Breeding method for improving quality of peucedanum praeruptorum dunn based on assistance of HPLC detection technology

Abstract

The invention discloses a breeding method for assisting in improving quality of peucedanum praeruptorum based on an HPLC (High Performance Liquid Chromatography) detection technology, and belongs to the technical field of improved variety breeding. The method comprises the following steps: screening from candidate materials to obtain an excellent group of which the yield and the content of methyl and ethyl elements (the contents of praeruptorin A and praeruptorin B, similarly hereinafter) both reach preset values; screening in the excellent population to obtain an excellent strain with fresh weight and methyl-ethyl element content reaching preset values; purifying and screening the progeny of the tissue culture seedling seeds to obtain a stable and excellent strain with fresh weight and methylethyl element content reaching preset values; and finally, carrying out tissue culture asexual propagation by taking the excellent strain as a material to produce tissue culture seedlings and breeding seeds. Compared with the prior art, the method has the advantages that quantitative auxiliary selection is carried out on key chemical components by utilizing HPLC (High Performance Liquid Chromatography) detection, and stable acquisition and rapid propagation of high-content strains are realized through a technical route of population optimization, strain optimization, multi-generation purification and tissue culture propagation, so that the quality of a radix peucedani medicinal material is improved, and the quality consistency is improved.

Description

Technical Field

[0001] This invention belongs to the field of improved variety breeding technology, specifically relating to a breeding method for improving the quality of Angelica dahurica based on HPLC detection technology. Background Technology

[0002] Peucedanum praeruptorum, a plant of the Apiaceae family, is known as white-flowered Peucedanum praeruptorum. Peucedanum prerupterum The dried root of *Peucedanum praeruptorum* (Dunhu) has the effects of lowering qi and resolving phlegm, dispelling wind and clearing heat. It is mainly used for phlegm-heat wheezing, slightly yellow and thick phlegm, and wind-heat cough with excessive phlegm. It is one of the commonly used Chinese medicinal materials. *Peucedanum praeruptorum* has a wide distribution area. In addition to the main producing areas of Zhejiang, Anhui, Hunan, and Sichuan, Guangxi, Jiangsu, Hubei, and Jiangxi also have high yields. Zhejiang has the highest yield and the best quality, with its products sold throughout the country and exported. Traditionally, white-flowered *Peucedanum praeruptorum* is divided into two authentic varieties: those produced in Zhejiang and Anhui are commonly called Ningqianhu; those produced in Guizhou, Hunan, Hubei, and Sichuan are commonly called Xinqianhu. Currently, Ningqianhu is mainly cultivated, accounting for about 80% of the national *Peucedanum praeruptorum*; Xinqianhu is mainly wild, accounting for about 20% of the national total. The domestication of wild *Peucedanum praeruptorum* has a history of more than 20 years, with Chun'an, Lin'an, and Pan'an in Zhejiang Province being the main producing areas. Originally, Chun'an County had an annual planting area of ​​about 10,000 mu (approximately 667 hectares) and a yield of 1,500 tons. With the increasing demand for Peucedanum praeruptorum (Qianhu) medicinal materials, the area under artificial cultivation has expanded rapidly. Cultivated Peucedanum praeruptorum is generally propagated by seed, with a cultivation period of one year. In the second year of artificial cultivation, almost all plants flower but are no longer medicinally viable, and there is significant field variation, unstable traits, and a high rate of quality defects, particularly in the content of glycoside B in white-flowered Peucedanum praeruptorum. Due to the relatively short cultivation period, research on the genetic characteristics, superior variety selection, and propagation techniques of Peucedanum praeruptorum has not yet been conducted. In production, farmers either save their own seeds or collect wild seeds to propagate seedlings for transplanting. Because seed quality is difficult to control, there are significant differences in the yield and quality of Peucedanum praeruptorum medicinal materials. The inconsistent quality of Peucedanum praeruptorum seeds and the low rate of qualified quality medicinal materials negatively impact the price of Peucedanum praeruptorum and the healthy development of the Peucedanum praeruptorum industry. Summary of the Invention

[0003] The purpose of this invention is to overcome the deficiencies in the existing technology and provide a rapid and efficient breeding method for improving the quality of Peucedanum praeruptorum based on HPLC (high performance liquid chromatography) detection technology. This invention involves the breeding of new Peucedanum praeruptorum strains, with the primary objective of improving the quality of the medicinal material, particularly increasing the content of purpurin B in white-flowered Peucedanum praeruptorum. Through collecting germplasm resources, conducting comparative experiments, and screening superior germplasm resources, and after years of systematic breeding, the content of purpurin A and purpurin B in white-flowered Peucedanum praeruptorum has been increased.

