7 g, the corn peptide: 0.45 g to 0.55 g, the glutathione: 0.05 g to 0.10 g, the astaxanthin: 0.01 g to 0.03 g; and / or Based on 1L, the differentiation medium comprises: MS basal medium, 6-benzylaminopurine: 1.5mg, α-naphthaleneacetic acid: 0.2mg, sucrose: 30g, agar: 7g, the glutathione: 0.095g~0.105g, and the astaxanthin: 0.0095g~0.0105g; and / or 1 L of the bulblet induction medium comprises: MS basal medium, 6-benzylaminopurine: 0.5 mg, α-naphthaleneacetic acid: 1.0 mg, sucrose: 50 g, agar: 7 g, the corn peptide: 0.5 g to 1.0 g, the astaxanthin: 0.02 g to 0.04 g, and the glutathione: 0.045 g to 0.055 g; and / or 1L of the rooting medium comprises: 1 / 2 MS basal medium, indolebutyric acid: 0.5mg, sucrose: 20g, agar: 7g, glutathione: 0.095g~0.105g, and astaxanthin: 0.0095g~0.0105g.

[0059] In these embodiments, the specific components of the embryogenic callus induction medium are defined as MS basal medium, 2,4-dichlorophenoxyacetic acid, α-naphthaleneacetic acid, vitamin C, sucrose, agar, polyvinylpyrrolidone, and glutathione. This promotes the dedifferentiation of squamous cells into embryogenic callus, rather than necrosis and browning, providing high-quality material for subsequent protoplast isolation. The specific components of the protoplast medium are defined as modified KM8P basal medium, 2,4-dichlorophenoxyacetic acid, 6-benzylaminopurine, mannitol, sucrose, polyvinylpyrrolidone, glutathione, and corn peptides. This improves protoplast survival rate and cell wall regeneration efficiency. The specific components of the callus proliferation medium are defined as MS basal medium, 6-benzylaminopurine, α-naphthaleneacetic acid, vitamin C, sucrose, agar, corn peptides, glutathione, and astaxanthin. This allows microcallus to rapidly expand into a large amount of callus tissue, laying a quantitative foundation for subsequent differentiation. The specific components of the differentiation medium are defined as MS basal medium, 6-benzylaminopurine, α-naphthaleneacetic acid, sucrose, agar, glutathione, and astaxanthin. This allows callus tissue to differentiate efficiently into adventitious buds, forming clustered buds. The specific components of the bulblet induction medium are defined as MS basal medium, 6-benzylaminopurine, α-naphthaleneacetic acid, sucrose, agar, corn peptide, astaxanthin, and glutathione. This allows the bud base to swell into bulblets, improving transplant survival rate. The specific components of the rooting medium are defined as 1 / 2 MS basal medium, indolebutyric acid, sucrose, agar, glutathione, and astaxanthin. This induces robust white roots with a rooting rate >85%, shortening seedling time.

[0060] Figure 2 A detailed flowchart of a method for propagating *Fritillaria purpurea* using protoplast culture, provided by an embodiment of this application, is illustrated by way of example. In some alternative implementations, such as Figure 2 As shown, the embryonic callus tissue is subjected to enzymatic digestion and purification processes sequentially to obtain protoplast samples, including the following steps: S501. Dissolve cellulase, ionizing enzyme and mannitol in CPW salt solution, and filter and sterilize them sequentially to obtain sterilized enzyme solution; S502. Polyvinylpyrrolidone and glutathione are added to the sterilization enzyme solution to obtain a composite enzymatic hydrolysate; S503. The embryonic callus is enzymatically hydrolyzed using the composite enzymatic hydrolysate to obtain an enzymatic hydrolysate mixture; S504. Filter the enzymatic hydrolysis mixture to obtain the enzymatic hydrolysis filtrate; S505. The enzymatic hydrolysis filtrate is centrifuged and washed to obtain the enzymatic hydrolysis wash; S506. The enzymatically hydrolyzed washings are subjected to gradient centrifugation using sucrose to obtain a protoplast sample; wherein the density of the protoplast sample is 1×10⁻⁶. 4 Cells / mL ~ 1×10 5 per mL.

[0061] In these embodiments, cellulase, ionizing enzyme, and mannitol are first dissolved in CPW salt solution, filtered, and sterilized to ensure uniform distribution of the enzymes. Then, polyvinylpyrrolidone and glutathione are added to ensure that cell membrane lipids are not oxidized during the enzymatic hydrolysis stage. Finally, after filtration, centrifugation, washing, and gradient centrifugation, a high-density protoplast sample can be obtained, which is beneficial for subsequent initial culture.

[0062] The density of this protoplast sample can be 1×10⁻⁶. 4 cells / mL, 2×10 4 cells / mL, 3×10 4 cells / mL, 4×10 4 cells / mL, 5×10 4 cells / mL or 1×10 5 per mL.

[0063] It should be noted that when the density of the protoplast sample is less than 1×10⁻⁶, 4 At a density of 1 × 10⁶ cells / mL, protoplast samples are difficult to initiate division; however, when the density of protoplast samples is greater than 1 × 10⁶ cells / mL... 5 At a concentration of 1 protoplast per mL, the accumulation of metabolic waste in protoplast samples can lead to browning.

[0064] In some optional embodiments, the composite enzymatic hydrolysate used in the enzymatic hydrolysis treatment includes polyvinylpyrrolidone, glutathione, cellulase, and cleavage enzyme; the mass ratio of the cellulase, the cleavage enzyme, the polyvinylpyrrolidone, and the glutathione is 150:50:(4.5~5.5):1.

