Method for inducing polyploidy of radix peucedani by using fasciculate buds and application thereof

Abstract

The application provides a method for inducing poly-podophylli rhizoma by using clump buds, comprising the following steps: S1, cutting stem segments from a podophylli rhizoma mother plant, soaking and cleaning to obtain explants; S2, disinfecting and cleaning the explants, inoculating into a clump bud induction medium to obtain podophylli rhizoma clump buds; S3, inoculating the clump buds into doubling liquid I, washing with a liquid clump bud induction medium after culture, and then transferring into doubling liquid II; S4, taking out the clump buds, cleaning, inoculating into a clump bud induction medium, and then inoculating into a bud proliferation medium; S5, waiting until 1-2 cm, screening out potential mutant strains through phenotypes, inoculating into the bud proliferation medium for subculture, and obtaining mutant plants; and S6, rooting culture of the mutant plants, and after root systems grow, planting and raising seedlings, and then poly-podophylli rhizoma plants are obtained. In the research, suitable concentration of colchicine is used for induction treatment twice, and through stage concentration switching and intermittent recovery strategies, the poly-ploid induction efficiency and explant survival are effectively considered.

Description

Technical Field

[0001] This invention belongs to the field of polyploid plant culture technology, specifically relating to a method for inducing polyploidy of Peucedanum praeruptorum using clustered buds and its application. Background Technology

[0002] Qianhu ( Peucedanum praeruptorum Dunn Peucedanum praeruptorum (also known as Angelica dahurica or Angiocarpus genus) is a perennial herb belonging to the Apiaceae family. It is a major traditional Chinese medicinal herb, primarily produced in the Yangtze River basin and areas south of it. Its dried root has expectorant, antitussive, and wind-heat dispersing effects, and is widely used in clinical prescriptions and the production of prepared Chinese medicines. With the increasing demand in the traditional Chinese medicine market, higher requirements are being placed on the yield, quality, and stress resistance of Peucedanum praeruptorum. Therefore, genetic improvement and new variety breeding are of great significance. However, current breeding methods for Peucedanum praeruptorum mainly rely on natural variation selection or conventional population selection, generally exhibiting a problem of "emphasizing selection over breeding," resulting in slow genetic progress and limited breeding efficiency.

[0003] Polyploidy is widespread in higher plants; studies suggest that approximately 50% of angiosperms have undergone polyploidization during their evolutionary history. Polyploid plants often exhibit desirable traits such as increased biomass, well-developed root systems, enhanced stress resistance, and increased accumulation of secondary metabolites. These characteristics hold significant application potential for *Peucedanum praeruptorum*, whose roots are used medicinally. Polyploid breeding can not only increase plant yield but also potentially enhance the content of its active ingredients, thereby improving the quality of the medicinal material. Therefore, polyploid induction using chromosome doubling technology has become an important approach to the genetic improvement of medicinal plants.

[0004] Although polyploid breeding has achieved significant results in various crops, systematic research on polyploid induction and identification in *Peucedanum praeruptorum* is still lacking. In practice, colchicine treatment is commonly used for chromosome doubling, but applying traditional concentration and time combinations in *Peucedanum praeruptorum* faces considerable problems: high concentrations for short periods easily lead to toxic death of clustered buds, while low concentrations for long periods result in low induction rates, making it difficult to obtain effective doubling populations. Therefore, establishing an efficient, stable, and reproducible method for inducing polyploids in *Peucedanum praeruptorum* is crucial for obtaining new polyploid germplasm with strong resistance, high yield, and excellent quality. Summary of the Invention

[0005] In view of this, the present invention provides a method for inducing polyploids of Peucedanum praeruptorum using clustered buds and its application, so as to obtain polyploid Peucedanum praeruptorum plants with stronger stress resistance and higher yield.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for inducing polyploidy in Peucedanum praeruptorum using clustered buds includes the following steps:

[0008] S1. Cut stem segments from the mother plant of Peucedanum praeruptorum, soak them in a mixed solution of citric acid and ascorbic acid, remove and wash them to obtain explants;

[0009] S2. After disinfecting, cleaning, and drying the explants, cut off both ends and inoculate them into the shoot induction medium to obtain shoot clusters.