[0004] The specific technical solution adopted in this invention is as follows: This invention provides a breeding method for improving the quality of Angelica dahurica based on HPLC detection technology, as detailed below: S1: Collect Peucedanum germplasm resources, establish a Peucedanum germplasm resource nursery, conduct Peucedanum variety comparison trials, and screen out superior populations whose yield, Peucedanum A content, and Peucedanum B content all reach the preset values. S2: Select the first superior strain from the superior population described in S1, where the single plant fresh weight, white flower angelica A content and white flower angelica B content all reach the preset values. S3: Using the first superior strain from S2 as propagation material, perform tissue culture asexual propagation to produce tissue culture seedlings; transplant the obtained tissue culture seedlings to propagate seeds, then sow the seeds, and perform selection work. Several purifications are carried out in the progeny of the tissue culture seedling seeds to screen out the second superior strain whose single plant fresh weight, white peucedanin A content, and white peucedanin B content all reach the preset values; then use the second superior strain as propagation material to perform tissue culture propagation to produce tissue culture seedlings.

[0005] Preferably, in step S1, half of the remaining underground fresh roots after the above-ground parts are cut off are used as propagation material, and the other half are used as the Peucedanum praeruptorum sample for HPLC content detection. The Peucedanum praeruptorum sample is dried at 50°C and then subjected to HPLC content detection. The detected contents of Peucedanum praeruptorum A and Peucedanum praeruptorum B are used as screening indicators.

[0006] Preferably, in S1 and S2, the contents of angelica dahurica A and angelica dahurica B are detected according to the HPLC detection method for angelica dahurica in the 2025 edition of the Chinese Pharmacopoeia.

[0007] Preferably, in S1, the preset value of the content of angelica dahurica A is not less than 0.90%, and the preset value of the content of angelica dahurica B is not less than 0.24%.

[0008] Preferably, in step S2, the preset value of the content of angelica dahurica A is not less than 1.0%, and the preset value of the content of angelica dahurica B is not less than 0.30%.

[0009] Preferably, in step S3, the purification is performed at least twice.

[0010] Preferably, in S3, the method for tissue culture propagation and production includes: asexually propagating tissue culture seedlings through tissue culture, transplanting the tissue culture seedlings to produce seeds, and using these seeds to produce Angelica dahurica medicinal materials.

[0011] Preferably, in step S3, the method for producing tissue culture seedlings through asexual propagation via tissue culture is as follows: Explants were rinsed thoroughly with sterile water and then soaked in 0.1% mercuric chloride solution for 6 minutes for disinfection. They were then rinsed repeatedly with sterile water five times to remove any residue. The callus induction medium consisted of 1 / 2 MS, 1 mg / L 2,4-D, 20 g / L sucrose, and 0.5 g / L activated charcoal for 20-30 days. Subsequently, differentiation was mediated using 1 / 2 MS, 1.0 mg / L 6-BA, 0.5 mg / L NAA, and 20 g / L sucrose for 30 days. Finally, rooting was mediated using 1 / 2 MS, 0.5 mg / L NAA, 0.2 mg / L 6-BA, and 20 g / L sucrose for 60 days. When the Peucedanum praeruptorum tissue culture seedlings reached a height of 4 cm or more and a root length of 3 cm or more, robust seedlings with good root systems and uniform height were selected for hardening-off for 2 days. The culture temperature was 25℃ and the relative humidity was 40%. 60%, light duration of 12 h / d, light intensity of 1500 ~ 2000 Lx; after hardening off, transplant into a substrate of humus:vermiculite = 3:1, keep moist, and provide shade to maintain 70% light transmittance.

[0012] Preferably, in step S3, the preset value of the content of angelica dahurica A is not less than 1.40%, and the preset value of the content of angelica dahurica B is not less than 0.40%.