[0065] In these embodiments, a complex enzymatic hydrolysate of cellulase, dissociative enzyme, polyvinylpyrrolidone, and glutathione in a mass ratio of 150:50:(4.5~5.5):1 is used to fully enzymatically hydrolyze the cell wall of embryogenic callus while avoiding the oxidation of cell membrane lipids, thus forming uniform protoplasts.

[0066] The mass ratio of cellulase, cleavage enzyme, polyvinylpyrrolidone, and glutathione can be 150:50:4.5:1, 150:50:4.6:1, 150:50:4.7:1, 150:50:4.8:1, 150:50:4.9:1, 150:50:5.0:1, or 150:50:5.5:1.

[0067] In some optional embodiments, the enzymatic hydrolysis is carried out in the dark and under stirring conditions, the temperature of the enzymatic hydrolysis is 22°C to 24°C, the time of the enzymatic hydrolysis is 4h to 5h, and the rotation speed of the enzymatic hydrolysis is 30rpm to 40rpm.

[0068] In these embodiments, enzymatic hydrolysis at a temperature of 22°C to 24°C, a time of 4 to 5 hours, and a rotation speed of 30 to 40 rpm can fully decompose the cell walls of embryogenic callus tissue while avoiding cell membrane oxidation, thus forming uniform protoplasts.

[0069] The temperature for this enzymatic hydrolysis treatment can be 22℃, 22.5℃, 23℃, 23.5℃, or 24℃.

[0070] The enzymatic hydrolysis treatment time is 4h, 4.1h, 4.2h, 4.3h, 4.4h, 4.5h, or 5h.

[0071] The enzymatic hydrolysis speed is 30 rpm, 31 rpm, 32 rpm, 33 rpm, 34 rpm, 35 rpm, or 40 rpm.

[0072] It should be noted that the enzymatic hydrolysis temperature needs to be below 25℃. This is because the dark purple fritillary is a high-altitude species, and the optimal temperature for the entire process needs to be controlled at around 22℃. Excessively high enzymatic hydrolysis temperatures will exacerbate browning and significantly reduce the protoplast division rate.

[0073] In some optional embodiments, the temperature of the low-temperature pretreatment is ≤4°C, and the time of the low-temperature pretreatment is 36h~54h.

[0074] In these embodiments, low-temperature pretreatment at ≤4°C for 36h~54h can inhibit the polyphenol oxidase activity of Fritillaria bulb samples, reducing the risk of subsequent browning from the source.

[0075] The low-temperature pretreatment time can be 36h, 38h, 40h, 42h, 44h, 46h, 48h, 50h, 52h or 54h.

[0076] In some optional embodiments, the explants are sequentially disinfected and subjected to antioxidant treatment to obtain detoxified and antioxidant explants, including the following steps: S301. Disinfect the explants with carbendazim to obtain virus-free explants; S302. The detoxified explants are subjected to antioxidant treatment in a sterile solution containing polyvinylpyrrolidone and glutathione to obtain detoxified antioxidant explants.

[0077] In these embodiments, disinfecting explants with carbendazim can prevent the accumulation of microorganisms on the explants. In addition, using a sterile solution containing polyvinylpyrrolidone and glutathione to perform antioxidant treatment on the detoxified explants can significantly reduce the proportion of explants discarded due to browning and necrosis through the dual defense of "adsorption + reduction" of polyvinylpyrrolidone and glutathione.

[0078] In some optional embodiments, the antioxidant treatment time is 10 min to 15 min.

[0079] In these embodiments, an antioxidant treatment lasting 10 to 15 minutes can significantly reduce the proportion of explants discarded due to browning and necrosis by using a dual defense of polyvinylpyrrolidone and glutathione.

[0080] The antioxidant treatment time can be 10 min, 11 min, 12 min, 13 min, 14 min or 15 min.

[0081] In some optional embodiments, the initial culture time is 20-25 days; and / or The temperature for adventitious bud differentiation is 18℃~22℃, and the time for adventitious bud differentiation is 3 weeks~4 weeks; and / or The rooting culture temperature is 18℃~22℃, and the rooting culture time is 10d~15d.

[0082] In these embodiments, an initial culture period of 20-25 days allows protoplast samples sufficient time to transform into microcallus. Additionally, adventitious bud differentiation at 18-22°C for 3-4 weeks allows callus tissue sufficient time and suitable temperature conditions to transform into adventitious buds. Furthermore, rooting culture at 18-22°C for 10-15 days allows bulblets sufficient time and suitable temperature conditions to induce robust white roots, shortening the seedling stage.

[0083] The initial culture period can be 20 days, 21 days, 22 days, 23 days, 24 days, or 25 days.

[0084] The temperature for adventitious bud differentiation can be 18℃, 18.5℃, 19℃, 19.5℃, 20℃, 20.5℃, 21℃, 21.5℃ or 22℃.

[0085] The rooting culture temperature can be 18℃, 18.5℃, 19℃, 19.5℃, 20℃, 20.5℃, 21℃, 21.5℃ or 22℃.

[0086] The rooting culture period can be 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days.

[0087] The present application is further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national / industry standards; if there is no corresponding national / industry standard, they are performed according to general international standards, conventional conditions, or conditions recommended by the manufacturer.