[0010] S3. Cut off the induced shoot clusters and inoculate them into double solution I for culture. After culture, rinse with liquid shoot cluster induction medium, let stand to recover culture, and then transfer to double solution II for culture.

[0011] The formula for the doubled solution I is: 0.05~0.1% colchicine + ascorbic acid 100mg / L;

[0012] The formula for the doubled solution II is: 0.1~0.15% colchicine + ascorbic acid 100mg / L;

[0013] S4. Remove the treated clustered buds, clean them, and inoculate them again into the clustered bud induction medium for recovery culture. After the culture is completed, inoculate them into the bud proliferation medium for culture.

[0014] S5. When the clustered buds grow to 1-2cm, potential mutant plants are initially screened out by the characteristics of dark green leaves, larger and thicker leaves with larger serrations on the leaf margins and thicker stems. Potential mutant plants are cut off and inoculated into bud proliferation medium for subculture. After 3-4 rounds of continuous screening and proliferation culture, a population of polyploid mutant plants with stable traits can be obtained.

[0015] S6. Place the mutant plant population in a rooting culture and cultivate it. After the roots grow, transplant the seedlings to obtain polyploid Peucedanum plants.

[0016] Furthermore, in step S1, the mother plant of Peucedanum praeruptorum is treated in the dark for 46-50 hours before cutting the stem segments; the length of the cut stem segments is 1.5-2 cm; the concentration of citric acid in the mixed solution of citric acid and ascorbic acid is 300 mg / L and the concentration of ascorbic acid is 200 mg / L.

[0017] Furthermore, the specific disinfection method in step S2 is as follows: place the explant in a 70% alcohol solution and shake to rinse for 12-18 seconds, then immediately rinse quickly with sterile water 3-5 times; then soak in a 1ppm ozone aqueous solution at 4°C for 3 minutes, and rinse with sterile water 2-3 times; finally, use a 0.05% (w / v) HgCl2 solution containing 0.1% (v / v) Tween-20, shake for 4 minutes, and finally rinse with sterile water 3-5 times.

[0018] In some specific embodiments, the preferred formulation of the shoot induction medium in step S2 is: MS + NAA 0.4 mg / L + KT 1.5 mg / L + 2-ip 0.2 mg / L + ascorbic acid 100 mg / L + sucrose 3% + agar 0.8%;

[0019] Culture conditions: Incubate in the dark at 26±1℃ for 7~10 days, then transfer to light and dark alternation at 1500~2000lx for 20~25 days, with light / dark alternation for 12h / 12h daily.

[0020] In some specific embodiments, preferably, the culture conditions in doubling solution I and doubling solution II in step S3 are the same: shake culture in a shaker at a speed of 70~90 r / min and a temperature of 24±1℃ for 12~48 h;

[0021] The static recovery culture time is 22-26 hours.

[0022] In some specific embodiments, preferably, the culture conditions are restored in step S4 by culturing at a temperature of 24±1℃ for 3 to 5 days.

[0023] In some specific embodiments, preferably, the bud proliferation medium formulation in step S4 is: MS + NAA 0.05 mg / L + KT 1.2 mg / L + 2-ip 0.1 mg / L + sucrose 3% + hydrolyzed casein 50 mg / L + agar 0.8%;

[0024] Shoot proliferation culture conditions: cultured under alternating light and dark conditions at a temperature of 23±2℃ and a light intensity of 1500~2000lx for 30~40 days.

[0025] In some specific embodiments, preferably, the rooting medium formulation in step S6 is: 1 / 2 MS + NAA 0.8 mg / L + KT 0.3 mg / L + sucrose 1.5% + hydrolyzed casein 50~150 mg / L + activated charcoal 0.03% + agar 0.8%;

[0026] Rooting culture conditions: Culture for 30-40 days under alternating light and dark conditions at a temperature of 24±2℃ and a light intensity of 1500~2000lx, with 10 hours of light per day.

[0027] Furthermore, step S6 before transplanting includes: when the seedlings grow to 10-12cm, peel off the lower epidermis to make a slide, place it under a microscope for observation, and preliminarily determine the polyploid of Peucedanum praeruptorum based on the size and density of stomata. Then, perform flow cytometry analysis on the Peucedanum praeruptorum leaves after stomatal identification to determine the DNA content and accurately determine the ploidy.