[0013] Compared with the prior art, the present invention has the following advantages: This invention utilizes HPLC-assisted technology to select a population of *Peucedanum praeruptorum* with high contents of both kaempferol A and kaempferol B. Then, within this high-content population, strains with high chemical component content are selected. After two generations of purification, seeds are produced using these high-content chemical component strains through tissue culture asexual propagation, thereby increasing the medicinal content of *Peucedanum praeruptorum*. Detailed Implementation

[0014] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention are described in detail below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The technical features in the various embodiments of the present invention can be combined accordingly without conflict.

[0015] This invention provides a breeding method for improving the quality of Peucedanum praeruptorum based on HPLC detection technology, offering a rapid and efficient new method for breeding new varieties of Peucedanum praeruptorum. The specific steps of this breeding method are as follows: S1: Collect Peucedanum germplasm resources, establish a Peucedanum germplasm resource nursery, conduct Peucedanum variety comparison trials, and screen out superior populations whose yield, Peucedanum A content, and Peucedanum B content all reach the preset values.

[0016] In a preferred embodiment of the present invention, in this step, half of the underground fresh root after the above-ground parts are cut off is used as propagation material, and the other half is used as the Peucedanum praeruptorum sample for HPLC content detection; the obtained Peucedanum praeruptorum sample is dried at 50°C, and then HPLC content detection is performed, and the content of white peucedanum praeruptorum A and white peucedanum praeruptorum B is used as screening indicators.

[0017] In a preferred embodiment of the present invention, the contents of angelica dahurica A and angelica dahurica B in this step are detected according to the HPLC detection method for angelica dahurica in the 2025 edition of the Chinese Pharmacopoeia.

[0018] In a preferred embodiment of the present invention, in this step, the preset value of the content of angelica dahurica A is not less than 0.90%, and the preset value of the content of angelica dahurica B is not less than 0.24%.

[0019] S2: Select the first superior strain from the superior population described in S1, where the single plant fresh weight, content of white angelica A, and content of white angelica B all reach the preset values.

[0020] In a preferred embodiment of the present invention, the contents of angelica sinensis A and angelica sinensis B in this step are measured according to the HPLC detection method for angelica sinensis in the 2025 edition of the Chinese Pharmacopoeia.

[0021] In a preferred embodiment of the present invention, in this step, the preset value of the content of angelica dahurica A is not less than 1.0%, and the preset value of the content of angelica dahurica B is not less than 0.30%.

[0022] S3: Using the first superior strain from S2 as propagation material, perform tissue culture asexual propagation to produce tissue culture seedlings; transplant the obtained tissue culture seedlings to propagate seeds, then sow the seeds, and perform selection work. Purify the tissue culture seedlings several times (at least twice) in the progeny of the seeds to screen out the second superior strain whose single plant fresh weight, white peucedanin A content, and white peucedanin B content all reach the preset values; then use the second superior strain as propagation material to perform tissue culture propagation to produce tissue culture seedlings.

[0023] In a preferred embodiment of the present invention, the tissue culture propagation production method in this step includes: asexual propagation of tissue culture seedlings through tissue culture, and / or transplanting tissue culture seedlings to propagate seeds and using these seeds to produce Peucedanum praeruptorum medicinal materials.

[0024] In a preferred embodiment of the present invention, the method for producing tissue culture seedlings by asexual propagation in this step is as follows: Explants were rinsed thoroughly with sterile water and then soaked in 0.1% mercuric chloride solution for 6 minutes for disinfection. They were then rinsed repeatedly with sterile water five times to remove any residue. The callus induction medium consisted of 1 / 2 MS, 1 mg / L 2,4-D, 20 g / L sucrose, and 0.5 g / L activated charcoal for 20-30 days. Subsequently, differentiation was mediated using 1 / 2 MS, 1.0 mg / L 6-BA, 0.5 mg / L NAA, and 20 g / L sucrose for 30 days. Finally, rooting was mediated using 1 / 2 MS, 0.5 mg / L NAA, 0.2 mg / L 6-BA, and 20 g / L sucrose for 60 days. When the Peucedanum praeruptorum tissue culture seedlings reached a height of 4 cm or more and a root length of 3 cm or more, robust seedlings with good root systems and uniform height were selected for hardening-off for 2 days. The culture temperature was 25℃ and the relative humidity was 40%. 60%, light duration of 12 h / d, light intensity of 1500 ~ 2000 Lx; after hardening off, transplant into a substrate of humus:vermiculite = 3:1, keep moist, and provide shade to maintain 70% light transmittance.