[0088] Example 1

[0089] A method for propagating *Fritillaria purpurea* using protoplast culture includes: S1. Select healthy, disease-free, fresh dark purple fritillary bulbs that are 2 years and 6 months old, and remove the inner tender scales to obtain fritillary bulb samples. S2. The fritillaria bulb samples were pretreated at low temperature to obtain explants; S301. Disinfect the explants with carbendazim to obtain virus-free explants; the specific procedure is as follows: Rinse the explant bulbs with running water for 3 hours to remove mud and sand. After separating individual scales, soak them in a 50% carbendazim solution diluted 500 times for 20 minutes. Then, disinfect them in a clean bench with 75% ethanol for 30 seconds, and rinse them with sterile water 2 to 3 times. Next, disinfect the virus-free explants in a 0.1% mercuric chloride solution containing 1 to 2 drops of Tween 80 for 8 minutes. Rinse them repeatedly with sterile water 5 to 6 times. Finally, use sterile filter paper to absorb the surface moisture. S302. Detoxified explants are subjected to antioxidant treatment in a sterile solution containing polyvinylpyrrolidone and glutathione to obtain detoxified antioxidant explants; the specific procedure is as follows: On a clean bench, the scales of the detoxified explants were immersed in a sterile solution containing PVP (K30) and reduced glutathione for 12 minutes to remove free radicals on the tissue surface in advance and reduce the initial browning caused by cutting damage. S4. In the embryogenic callus induction medium, detoxified antioxidant explants were sequentially induced, inoculated, and subcultured for purification to obtain embryogenic callus tissue; the specific procedure is as follows: The scales of the detoxified and antioxidant explants were cut into small pieces of 4 mm to 6 mm square. The small pieces were inoculated into embryogenic callus induction medium with the base facing down. They were then cultured at a constant temperature of 20°C and in complete darkness for 17 days, allowing the base of the scales to gradually dedifferentiate and form pale yellow, loose, granular primary embryogenic callus. The primary embryogenic callus was divided and transferred, and the embryogenic callus with vigorous growth and uniform granules was selected. The callus was subcultured every 5 days. After 2 or 3 consecutive subcultures, stable embryogenic callus was obtained for subsequent protoplast isolation. S501. Dissolve cellulase, ionizing enzyme and 0.6 mol / L mannitol in CPW salt solution, filter and sterilize using a 0.22 μm filter membrane to obtain sterilized enzyme solution; S502. Add polyvinylpyrrolidone and glutathione to the sterilization enzyme solution to obtain 1L of composite enzymatic hydrolysate; S503. Enzymatic hydrolysis of embryogenic callus tissue using a compound enzymatic hydrolysate to obtain an enzymatic hydrolysate mixture; the specific process is as follows: Using 10-15 day logarithmic growth stage embryogenic callus as material, the embryogenic callus was completely immersed in a compound enzymatic hydrolysis solution for enzymatic hydrolysis. After enzymatic hydrolysis, it was examined under a microscope. Enzymatic hydrolysis was terminated when a large number of spherical protoplasts were released. S504. Filter the enzymatic hydrolysis mixture to obtain the enzymatic hydrolysis filtrate; the specific process is as follows: The enzymatic hydrolysate mixture was filtered through a 300-mesh sterile nylon mesh to remove undegraded callus fragments, yielding an enzymatic hydrolysate filtrate. S505. The enzymatic hydrolysis filtrate is centrifuged and washed to obtain the enzymatic hydrolysis wash; the specific process is as follows: Centrifuge the enzymatic hydrolysis filtrate at 650 rpm for 3 min and discard the supernatant; then resuspend the precipitate with protoplast washing buffer containing glutathione, and repeat the centrifugation and washing 2 to 3 times to completely remove residual enzyme solution and obtain the enzymatic hydrolysis wash. S506. The enzymatically hydrolyzed washings were subjected to gradient centrifugation using sucrose to obtain protoplast samples; wherein the density of the protoplast samples was 1×10⁻⁶.4 Cells / mL ~ 1×10 5 cells / mL; the specific procedure is as follows: Using sucrose with a mass concentration of 20%–25%, gradient centrifugation was performed at different sucrose densities of 20%, 21%, 22%, 23%, 24%, and 25%. Each gradient centrifugation was performed at 800 rpm for 5 minutes. The supernatant high-viability protoplast bands were collected and analyzed by fluorescein diacetate (FDA) staining until protoplast viability was ≥70%. The protoplast density was then adjusted to 1 × 10⁻⁶ using a hemocytometer. 4 Cells / mL ~ 1×10 5 Units / mL, forming protoplast samples; S6. Initially culture protoplast samples in protoplast culture medium to obtain microcall tissue; the specific procedure is as follows: A solid-liquid bilayer culture method was adopted, with a solid protoplast culture medium containing agar laid in the lower layer and protoplast samples inoculated in the upper layer, which takes into account both aeration and nutrient supply. PVP (K30), glutathione, and corn peptide were added to the protoplast culture medium, which have the functions of free radical scavenging, polyphenol adsorption, and organic nutrient supplementation. No fat-soluble astaxanthin was added throughout the process to avoid damaging the protoplast cell membrane. Then, the protoplasts were initially cultured statically at a constant temperature of 20°C and in complete darkness. Shaking was avoided in the early stage of the initial culture to prevent mechanical rupture of the protoplasts. Initial culture 2-3 days: protoplasts begin to regenerate cell walls, and their morphology changes from regular spherical to elliptical. Cell wall formation can be identified by staining with a fluorescent whitening agent. Initial culture 5-7 days: the first cell division is completed, and then the cells gradually enter a continuous division state; Initial culture 10-15 days: multi-cell clusters are formed. At this time, fresh protoplast culture medium is added every 7 days, and the mannitol concentration is gradually reduced (by 1 / 3 each time), while the concentration of antioxidants is gradually reduced. Initial culture for 20-25 days: forming micro-callus tissue with a diameter of 0.5 mm to 1 mm that is visible to the naked eye; S7. In the callus proliferation medium, the micro-callus tissue is proliferated to obtain callus tissue; the specific procedure is as follows: When the microcallus grows to 1 mm to 2 mm, it is transferred to a solid callus proliferation medium and then subcultured every 20 days under constant temperature of 20℃ and light conditions of 16 h / d (light intensity of 1500 lx). Light yellow, granular, and vigorously growing callus tissues are selected for differentiation culture to expand the culture and maintain the embryogenic state, laying the foundation for subsequent plant regeneration. S8. In the differentiation medium, the callus tissue is subjected to adventitious shoot differentiation to obtain clustered shoots; the specific procedure is as follows: Healthy callus tissue was transferred to differentiation medium and cultured at a constant temperature of 20℃ with a light intensity of 12 h / d for 3 to 4 weeks, allowing the callus tissue to gradually differentiate into adventitious shoots, forming... Figure 3 The clustered buds shown; S9. In the bulblet induction medium, the clustered buds are induced to produce bulblets; the specific procedure is as follows: The clustered buds were transferred into a bulblet induction medium and cultured under diurnal temperature variations of 18℃~20℃ during the day and 8℃~12℃ at night. After 30 to 40 days, sterile bulblets with a diameter of 0.5cm~1cm were formed. S10. Rooting culture of small bulbs in rooting medium yields dark purple fritillary seedlings; the specific procedure is as follows: Select single buds (or bulbs with a diameter of more than 0.5 cm) from healthy small bulbs with a height of 2cm to 3cm, transfer them into culture bottles containing rooting medium, and culture them for 10 to 15 days under constant temperature of 20℃ and light conditions of 12h / d. White and healthy roots will grow, and dark purple fritillary seedlings will be obtained. S11. The seedlings of *Fritillaria purpurea* are successively hardened off, transplanted, and nurtured to obtain mature *Fritillaria purpurea* plants; the specific process is as follows: After the rooting culture is completed, open the sealing film of the culture bottle and harden the seedlings at room temperature for 3 to 5 days to gradually adapt to the external humidity and light environment. Then wash away the residual culture medium from the roots of the dark purple fritillary seedlings, transplant them into sterilized substrate (humus and perlite in a mass ratio of 3:1), water thoroughly to settle the roots, keep moist and shaded. Maintain an ambient temperature of 15℃~20℃ and an air humidity of 70%~80%, gradually increasing the light intensity to normal; after 2 months of cultivation, transplant to a location where... Figure 4 The fields or semi-wild cultivation environments shown are depicted.