[0028] The above method is applied in obtaining polyploid Peucedanum praeruptorum plants.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] (1) In this invention, the use of dark culture in the pretreatment of explants can significantly inhibit the activity of polyphenol oxidase and avoid light-driven oxidation reactions, thereby effectively slowing down the conversion of phenolic substances into quinones and reducing the browning rate. Further combined treatment with a mixed solution of citric acid and ascorbic acid, through citric acid chelating copper cofactor to inhibit polyphenol oxidase activity and ascorbic acid reducing oxidized quinones, forms a "prevention-repair" dual-effect antioxidant mechanism, synergistically inhibiting phenolic oxidation. This combined treatment strategy significantly reduces explant browning and improves initial survival rate and the induction efficiency of shoot clusters.

[0031] (2) In the disinfection treatment of stem segments, this invention significantly reduces explant browning and mitigates disinfectant damage by synergistically regulating the physical and chemical environment. A short rinse with 70% ethanol aims to quickly penetrate the cell wall, kill some surface microorganisms, and fix the epidermis, avoiding prolonged treatment that could lead to cell death and increased phenolic leakage. Subsequent low-temperature soaking with low-concentration ozone water effectively sterilizes the explants while utilizing its strong oxidizing properties to degrade oxidized phenolic substances, and the low temperature inhibits enzyme activity, reducing toxicity. Finally, combined with short-term treatment with low-concentration HgCl2, the cytotoxicity of heavy metal ions to the explants is significantly reduced while ensuring broad-spectrum sterilization efficiency. The entire process employs a phased, short-term, and low-concentration composite disinfection strategy, maintaining a sterile environment while minimizing the metabolic stress on the explants, thereby inhibiting the oxidation of phenolic compounds and subsequent browning reactions. This provides crucial technical support for the initial survival and regeneration of easily browning explants.

[0032] (3) Based on the traditional colchicine induction method, this invention establishes a gradient concentration culture system, optimizes the treatment concentration and time at each stage, and innovatively adopts a two-stage induction strategy, effectively solving the contradiction between "high concentration toxicity" and "low concentration inefficiency". This system combines concentration switching with intermittent recovery, ensuring a 91.98% survival rate of clustered shoots while increasing the polyploid induction rate to 43.88%, and maintaining stable growth of clustered shoots with no inhibition of proliferation and rooting. The obtained polyploid plants exhibit excellent traits, possessing typical polyploid advantages such as robust rhizomes, thick leaves, high biomass, and increased content of medicinal components, providing a stable and efficient technical approach for breeding new Angelica dahurica germplasm with strong resistance, high yield, and excellent quality. Attached Figure Description

[0033] Figure 1 This is a diagram showing the state of Peucedanum praeruptorum after 20 days of cultivation in a bud induction medium, as described in Example 1 of this invention.

[0034] Figure 2 This is a diagram showing the recovery state of the clustered buds of Peucedanum praeruptorum after doubling induction in Example 1 of the present invention.

[0035] Figure 3 This is a diagram showing the state of the proliferation culture of the clustered buds of Peucedanum praeruptorum after doubling induction in Example 1 of the present invention.

[0036] Figure 4 The images show the state of the Peucedanum praeruptorum bud mutant strain in Example 1 of this invention after multiple rounds of screening and proliferation culture; the left image shows the first round of screening and proliferation culture, and the right image shows the third round of screening and proliferation culture.

[0037] Figure 5 This is a diagram showing the state of Peucedanum praeruptorum after 35 days of cultivation in rooting medium in Example 1 of the present invention.

[0038] Figure 6 The images show the morphology of stomata on leaves of wild-type and polyploid Peucedanum praeruptorum in Example 2 of this invention; the left image shows wild-type Peucedanum praeruptorum, and the right image shows polyploid Peucedanum praeruptorum.

[0039] Figure 7 The image shows the DNA content analysis of wild-type and polyploid Peucedanum cells obtained by doubling induction in Example 2 of this invention; the left image is wild-type Peucedanum, and the right image is polyploid Peucedanum.