[0025] In a preferred embodiment of the present invention, in this step, the preset value of the content of white peucedanum praeruptorum A is not less than 1.40%, and the preset value of the content of white peucedanum praeruptorum B is not less than 0.40%.

[0026] The methods and effects of the present invention will be specifically illustrated below through examples.

[0027] Example This embodiment provides a breeding method for improving the quality of Peucedanum praeruptorum based on HPLC detection technology. The method selects the No. 9 germplasm resource group from 16 Peucedanum praeruptorum germplasm resource groups, which has better yield, content of white peucedanin A and white peucedanin B. Then, combined with the fresh weight of single plants and the content of chemical components, the 9-43 strain with better fresh weight of single plants, content of white peucedanin A and white peucedanin B is screened from the No. 9 group. The 9-43 strain was used as propagation material to cultivate tissue culture seedlings. These seedlings were then transplanted for seed production, and the seeds were harvested. After sowing, based on individual plant fresh weight and chemical composition, the 9-43-52 strain was selected from the 9-43 progeny population. This 9-43-52 strain had superior individual plant fresh weight, content of white peucedanin A, and content of white peucedanin B. This 9-43-52 strain was then used as propagation material to cultivate tissue culture seedlings. These seedlings were then transplanted for seed production. Subsequently, the 9-43-52 tissue culture seedlings were propagated annually. The cultivated 9-43-52 seedlings were then transplanted for seed production, and the harvested seeds were used as seeds for the production of new peucedanum medicinal materials. Details are as follows: 1. Germplasm resource collection and variety comparison trials Table 1 Germplasm resources of Peucedanum praeruptorum 1.1 Materials and Methods In 2016, germplasm resources of Peucedanum praeruptorum (No. 1-16) from 16 different locations in Zhejiang and Anhui provinces were collected. The sources of the germplasm resources are shown in Table 1. The collected Peucedanum praeruptorum germplasm resources were used to establish a Peucedanum praeruptorum germplasm resource nursery in Fengshuling Town, Chun'an County, and comparative trials of Peucedanum praeruptorum germplasm resources from different locations were carried out.

[0028] Sampling method: Samples for yield testing were randomly collected from each plot, with a sampling depth of 5m. 2 The contents of angelica root extract A, angelica root B and angelica root C were determined by randomly selecting 20 individual plants from each germplasm resource.

[0029] Yield assessment: Cut off the above-ground parts, dry at 50℃, calculate the yield per acre of dried product, and compare yields.

[0030] Pre-processing method: Cut off the above-ground parts, dry the medicinal materials at 50℃, grind them into powder and pass them through a No. 3 sieve, and store them at low temperature in a desiccator for later use.

[0031] Chemical composition detection method: According to the detection method of angelica root in Part I of the 2025 edition of the Chinese Pharmacopoeia, the contents of angelica A, angelica B and angelica C in different germplasm resources were determined.

[0032] 1.2 Results and Analysis Table 2. Statistical analysis of average yield and chemical composition content among various mineral resources. Table 3. Coefficients of variation of yield and chemical composition content of various mineral resources Yield traits are an important economic indicator for the production of Angelica dahurica. As can be seen from Tables 2 and 3, there are some differences in the yield per mu of dried product among various germplasm resources. Yield analysis shows that the yields of No. 3 and No. 9 are significantly higher than those of other germplasm resources, with yields of over 160 kg per mu. The intraspecific yield variability of various germplasm resources is relatively small.