[0090] The antioxidants used in the antioxidant treatment had a mass concentration of 8 g / L for polyvinylpyrrolidone and a mass concentration of 0.4 g / L for glutathione.

[0091] The embryogenic callus induction medium, per 1L, includes: MS basal medium, 2,4-dichlorophenoxyacetic acid: 1.0 mg, α-naphthaleneacetic acid: 0.5 mg, vitamin C: 0.1 g, sucrose: 30 g, agar: 7 g, polyvinylpyrrolidone: 0.7 g, and glutathione: 0.15 g. 1L of protoplast culture medium includes: modified KM8P basal medium (PM90531102, Crown Bioscience), 2,4-dichlorophenoxyacetic acid: 0.8mg, 6-benzylaminopurine: 0.3mg, mannitol: 0.45mol, sucrose: 20g, polyvinylpyrrolidone: 0.5g, glutathione: 0.1g, and corn peptide: 0.3g; 1L of callus proliferation medium includes: MS basal medium, 6-benzylaminopurine: 1.6mg, α-naphthaleneacetic acid: 1.2mg, vitamin C: 0.1g, sucrose: 30g, agar: 7g, corn peptide: 0.5g, glutathione: 0.08g, astaxanthin: 0.02g; The differentiation medium, per 1L, includes: MS basal medium, 6-benzylaminopurine: 1.5mg, α-naphthaleneacetic acid: 0.2mg, sucrose: 30g, agar: 7g, glutathione: 0.1g, and astaxanthin: 0.01g; The bulblet induction medium, per 1L, includes: MS basal medium, 6-benzylaminopurine: 0.5mg, α-naphthaleneacetic acid: 1.0mg, sucrose: 50g, agar: 7g, corn peptide: 0.8g, astaxanthin: 0.03g and glutathione: 0.050g; 1L of rooting medium consists of: 1 / 2 MS basal medium, indolebutyric acid: 0.5mg, sucrose: 20g, agar: 7g, glutathione: 0.1g and astaxanthin: 0.01g.

[0092] The compound enzymatic hydrolysate used in the enzymatic hydrolysis treatment includes polyvinylpyrrolidone, glutathione, cellulase, and cleavage enzyme; the mass ratio of cellulase, cleavage enzyme, polyvinylpyrrolidone, and glutathione in 1L of the compound enzymatic hydrolysate is 15g:5g:0.5g:0.1g.

[0093] The enzymatic hydrolysis was carried out in the dark and under stirring conditions. The temperature of the enzymatic hydrolysis was 22℃~24℃, the time of the enzymatic hydrolysis was 4.5h, and the rotation speed of the enzymatic hydrolysis was 35rpm.

[0094] The low-temperature pretreatment temperature was 4℃, and the low-temperature pretreatment time was 48h.

[0095] The temperature for adventitious bud differentiation was 20℃, and the differentiation time was 3 weeks. The rooting culture temperature is 20℃, and the rooting culture time is 10-15 days.