[0040] Figure 8 The images show a morphological comparison of polyploid and wild-type Peucedanum praeruptorum tissue culture seedlings. The left image shows the polyploid Peucedanum praeruptorum tissue culture seedlings, while the right image shows the wild-type Peucedanum praeruptorum tissue culture seedlings. Detailed Implementation

[0041] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0042] Example 1

[0043] This embodiment provides a method for inducing polyploidy in Peucedanum praeruptorum using clustered buds. The specific steps are as follows:

[0044] 1. Material selection and pretreatment

[0045] From healthy, disease-free Peucedanum praeruptorum mother plants, select vigorous, current-year stem segments from the middle and upper parts as explants. Remove the leaves, tightly wrap the target stem segments in black polyethylene bags with 100% light-blocking capacity, and pre-treat at 15℃ for 48 hours. Under low-light conditions, use sterile scissors to cut stem segments of about 2 cm, soak them in 300 mg / L citric acid + 200 mg / L ascorbic acid for 10 minutes, rinse with running water for 20 minutes, and gently brush the surface with a soft brush to remove dust and some microorganisms, thus obtaining the explants.

[0046] 2. Explant disinfection

[0047] The explants were placed in a 70% alcohol solution and shaken for 15 seconds, then immediately transferred to sterile water and rinsed 4 times. Subsequently, they were soaked in a 1 ppm ozone solution at 4°C for 3 minutes and rinsed 3 times with sterile water. Finally, they were treated with a 0.05% (w / v) HgCl2 solution (containing 0.1% (v / v) Tween-20 as a surfactant) and shaken for 4 minutes. Finally, they were rinsed 4 times with sterile distilled water to completely remove sterilizing agent residue.

[0048] 3. Inducing clustered buds

[0049] The explants were placed on sterile filter paper to absorb surface moisture, both ends were cut off, and the stem segments were cut into 1cm pieces. These segments were then inoculated into shoot induction medium and cultured in the dark at 26±1℃ for 8 days. They were then transferred to a light-dark cycle at 1800lx for 24 days, with daily light / dark alternation of 12h / 12h (see [reference to induced shoot induction medium]). Figure 1 ).

[0050] The formula for the shoot induction medium is: MS + NAA 0.4 mg / L + KT 1.5 mg / L + 2-ip 0.2 mg / L + ascorbic acid 100 mg / L + sucrose 3% + agar 0.8%.

[0051] 4. Doubling of induction of clustered buds

[0052] The induced shoot clusters were cut off and inoculated into doubled solution I, and cultured in a shaker at 80 r / min and 24 ± 1 ℃ for 48 h. Then, they were washed 4 times with shoot cluster induction liquid medium and allowed to stand for 24 h to recover. After that, they were transferred to doubled solution II and cultured for another 12 h at the same temperature and shaking frequency.

[0053] The formula for double-strength solution I is: 0.05% colchicine + 100mg / L ascorbic acid; the formula for double-strength solution II is: 0.125% colchicine + 100mg / L ascorbic acid.

[0054] 5. Regeneration culture of clustered buds

[0055] The doubled-treated shoot clusters were washed four times with sterile water and then inoculated again into the shoot cluster induction medium. They were then cultured at 24±1℃ for four days (see the image below for the recovery state of the shoot clusters after doubled induction). Figure 2 )

[0056] The formula for the shoot induction medium is: MS + NAA 0.4 mg / L + KT 1.5 mg / L + 2-ip 0.2 mg / L + ascorbic acid 100 mg / L + sucrose 3% + agar 0.8%.

[0057] 6. Bud proliferation culture

[0058] The recovered bud clusters were inoculated into bud proliferation medium and cultured for 35 days under alternating light and dark conditions at a temperature of 23±2℃ and a light intensity of 1800 lx (see the state of the bud clusters after doubling induction proliferation culture). Figure 3 ).

[0059] The bud proliferation medium formula is as follows: MS + NAA 0.05 mg / L + KT 1.2 mg / L + 2-ip 0.1 mg / L + sucrose 3% + hydrolyzed casein 50 mg / L + agar 0.8%.

[0060] 7. Screening and Propagation of Mutant Plants

[0061] When the newly sprouted shoots of *Hylocereus undatus* reach 1.5 cm in length, mutant plants with potential polyploid characteristics are systematically screened. The identification criteria include: significantly deepened leaf color to a dark green, noticeably larger and thicker leaves with increased serrations along the leaf margins, and thicker stems, among other typical polyploid morphological characteristics. After cutting off the mutant shoots that meet the criteria, they are further inoculated into a shoot proliferation medium for subculture. After three rounds of continuous screening and proliferation culture, a stable population of polyploid mutant plants can be obtained (see the status of the newly sprouted shoot mutants after multiple rounds of screening and proliferation culture for details). Figure 4 ).