[0033] Quality characteristics are the most important aspects to consider when selecting Peucedanum praeruptorum (Qianhu) medicinal materials. The primary goal of this stage of breeding is to select varieties that meet the requirements of the 2025 edition of the Chinese Pharmacopoeia for both white-flowered peucedanin A and white-flowered peucedanin B. The 2025 edition of the Chinese Pharmacopoeia stipulates that the content of white-flowered peucedanin A in Peucedanum praeruptorum medicinal materials should not be less than 0.90%, and the content of white-flowered peucedanin B should not be less than 0.24%. As can be seen from Tables 2 and 3, for white-flowered peucedanin A: except for Peucedanum praeruptorum A in variety No. 7... Aside from the substandard content of certain purslane compounds, the content of purslane A in other germplasm resources met the pharmacopoeia standard, with a pass rate of 94%. Germplasm No. 14 had the highest purslane A content at 1.595%. For purslane B, only germplasm resources No. 7 and No. 9 met the pharmacopoeia standard, with a pass rate of 13%. Germplasm No. 7 had the highest purslane B content at 0.377%. For purslane C, Germplasm No. 12 had the highest C content at 0.318%. The main reason for the substandard quality of the purslane medicinal material was the substandard content of purslane B, while the pass rate for purslane A was relatively high. As shown in Table 3, the variation in the content of white-flowered angelica ethyl glycosides was the largest among the various angelica germplasm resources, reaching 91%, indicating that the content of white-flowered angelica ethyl glycosides varied greatly among the different strains within the population; the variation in white-flowered angelica propyl glycosides was the second largest, at 60%; and the variation in white-flowered angelica alpha glycosides was the smallest, at 40%, indicating that the content of white-flowered angelica alpha glycosides showed relatively small variation and good stability.

[0034] Based on the results of comparative experiments on different germplasm resources of Peucedanum praeruptorum, there are significant differences in yield and chemical composition content among and within different Peucedanum praeruptorum germplasm resources, especially in the content of white-flowered Peucedanum praeruptorum B. This provides breeding materials for the selection of new Peucedanum praeruptorum varieties. Combining the yield of Peucedanum praeruptorum, the content of white-flowered Peucedanum praeruptorum A and B in Peucedanum praeruptorum A and B in Peucedanum praeruptorum A and B in Peucedanum praeruptorum germplasm resources from Shuangxikou Village, Shuangxikou Township, Jinyun County, meets the requirements of the Chinese Pharmacopoeia and has a relatively high yield. Therefore, Peucedanum praeruptorum ...

[0035] 2. Screening of superior strains, rapid propagation through tissue culture, and stable system 2.1 Screening of superior strains in population No. 9 2.1.1 Materials and Methods In 2016, 100 individual plants from group 9 were collected. After each plant was numbered, the lower half of the rhizome was used for content determination, while the upper half was numbered and preserved. Fresh rhizomes were dried at 50℃ to constant weight, and the content of purslane A and purslane B was determined according to the 2025 edition of the Chinese Pharmacopoeia. Superior strains with high contents of both purslane A and purslane B were selected.

[0036] 2.1.2 Results and Analysis The results of the fresh weight and content of purpureusin A and purpureusin B of the strains are shown in Table 4. As can be seen from Table 4, there are significant differences in fresh weight and chemical composition content among different strains. The pass rate for purpureusin A content is 64%, the pass rate for purpureusin B content is 55%, and the proportion of strains with both purpureusin A and purpureusin B content that are qualified is 20%. Strains 9-21, 9-39, and 9-43, which have relatively high levels of both purpureusin A and purpureusin B, were selected as backup materials. Based on the fresh weight of individual plants, 9-43 was selected as the superior strain for tissue culture propagation.

[0037] Table 4. Content and fresh weight of peucedanin A and peucedanin B in 100 individual plants from population 9 2.2 Tissue Culture Propagation 2.2.1 Materials and Methods Tissue culture material: The explants were tender shoots from the tip of *Peucedanum praeruptorum*. Stock solutions (macroelements, microelements, organic components, and iron salts) used to prepare MS medium were all prepared in the laboratory. Plant growth regulators: 6-benzyladenine (6-BA), kinetin (KT), α-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and thidiazuron (TDZ) were all products of Sigma-Aldrich (USA); sucrose, agar, anhydrous ethanol, Tween-20, and other experimental reagents were all purchased from Sinopharm Chemical Reagent Co., Ltd.