[0096] Example 2

[0097] Compared to Example 1, the differences in this example are as follows, while the rest are the same: The antioxidants used in the antioxidant treatment contained polyvinylpyrrolidone at a mass concentration of 5 g / L and glutathione at a mass concentration of 0.2 g / L.

[0098] The embryogenic callus induction medium, per 1L, includes: polyvinylpyrrolidone: 0.5g and glutathione: 0.1g; 1L of protoplast culture medium includes: glutathione: 0.05g and corn peptide: 0.25g; The callus proliferation culture medium, per 1L, includes: corn peptide: 0.45g, glutathione: 0.05g, and astaxanthin: 0.01g. The bulblet induction medium, per 1L, includes: corn peptide: 0.5g, astaxanthin: 0.02g, and glutathione: 0.045g; The mass ratio of cellulase, ionizing enzyme, polyvinylpyrrolidone, and glutathione was 15g:5g:0.45g:0.1g.

[0099] The low-temperature pretreatment time is 50 hours.

[0100] The antioxidant treatment time is 10 minutes.

[0101] Example 3

[0102] Compared to Example 1, the differences in this example are as follows, while the rest are the same: The antioxidants used in the antioxidant treatment contained polyvinylpyrrolidone at a mass concentration of 10 g / L and glutathione at a mass concentration of 0.5 g / L.

[0103] 1 L of embryogenic callus induction medium contains: polyvinylpyrrolidone: 1.0 g and glutathione: 0.2 g;

[0104] 1L of protoplast culture medium includes: 0.15g of glutathione and 0.35g of corn peptide; The callus proliferation culture medium, per 1L, includes: corn peptide: 0.55g, glutathione: 0.10g, and astaxanthin: 0.03g. The bulblet induction medium, per 1L, includes: corn peptide: 1.0g, astaxanthin: 0.04g, and glutathione: 0.055g; The mass ratio of cellulase, ionizing enzyme, polyvinylpyrrolidone, and glutathione was 15g:5g:0.55g:0.1g.

[0105] The low-temperature pretreatment time is 54 hours.

[0106] The antioxidant treatment time is 15 minutes.

[0107] Comparative Example 1

[0108] Compared to Example 1, the differences in this comparative example are as follows, while the rest are the same: No antioxidant treatment is applied.

[0109] No polyvinylpyrrolidone or glutathione are added to the compound enzymatic hydrolysate.

[0110] Meanwhile, no antioxidants (polyvinylpyrrolidone, glutathione, corn peptides, and astaxanthin) are added to the embryogenic callus induction medium, protoplast medium, callus proliferation medium, differentiation medium, bulblet induction medium, and rooting medium.

[0111] Comparative Example 2

[0112] Compared to Example 1, the differences in this comparative example are as follows, while the rest are the same: No polyvinylpyrrolidone or glutathione are added to the compound enzymatic hydrolysate.

[0113] Meanwhile, no antioxidants (polyvinylpyrrolidone, glutathione, corn peptides, and astaxanthin) are added to the embryogenic callus induction medium, protoplast medium, callus proliferation medium, differentiation medium, bulblet induction medium, and rooting medium.

[0114] Comparative Example 3

[0115] Compared to Example 1, the differences in this comparative example are as follows, while the rest are the same:

[0116] Antioxidants (polyvinylpyrrolidone, glutathione, corn peptides, and astaxanthin) are not added to the embryogenic callus induction medium, protoplast medium, callus proliferation medium, differentiation medium, bulblet induction medium, and rooting medium.

[0117] Comparative Example 4

[0118] Compared to Example 1, the differences in this comparative example are as follows, while the rest are the same: The embryogenic callus induction medium without polyvinylpyrrolidone served as control group 1 for comparative example 4. The embryogenic callus induction medium without glutathione served as control group 2 for comparison example 4. The embryogenic callus induction medium without the addition of polyvinylpyrrolidone and glutathione served as the control group 3 for comparative example 4.

[0119] Comparative Example 5

[0120] Compared to Example 1, the differences in this comparative example are as follows, while the rest are the same: The protoplast culture medium without glutathione served as control group 1 in comparison example 5. The protoplast culture medium without corn peptides served as control group 2 for comparison example 5. The protoplast culture medium without glutathione and corn peptide served as control group 3 for comparison example 5.

[0121] Comparative Example 6

[0122] Compared to Example 1, the differences in this comparative example are as follows, while the rest are the same: No corn peptide was added to the callus proliferation culture medium, serving as control group 1 for comparison example 6; The callus proliferation culture medium without glutathione served as control group 2 for comparison example 6. Astaxanthin was not added to the callus proliferation culture medium, which served as the control group 3 for comparison example 6. The callus proliferation culture medium without corn peptide, glutathione and astaxanthin was used as the control group 4 of the control group 6.

[0123] Comparative Example 7

[0124] Compared to Example 1, the differences in this comparative example are as follows, while the rest are the same: No glutathione was added to the differentiation medium, which served as control group 1 for comparison example 7; Astaxanthin was not added to the differentiation medium, which served as control group 2 for comparison example 7. The differentiation medium without glutathione and astaxanthin served as the control group 3 for comparison example 7.

[0125] Comparative Example 8

[0126] Compared to Example 1, the differences in this comparative example are as follows, while the rest are the same: No corn peptide was added to the bulblet induction medium, serving as control group 1 for comparison example 8; Astaxanthin was not added to the bulblet induction medium, which served as control group 2 for comparison example 8. No glutathione was added to the bulblet induction medium, which served as the control group 3 for comparison example 8. The bulblet induction medium without corn peptide, astaxanthin, and glutathione served as the control group 4 for comparison ratio 8.