[0062] 8. Rooting Culture

[0063] The initially screened induced variants were subjected to rooting culture at a temperature of 24±2℃ and a light intensity of 1800 lx under alternating light and dark conditions for 35 days, with 10 hours of light per day (see the results of 35 days of culture in the rooting medium). Figure 5 ).

[0064] The rooting medium formula is as follows: 1 / 2 MS + NAA 0.8 mg / L + KT 0.3 mg / L + sucrose 1.5% + hydrolyzed casein 100 mg / L + activated carbon 0.03% + agar 0.8%.

[0065] Finally, after the roots have grown, they are transplanted and raised into seedlings, which are then polyploid Peucedanum plants.

[0066] Furthermore, to determine the most suitable doubling liquid induction conditions, this embodiment employs a four-factor, three-level orthogonal experimental design, using induced shoot clusters as experimental material to investigate the effects of different concentrations of doubling liquid and their treatment times on chromosome doubling efficiency. The experimental design includes four factors: Factor A (concentration of doubling liquid I), Factor B (first treatment time), Factor C (concentration of doubling liquid II), and Factor D (second treatment time), with three levels for each factor. Using the ploidy identification method of this invention, the polyploidy rate and survival rate of each experimental group were statistically analyzed. The specific experimental design and results are shown in Table 1.

[0067] Table 1. Results of orthogonal experiments on polyploid induction of buds from the clustered shoots of *Peucedanum praeruptorum*

[0068]

[0069] Table 1 shows that colchicine concentration and treatment time significantly affect the survival rate and polyploid induction efficiency of *Peucedanum praeruptorum* shoot clusters. High concentrations of colchicine or prolonged treatment have a strong toxic effect on shoot clusters, significantly reducing their survival rate; while low concentrations of colchicine or short treatment times are insufficient to achieve effective polyploid induction. The experimental results indicate that two induction treatments using colchicine at appropriate concentration gradients can significantly improve polyploid induction efficiency while maximizing the survival rate of shoot clusters.

[0070] Example 2

[0071] This embodiment further verifies the polyploid Peucedanum praeruptorum obtained in Example 1, as follows:

[0072] When the seedlings reached 10cm in height, polyploid phenotypic screening was conducted. Plants exhibiting typical polyploid characteristics (such as larger and thicker leaves, deeper leaf color, and robust root systems) were selected. Temporary slides of the lower epidermis of the leaves were prepared using the epidermal peeling method, and stomatal morphology was observed under an optical microscope. Preliminary ploidy identification was performed by comparing stomatal size and other indicators with diploid controls (results are shown in Table 2). Figure 6 Furthermore, plants selected based on stomatal characteristics were analyzed using flow cytometry to determine their DNA content and accurately assess ploidy (results are shown in [link to results]). Figure 7 This identification process combines phenotypic observation with flow cytometry analysis to ensure the accuracy of ploidy identification results.

[0073] Table 2 Comparison of stomatal morphology in diploid and tetraploid Peucedanum praeruptorum.

[0074]

[0075] Macroscopic morphological analysis ( Figure 8 The results showed that polyploid plants exhibited typical characteristics such as enlarged leaves, darker leaf color, and robust root systems. Microscopic observation indicated that... Figure 6 Table 2: The stomatal length and width of polyploid *Angelica dahurica* leaves were significantly increased by 27.59% and 40.56% respectively compared with the diploid control (P<0.05), while the stomatal density was significantly decreased by 21.42% (P<0.05). DNA content detection results showed ( Figure 7 The DNA content in the leaves of the polyploid plant was twice that of the diploid wild-type plant, confirming that the chromosomes had been successfully doubled.

[0076] Based on the combined morphological observations, stomatal parameter analysis, and DNA content determination, the results of these three studies corroborated each other, confirming that the obtained polyploid Peucedanum plants possess stable tetraploid characteristics.