[0038] Tissue culture methods: Explants were rinsed thoroughly with sterile water and soaked in 0.1% mercuric chloride solution for 6 minutes for disinfection. They were then rinsed repeatedly with sterile water 5 times to remove any residue. The callus induction medium was 1 / 2 MS + 1 mg / L 2,4-D + 20 g / L sucrose + 0.5 g / L activated charcoal for 20-30 days. Differentiation medium was 1 / 2 MS + 1.0 mg / L 6-BA + 0.5 mg / L NAA + 20 g / L sucrose for 30 days. Rooting medium was 1 / 2 MS + 0.5 mg / L NAA + 0.2 mg / L 6-BA + 20 g / L sucrose for 60 days. When the Peucedanum praeruptorum tissue culture seedlings reached a height of 4 cm or more and a root length of 3 cm or more, robust seedlings with good root systems and uniform height were selected for hardening off for 2 days. The cultivation conditions were as follows: the temperature in the cultivation room was 25℃, the relative humidity was maintained at 40%~60%, the light duration was 12 h / d, and the light intensity was 1500~2000 Lx. After hardening off, the seedlings were transplanted into a substrate of humus:vermiculite = 3:1 at the appropriate time, kept moist, and shaded to maintain 70% light transmittance.

[0039] In 2016, the dominant strain 9-43 from population 9 was collected as a single plant material for tissue culture propagation.

[0040] 2.2.2 Results and Analysis Tissue culture experiment results: Under the above propagation system, the propagation ratio of individual plants of Peucedanum praeruptorum was between 1:10 and 1:30.

[0041] 2.3 Screening and rapid propagation of superior strains in population 9-43 2.3.1 Materials and Methods In the first half of 2018, tissue culture seedlings of size 9-43 were planted individually, and seeds were harvested at the end of 2018. Seeds were sown in 2019, and at the end of 2019, 100 well-grown lines were harvested, numbered, and the lower half of the rhizome of each line was numbered for content determination, while the upper half was numbered and preserved for a second round of purification.

[0042] 2.3.2 Methods and Analysis Table 5 shows the content of purpuricin A, purpuricin B, and fresh weight of 100 strains in population 9-43. The average content of purpuricin A in 100 strains in population 9-43 was 1.24%, the average content of purpuricin B was 0.31%, and the fresh weight of a single strain was 31g. All of these were better than the average content of purpuricin A, purpuricin B, and fresh weight of 100 strains in population 9. The RSD of purpuricin A, purpuricin B, and fresh weight also decreased from 34.29%, 69.08%, and 28.00% to 23.91%, 29.40%, and 23.92%, respectively. The consistency of purpuricin A, purpuricin B, and fresh weight was improved, especially the consistency of purpuricin B, which was greatly improved, thus improving the quality stability of the purpuricin medicinal material. From population 9-43, a strain 9-43-52 was obtained, exhibiting high yield per plant and excellent levels of angelica dahurica A and B. This individual strain was then further propagated through tissue culture. In early 2020, the tissue-cultured seedlings were planted individually, and seeds were harvested at the end of 2020. Population quality evaluation and seed propagation were conducted in 2021.

[0043] Table 5. Content and fresh weight of peucedanin A and peucedanin B in 100 strains of population 9-43 From 16 germplasm resources from different origins, germplasm resource No. 9 was selected, with qualified contents of both angelica A and angelica B. From germplasm resource No. 9, 100 lines were selected for content testing and single-plant fresh weight measurement. Based on single-plant yield, angelica A and angelica B content, the superior line 9-43 was selected. Then, the superior single plants of 9-43 were propagated through tissue culture, and the tissue-cultured seedlings of 9-43 were planted individually, seeds were harvested, and purified. After multiple generations of purification, a line 9-43-52 with high single-plant fresh weight and excellent angelica A and angelica B content was obtained. Single plants of 9-43 were propagated through tissue culture, and the tissue-cultured seedlings were planted individually in early 2020, with seeds harvested at the end of 2020. Population quality evaluation and seed propagation were conducted in 2021. A new angelica dahurica line was developed.

[0044] 3. Comparison Test of Different Areas 3.1 Materials and Methods Purified seeds were selected for a plot-based variety comparison trial. The trial was conducted in mountainous areas of Fengshuling Town, Chun'an County. The comparison materials were the new variety, the original population No. 9, and a local variety from Linqi Town, Chun'an (control). Each plot had a trial area of ​​20m² for each variety. 2 Repeat the planting process three times, with protective rows around the perimeter. The beds are 1.2m wide, with shallow furrows spaced 25-30cm apart. Sow seeds in rows, moistening them with warm water before sowing, then mixing them with organic fertilizer and fine soil (1:1:1). Scatter the seeds evenly in the furrows, then lightly sweep and compact the soil. Sowing time was February 6, 2021. Seedlings emerged approximately 55 days after sowing. Thin the seedlings as needed, maintaining a planting density of approximately 8000 plants per mu (approximately 667 square meters). Fertilization methods included applying 500kg of organic fertilizer per mu as base fertilizer during land preparation before planting, and applying 200kg of organic fertilizer per mu as top dressing on August 28th. Daily management followed the Green Production Technical Regulations for Angelica dahurica (DB 33 / T 2280-2020). Record phenological stages such as sowing, emergence, and harvesting.