[0127] Comparative Example 9

[0128] Compared to Example 1, the differences in this comparative example are as follows, while the rest are the same: The rooting medium without glutathione served as control group 1 for comparison example 9. Astaxanthin was not added to the rooting medium, which served as control group 2 for comparison example 9; The rooting medium without glutathione and astaxanthin served as the control group 3 for comparison 9.

[0129] Relevant experimental and effect data: The data of the cultivation stages of *Fritillaria purpurea* in each embodiment and comparative example were statistically analyzed, and the results are shown in Table 1.

[0130] Table 1. Data distribution of *Fritillaria purpurea* at different culture stages in various examples and comparative examples.

[0131] The overall propagation rate = protoplast division rate × microcallus formation rate × adventitious bud differentiation rate × bulblet induction rate × rooting rate × transplant survival rate × 100%, representing the probability that a single protoplast will eventually develop into a complete plant.

[0132] As shown in Table 1, the method for propagating *Fritillaria purpurea* using protoplast culture provided in this application systematically improves the propagation efficiency of *Fritillaria purpurea* protoplast culture while controlling costs through mechanisms such as PVP+GSH for full-process browning prevention, corn peptides replacing some expensive nutrients and antioxidant components, precise micro-injection of astaxanthin, and the protoplast single-cell regeneration pathway. Specifically: (1) The overall protoplast proliferation rate of Example 1 reached 3.31%, meaning that approximately 1.72 × 10⁻⁶ cells / gram of embryogenic callus could be obtained on average through protoplast culture. 4 One complete plant (based on protoplast yield of 5.2 × 10⁶) 5 (Calculated based on 13790 times per g and a comprehensive reproduction rate of 3.31%), compared to Comparative Example 1 (0.00024%), and compared to Comparative Example 3 (0.016%), it is about 207 times higher, achieving efficient regeneration of protoplast culture of *Fritillaria thunbergii*.

[0133] (2) Antioxidant treatment (PVP+GSH) during the explant stage is a key control point for protoplast quality: the protoplast yield of Comparative Example 1 (without antioxidant treatment) was only 2.1×10 5 The number of bulbs with a diameter of 2-3 cm per gram was 35%, the division rate was 5%, and the overall propagation rate was only 0.00024%. This indicates that explant browning directly leads to the deterioration of subsequent protoplast yield and vigor, making plant regeneration almost impossible and is the primary bottleneck in the entire cultivation process.

[0134] (3) PVP+GSH in the embryogenic callus induction stage is crucial for establishing high-quality donor materials: In control group 3 (which simultaneously lacks PVP and glutathione) of Comparative Example 4, the protoplast division rate decreased to 25% (35% in Example 1), the microcallus formation rate decreased to 18% (28% in Example 1), and the overall proliferation rate decreased to 0.16%, a decrease of 95.2% compared to Example 1. The overall proliferation rates of PVP alone (control group 1) or glutathione alone (control group 2) were 0.62% and 0.91%, respectively, indicating that the two have a synergistic effect in preventing browning and maintaining cell viability.

[0135] (4) GSH + corn peptide in protoplast culture significantly promotes cell wall regeneration and early division: Comparative Example 5 only adjusted the composition of the protoplast culture medium, and the initial protoplast yield and viability were completely consistent with Example 1; among them, the protoplast division rate of Control Group 3 (which lacked both GSH and corn peptide) decreased to 12%, the microcallus formation rate was only 7%, and the overall proliferation rate decreased to 0.061%. The overall proliferation rates of GSH alone (Control Group 1) or corn peptide alone (Control Group 2) were 0.49% and 0.33%, respectively, indicating that GSH maintaining the reduced state of membrane lipids and corn peptide providing nutritional support are both indispensable conditions for protoplast regeneration.

[0136] (5) The antioxidant synergy (corn peptide + GSH + astaxanthin) during the callus proliferation stage is crucial for maintaining cell division activity: Comparative Example 6 only adjusted the composition of the callus proliferation culture medium, and the protoplast stage indicators were completely consistent with those in Example 1; among them, the callus proliferation fold of control group 4 (which lacked all three) decreased to 3.5 (8.5 in Example 1), and the overall reproduction rate decreased to 0.87%. The proliferation fold of corn peptide alone (control group 1) or astaxanthin alone (control group 3) decreased to 4.2 and 6.8, respectively, and the overall reproduction rates were 1.24% and 1.63%, respectively, indicating that the nutritional supplementation effect of corn peptide has the greatest impact on the proliferation stage.

[0137] (6) GSH and astaxanthin during the differentiation stage significantly affected the differentiation efficiency of adventitious buds: Comparative Example 7 only adjusted the composition of the differentiation medium, and all indicators in the early stages were completely consistent with those in Example 1; among them, the adventitious bud differentiation rate of Control Group 3 (which lacked both GSH and astaxanthin) decreased to 30% (65% in Example 1), and the overall reproduction rate decreased to 0.58%. The differentiation rates of GSH alone (Control Group 1) or astaxanthin alone (Control Group 2) were 40% and 48%, respectively, and the overall reproduction rates were 0.96% and 1.33%, respectively, indicating that GSH plays a more critical role in maintaining the redox signal required for differentiation.

[0138] (7) The combination of corn peptide, astaxanthin, and GSH during the bulblet induction stage has a synergistic promoting effect on bulb enlargement: Comparative Example 8 only adjusted the composition of the bulblet induction culture medium, and the indicators before differentiation were completely consistent with those in Example 1; among them, the bulblet induction rate of control group 4 (which lacked all three components) decreased to 32% (72% in Example 1), and the overall reproduction rate decreased to 0.86%. The induction rate of corn peptide alone (control group 1) decreased to 42%, and the overall reproduction rate was 1.30%, indicating that the synergistic effect of corn peptide and sucrose in promoting dry matter accumulation was the most prominent.