[0077] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for inducing polyploidy in Peucedanum praeruptorum using clustered buds, characterized in that, Includes the following steps: S1. Cut stem segments from the mother plant of Peucedanum praeruptorum, soak them in a mixed solution of citric acid and ascorbic acid, remove and wash them to obtain explants; S2. After disinfecting, cleaning, and drying the explants, cut off both ends and inoculate them into the shoot induction medium to obtain shoot clusters. S3. Cut off the induced shoot clusters and inoculate them into double solution I for culture. After culture, rinse with liquid shoot cluster induction medium, let stand to recover culture, and then transfer to double solution II for culture. The formula for the doubled solution I is: 0.05~0.1% colchicine + ascorbic acid 100mg / L; The formula for the doubled solution II is: 0.1~0.15% colchicine + ascorbic acid 100mg / L; The culture conditions for doubled solution I and doubled solution II are the same: shake culture in a shaker at a speed of 70~90 r / min and a temperature of 24±1℃ for 12~48 h; The static recovery culture time is 22-26 hours; S4. Remove the clustered buds, clean them, and inoculate them again into the clustered bud induction medium for recovery culture. After the culture is completed, inoculate them into the bud proliferation medium for culture. S5. When the clustered buds grow to 1-2cm, potential mutant plants are initially screened out by the characteristics of dark green leaves, larger and thicker leaves with larger serrations on the leaf margins and thicker stems. Potential mutant plants are cut off and inoculated into bud proliferation medium for subculture. After 3-4 rounds of continuous screening and proliferation culture, a population of polyploid mutant plants with stable traits can be obtained. S6. Place the mutant plant population in a rooting medium for cultivation. After the roots have grown, transplant them for seedling cultivation to obtain polyploid Peucedanum praeruptorum plants. Step S6 before transplanting also includes: when the seedlings grow to 10-12cm, peel off the epidermis to make a slide, place it under a microscope for observation, and preliminarily determine the polyploid Peucedanum based on the size and density of stomata. Then, perform flow cytometry analysis on the Peucedanum leaves after stomatal identification to determine the DNA content and accurately determine the ploidy. Formula for shoot induction medium: MS + NAA 0.4 mg / L + KT 1.5 mg / L + 2-ip 0.2 mg / L + ascorbic acid 100 mg / L + sucrose 3% + agar 0.8%; The culture conditions in step S2 are as follows: culture in the dark at 26±1℃ for 7~10 days, and then culture in alternating light and dark conditions at 1500~2000lx for 20~25 days, with 12h / 12h light / dark alternations per day; Shoot proliferation medium formula: MS + NAA 0.05 mg / L + KT 1.2 mg / L + 2-ip 0.1 mg / L + sucrose 3% + hydrolyzed casein 50 mg / L + agar 0.8%; In step S4, the bud proliferation culture conditions are as follows: cultured under alternating light and dark conditions at a temperature of 23±2℃ and a light intensity of 1500~2000lx for 30~40 days. The rooting medium formula in step S6 is: 1 / 2 MS + NAA 0.8 mg / L + KT 0.3 mg / L + sucrose 1.5% + hydrolyzed casein 50~150 mg / L + activated charcoal 0.03% + agar 0.8%; Rooting culture conditions: Culture for 30-40 days under alternating light and dark conditions at a temperature of 24±2℃ and a light intensity of 1500~2000lx, with 10 hours of light per day.

2. The method according to claim 1, characterized in that, In step S1, the mother plant of Peucedanum praeruptorum is protected from light for 46-50 hours before the stem segments are cut; the length of the stem segments is 1.5-2 cm; the concentration of citric acid in the mixed solution of citric acid and ascorbic acid is 300 mg / L and the concentration of ascorbic acid is 200 mg / L.

3. The method according to claim 1, characterized in that, The specific disinfection method in step S2 is as follows: place the explant in a 70% alcohol solution and shake to rinse for 12-18 seconds, then immediately rinse quickly in sterile water 3-5 times; then soak in a 1ppm ozone aqueous solution at 4°C for 3 minutes, and rinse with sterile water 2-3 times; finally, treat with a 0.05% (w / v) HgCl2 solution containing 0.1% (v / v) Tween-20 and shake for 4 minutes, and finally rinse with sterile water 3-5 times.

4. The method according to claim 1, characterized in that, In step S4, restore the culture conditions: incubate at 24±1℃ for 3~5 days.

5. The application of the method according to any one of claims 1-4 in obtaining polyploid Peucedanum praeruptorum plants.

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