[0045] On December 5, 2021, the variety was tested. A 5㎡ area was randomly selected from each plot, and the yield of all individual plants within the harvested area was determined for the corresponding strain, with three replicates. The rhizomes of 12 randomly selected individual plants were dried at 50℃ until constant weight after removing the rhizome heads. The content of angelica dahurica A and angelica dahurica B was determined according to the 2025 edition of the Chinese Pharmacopoeia.

[0046] 3.2 Results and Analysis Table 6 Phenological Period Survey Table 7. Comparison of phenotypic characteristics of different Angelica dahurica germplasm resources (2021, n=12) Table 8. Comparison of Yield and Quality in the 2020 Plot Variety Comparison Test Table 9. Variation of species in the 2020 local variety comparison test Phenological stages and growth characteristics: The phenological survey results showed that the new strain, the original population, and the Chun'an local variety were basically the same in terms of emergence period, morphology, uniformity, and harvest period. The new strain was slightly shorter than the Chun'an local variety in plant height, but its root length was similar to that of the Chun'an local variety. The average fresh weight of a single plant was slightly heavier than that of the Chun'an local variety, and the average diameter at root was slightly smaller than that of the Chun'an local variety.

[0047] Growth period: The sowing date for the variety comparison test was uniformly set as February 6, 2021. The emergence period of the Chun'an local variety was 2 days later than that of the new line and the original population. The harvest period was uniformly set as December 5. The entire growth period of the new line, the original population and the Chun'an local variety was the same, which was 303 days.

[0048] Resistance characteristics: No serious pests or diseases occurred during the variety comparison trials. The new strain, the original population, and the Chun'an local variety all exhibited good resistance.

[0049] Agronomic traits: The survey results showed that the developmental characteristics, plant morphology, and leaf traits observed in the comparative experiment of Angelica dahurica were basically consistent. The results showed that there were some differences in plant height and root type among the new strain, the original population, and the Chun'an local variety. The Chun'an local variety was slightly taller than the new strain and the original population, while the root length of the original population was slightly longer than that of the new strain and the Chun'an local variety. The new strain had the heaviest average fresh root weight per plant, while the original population had a lighter average fresh root weight per plant. The Chun'an local variety had the thickest average diameter at root, while the original population had the thinnest average diameter at root.

[0050] Yield: The new strain yielded 173 kg per mu, which is 4.01% higher than the original population and 11.46% higher than the local variety from Chun'an.

[0051] Key components: Peucedanum praeruptorum glycoside A: The new strain contains 1.36% peucedanum praeruptorum glycoside A, which is 5.43% higher than the original population and 7.09% higher than the Chun'an local variety; Peucedanum praeruptorum glycoside B: The new strain contains 0.31% peucedanum praeruptorum glycoside A, which is 63.16% higher than the original population and 181.82% higher than the Chun'an local variety.

[0052] After systematic breeding, the new cultivar of Peucedanum praeruptorum has shown more stable agronomic traits, yield, and chemical composition compared to the original population. The yield and index component content have been improved compared to the original population and the Chun'an local variety (control). The yield per mu of the new cultivar is 11.46% higher than that of the Chun'an local variety. The contents of white-flowered Peucedanum praeruptorum A and B are higher than those of the original population and the Chun'an local variety. In particular, the content of white-flowered Peucedanum praeruptorum B is 63.16% higher than that of the original population and 181.82% higher than that of the Chun'an local variety. The contents of white-flowered Peucedanum praeruptorum A and B meet the requirements of the Chinese Pharmacopoeia.

[0053] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.