[0139] (8) GSH and astaxanthin during the rooting stage have a significant impact on root development and transplant survival: Comparative Example 9 only adjusted the composition of the rooting medium, and the indicators before bulb induction were completely consistent with those in Example 1; among them, the rooting rate of Control Group 3 (which lacked both) decreased to 45% (88% in Example 1), the transplant survival rate decreased to 35%, and the overall propagation rate decreased to 0.72%. The rooting rates of GSH alone (Control Group 1) or astaxanthin alone (Control Group 2) were 55% and 62%, respectively, and the transplant survival rates were 45% and 52%, respectively, indicating that GSH plays a more crucial role in promoting root elongation and root hair formation.

[0140] In summary, the method for propagating *Fritillaria purpurea* using protoplast culture provided in this application combines a low-cost antioxidant system (PVP+GSH) + agricultural by-product-derived polypeptides (corn peptides) + precise micro-addition (astaxanthin) + a comprehensive loss prevention strategy, achieving a comprehensive propagation rate of 1.43%~3.31%, with Example 1 reaching 3.31%, significantly higher than the comparative examples (0.00024%~1.69%). This method precisely adds antioxidants at each stage, specifically addressing the species characteristics of *Fritillaria purpurea*, such as easy browning of tissues, low protoplast viability, and high regeneration difficulty. Furthermore, it uses low-cost PVP and GSH to replace expensive antioxidants throughout the process, and corn peptides to replace some nutrients and antioxidant components, achieving efficient propagation of *Fritillaria purpurea* protoplast culture while controlling costs.

[0141] Furthermore, this application provides a method for propagating *Fritillaria purpurea* using protoplast culture. This method uses *Fritillaria purpurea* bulbs as the starting explant and protoplast isolation and culture as the essential core pathway. Through a complete technical system encompassing embryogenic callus induction, protoplast preparation and purification, protoplast culture and regeneration, and callus differentiation into seedlings, it achieves the cultivation from single cells to complete plants. This method incorporates four types of antioxidants throughout the process: reduced glutathione, corn peptides, astaxanthin, and polyvinylpyrrolidone. These antioxidants are added precisely in stages, specifically addressing the species characteristics of *Fritillaria purpurea*, such as easy browning of tissues, low protoplast viability, and high regeneration difficulty.

[0142] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed in this application.

Claims

1. A method for propagating *Fritillaria purpurea* using protoplast culture, characterized in that, The method includes: Fresh, dark purple fritillary bulbs that are 2 to 3 years old, healthy, and free from pests and diseases are selected, and the inner tender scales are removed to obtain fritillary bulb samples. The fritillaria bulb samples were pretreated at low temperature to obtain explants; The explants were sequentially disinfected and subjected to antioxidant treatment to obtain detoxified and antioxidant explants. In the embryogenic callus induction medium, the detoxified antioxidant explants were sequentially induced, inoculated, and subcultured for purification to obtain embryogenic callus tissue. The embryonic callus was sequentially subjected to enzymatic hydrolysis and purification to obtain protoplast samples. The protoplast sample was initially cultured in protoplast culture medium to obtain microcall tissue; The micro-callus tissue was proliferated in a callus proliferation culture medium to obtain callus tissue; In a differentiation medium, the callus tissue was subjected to adventitious bud differentiation to obtain clustered buds; In a bulblet induction medium, the clustered buds were induced to form bulblets to obtain bulblets; The small bulbs were rooted in a rooting medium to obtain dark purple Fritillaria seedlings; The seedlings of the dark purple Fritillaria were successively hardened, transplanted and nurtured to obtain mature dark purple Fritillaria. The antioxidants used in the antioxidant treatment include polyvinylpyrrolidone and glutathione; The composite enzymatic hydrolysate used in the enzymatic hydrolysis process includes polyvinylpyrrolidone and glutathione. The embryogenic callus induction medium includes polyvinylpyrrolidone and glutathione; The protoplast culture medium includes polyvinylpyrrolidone, glutathione, and corn peptides; The callus proliferation culture medium includes corn peptides, glutathione, and astaxanthin; The differentiation culture medium includes glutathione and astaxanthin; The bulblet induction culture medium includes corn peptide, astaxanthin, and glutathione. The rooting medium includes glutathione and astaxanthin.

2. The method according to claim 1, characterized in that, In the antioxidants used in the antioxidant treatment, the mass concentration of polyvinylpyrrolidone is 5 g / L to 10 g / L, and the mass concentration of glutathione is 0.2 g / L to 0.5 g / L; and / or In the embryogenic callus induction medium, the mass concentration of polyvinylpyrrolidone is 0.5 g / L to 1.0 g / L, and the mass concentration of glutathione is 0.1 g / L to 0.2 g / L; and / or In the protoplast culture medium, the mass concentration of the polyvinylpyrrolidone is 0.45 g / L to 0.55 g / L, the mass concentration of the glutathione is 0.05 g / L to 0.15 g / L, and the mass concentration of the corn peptide is 0.25 g / L to 0.35 g / L; and / or In the callus proliferation medium, the mass concentration of the corn peptide is 0.45 g / L to 0.55 g / L, the mass concentration of the glutathione is 0.05 g / L to 0.10 g / L, and the mass concentration of the astaxanthin is 0.01 g / L to 0.03 g / L; and / or In the differentiation medium, the mass concentration of glutathione is 0.095 g / L to 0.105 g / L, and the mass concentration of astaxanthin is 0.0095 g / L to 0.0105 g / L; and / or In the bulblet induction medium, the mass concentration of the corn peptide is 0.5 g / L to 1.0 g / L, the mass concentration of the astaxanthin is 0.02 g / L to 0.04 g / L, and the mass concentration of the glutathione is 0.045 g / L to 0.055 g / L; and / or In the rooting medium, the mass concentration of glutathione is 0.095 g / L to 0.105 g / L, and the mass concentration of astaxanthin is 0.0095 g / L to 0.0105 g / L.

3. The method according to claim 1, characterized in that, Based on 1L, the embryogenic callus induction medium comprises: MS basal medium, 2,4-dichlorophenoxyacetic acid: 1.0 mg, α-naphthaleneacetic acid: 0.5 mg, vitamin C: 0.1 g, sucrose: 30 g, agar: 7 g, the polyvinylpyrrolidone: 0.5 g to 1.0 g, and the glutathione: 0.1 g to 0.2 g; and / or 1 L of the protoplast culture medium comprises: modified KM8P basal medium, 2,4-dichlorophenoxyacetic acid: 0.8 mg, 6-benzylaminopurine: 0.3 mg, mannitol: 0.45 mol, sucrose: 20 g, polyvinylpyrrolidone: 0.45 g to 0.55 g, glutathione: 0.05 g to 0.15 g, and corn peptide: 0.25 g to 0.35 g; and / or 1 L of the callus proliferation medium comprises: MS basal medium, 6-benzylaminopurine: 1.6 mg, α-naphthaleneacetic acid: 1.2 mg, vitamin C: 0.1 g, sucrose: 30 g, agar: 7 g, the corn peptide: 0.45 g to 0.55 g, the glutathione: 0.05 g to 0.10 g, the astaxanthin: 0.01 g to 0.03 g; and / or Based on 1L, the differentiation medium comprises: MS basal medium, 6-benzylaminopurine: 1.5mg, α-naphthaleneacetic acid: 0.2mg, sucrose: 30g, agar: 7g, the glutathione: 0.095g~0.105g, and the astaxanthin: 0.0095g~0.0105g; and / or 1 L of the bulblet induction medium comprises: MS basal medium, 6-benzylaminopurine: 0.5 mg, α-naphthaleneacetic acid: 1.0 mg, sucrose: 50 g, agar: 7 g, the corn peptide: 0.5 g to 1.0 g, the astaxanthin: 0.02 g to 0.04 g, and the glutathione: 0.045 g to 0.055 g; and / or 1L of the rooting medium comprises: 1 / 2 MS basal medium, indolebutyric acid: 0.5mg, sucrose: 20g, agar: 7g, glutathione: 0.095g~0.105g, and astaxanthin: 0.0095g~0.0105g.

4. The method according to claim 1, characterized in that, The embryonic callus tissue was subjected to enzymatic digestion and purification processes sequentially to obtain protoplast samples, including the following steps: Cellulase, dissociation enzyme, and mannitol were dissolved in CPW salt solution and then filtered and sterilized sequentially to obtain sterilized enzyme solution; Polyvinylpyrrolidone and glutathione were added to the sterilized enzyme solution to obtain a composite enzymatic hydrolysate. The embryonic callus was enzymatically hydrolyzed using the composite enzymatic hydrolysate to obtain an enzymatic hydrolysate mixture; The enzymatic hydrolysate was filtered to obtain the enzymatic hydrolysate filtrate. The enzymatic hydrolysis filtrate was centrifuged and washed to obtain the enzymatic hydrolysis wash. The enzymatically hydrolyzed washings were subjected to gradient centrifugation using sucrose to obtain protoplast samples; wherein the density of the protoplast samples was 1 × 10⁻⁶. 4 Cells / mL ~ 1×10 5 per mL.

5. The method according to claim 1 or 4, characterized in that, The compound enzymatic hydrolysate used in the enzymatic hydrolysis treatment includes polyvinylpyrrolidone, glutathione, cellulase, and ionizing enzyme; the mass ratio of the cellulase, the ionizing enzyme, the polyvinylpyrrolidone, and the glutathione is 150:50:(4.5~5.5):

1.

6. The method according to claim 1 or 4, characterized in that, The enzymatic hydrolysis is carried out in the dark and under stirring conditions. The temperature of the enzymatic hydrolysis is 22℃~24℃, the time of the enzymatic hydrolysis is 4h~5h, and the rotation speed of the enzymatic hydrolysis is 30rpm~40rpm.

7. The method according to claim 1, characterized in that, The temperature of the low-temperature pretreatment is ≤4℃, and the time of the low-temperature pretreatment is 36h~54h.

8. The method according to claim 1, characterized in that, The explants were sequentially disinfected and subjected to antioxidant treatment to obtain detoxified and antioxidant explants, including the following steps: The explants were disinfected using carbendazim to obtain virus-free explants; The detoxified explants were subjected to antioxidant treatment in a sterile solution containing polyvinylpyrrolidone and glutathione to obtain detoxified antioxidant explants.

9. The method according to claim 1 or 8, characterized in that, The antioxidant treatment time is 10 min to 15 min.

10. The method according to claim 1, characterized in that, The initial culture time is 20-25 days; and / or The temperature for adventitious bud differentiation is 18℃~22℃, and the time for adventitious bud differentiation is 3 weeks~4 weeks; and / or The rooting culture temperature is 18℃~22℃, and the rooting culture time is 10d~15d.

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