Claims

1. A breeding method for improving the quality of Angelica dahurica based on HPLC detection technology, characterized in that, Specifically as follows: S1: Collect Peucedanum germplasm resources, establish a Peucedanum germplasm resource nursery, conduct Peucedanum variety comparison trials, and screen out superior populations whose yield, Peucedanum A content, and Peucedanum B content all reach the preset values. S2: Select the first superior strain from the superior population described in S1, where the single plant fresh weight, white flower angelica A content and white flower angelica B content all reach the preset values. S3: Using the first superior strain from S2 as propagation material, perform tissue culture asexual propagation to produce tissue culture seedlings; transplant the obtained tissue culture seedlings to propagate seeds, then sow the seeds, and perform selection work. Purify the progeny of the tissue culture seedling seeds again to screen out the second superior strain with the same fresh weight, white peucedanin A content, and white peucedanin B content reaching the preset values; then use the second superior strain as propagation material to perform tissue culture propagation to produce tissue culture seedlings.

2. The breeding method for improving the quality of Angelica dahurica based on HPLC detection technology according to claim 1, characterized in that, In step S1, half of the underground fresh roots after the above-ground parts are cut off are used as propagation material, and the other half are used as samples for HPLC content detection of Peucedanum praeruptorum. The Peucedanum praeruptorum samples are dried at 50°C and then subjected to HPLC content detection. The detected contents of Peucedanum praeruptorum A and Peucedanum praeruptorum B are used as screening indicators.

3. The breeding method for improving the quality of Peucedanum praeruptorum based on HPLC detection technology according to claim 1, characterized in that, In S1 and S2, the contents of angelica dahurica A and angelica dahurica B were determined by HPLC according to the detection method for angelica dahurica medicinal materials in the 2025 edition of the Chinese Pharmacopoeia.

4. The breeding method for improving the quality of Angelica dahurica based on HPLC detection technology according to claim 1, characterized in that, In S1, the preset value of the content of angelica dahurica A is not less than 0.90%, and the preset value of the content of angelica dahurica B is not less than 0.24%.

5. The breeding method for improving the quality of Peucedanum praeruptorum based on HPLC detection technology according to claim 1, characterized in that, In S2, the preset value of the content of angelica dahurica A is not less than 1.0%, and the preset value of the content of angelica dahurica B is not less than 0.30%.

6. The breeding method for improving the quality of Peucedanum praeruptorum based on HPLC detection technology according to claim 1, characterized in that, In step S3, the purification process is performed at least twice.

7. The breeding method for improving the quality of Angelica dahurica based on HPLC detection technology according to claim 1, characterized in that, In S3, the method of tissue culture propagation and production includes: asexually propagating tissue culture seedlings through tissue culture, transplanting the tissue culture seedlings to produce seeds, and using these seeds to produce Angelica dahurica medicinal materials.

8. The breeding method for improving the quality of Peucedanum praeruptorum based on HPLC detection technology according to claim 1, characterized in that, In S3, the method for producing tissue culture seedlings through asexual propagation via tissue culture is as follows: Explants were rinsed thoroughly with sterile water and then soaked in 0.1% mercuric chloride solution for 6 minutes for disinfection. They were then rinsed repeatedly with sterile water five times to remove any residue. Callus induction was performed using 1 / 2 MS medium, 1 mg / L 2,4-D, 20 g / L sucrose, and 0.5 g / L activated charcoal for 20-30 days. Subsequently, differentiation was mediated using 1 / 2 MS medium, 1.0 mg / L 6-BA, 0.5 mg / L NAA, and 20 g / L sucrose for 30 days. Finally, rooting was performed using 1 / 2 MS medium, 0.5 mg / L NAA, 0.2 mg / L 6-BA, and 20 g / L sucrose for 60 days. When the Peucedanum praeruptorum tissue culture seedlings reached a height of 4 cm or more and a root length of 3 cm or more, robust seedlings with good root systems and uniform height were selected for hardening-off for 2 days. The culture temperature was 25℃ and the relative humidity was 40%. 60%, light duration of 12 h / d, light intensity of 1500 ~2000 Lx; after hardening off, transplant into a substrate of humus:vermiculite = 3:1, keep moist, and provide shade to maintain 70% light transmittance.

9. The breeding method for improving the quality of Peucedanum praeruptorum based on HPLC detection technology according to claim 1, characterized in that, In S3, the preset value of the content of angelica dahurica A is not less than 1.40%, and the preset value of the content of angelica dahurica B is not less than 0.40%.

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