In-vitro rapid propagation bud cultivation method for bupleurum yutai

Abstract

The invention discloses an in-vitro rapid propagation bud cultivation method for bupleurum yutai, and belongs to the field of traditional Chinese medicinal material plant breeding. The method is realized by five steps of explant surface sterilization, cluster bud induction, proliferation and subculture, rooting and seedling hardening. Selecting a stem base bud as an explant, and sterilizing with 75% alcohol and mercury bichloride; different combinations and proportions of hormones such as 6-BA, IBA, NAA and ZT are utilized to improve the propagation coefficient of buds and realize rapid rooting of tissue culture seedlings, and seedling hardening is performed through peat and perlite (2: 1). According to the method, an efficient vegetative propagation system is established, the method is not limited by natural environment, cluster buds can be directly induced, the production period is shortened, the production cost is reduced, the plant inheritable character is stable, and the excellent quality and characteristics of stock plants are reserved. The method can rapidly breed a large number of aseptic seedlings, solves the difficult propagation problem of bupleurum yutai, meets the market demand, and has high application value.

Description

Technical Field

[0001] This invention belongs to the field of propagation and cultivation of Chinese medicinal plants, and specifically relates to a method for rapid in vitro propagation of young shoots of Bupleurum chinense from Yantai. This method can establish an efficient asexual propagation system for Bupleurum chinense from Yantai, providing technical support for meeting market demand for Bupleurum chinense from Yantai. Background Technology

[0002] *Bupleurum chinese* DC.f.vanheuckii (Muell.-Arg.) ShanetY.Li., also known as Qunhu, is a variety of *Bupleurum chinense*. Its main production area is Yantai, Shandong Province, and it is widely distributed in areas such as Yashan, Aishan, and Kunyushan in Yantai City. It grows primarily on grassy mountain slopes at altitudes of 200-950 meters, making it a unique medicinal herb species of Shandong Province. Small quantities are also found in other parts of China, such as Jilin, Shanxi, Liaoning, and Jiangsu. The entire *Bupleurum chinense* plant can be used medicinally. The root is cylindrical or oblong-conical, branched, and its surface color ranges from brownish-brown to dark brown, with longitudinal wrinkles and protruding transverse or dotted lenticels. It is fragile and easily broken, with a relatively flat cross-section, yellowish-white in color, and has a slightly fragrant odor and mild taste. Furthermore, the stems and leaves of *Bupleurum chinense* have been used for many years as a raw material for the pediatric pneumonia preparation, Chaitong tablets, with proven efficacy.

[0003] Currently, due to over-harvesting, the wild resources of *Bupleurum yantaiense* have suffered severe damage, and germplasm resources are becoming increasingly scarce. This makes it impossible to establish a complete cultivation system. However, as an important raw material for *Bupleurum yantaiense* tablets, the market demand for *Bupleurum yantaiense* is increasing rapidly. Although tissue culture technology has been widely used in many rare and endangered medicinal plants in recent years, there are still no reports on tissue culture of *Bupleurum yantaiense*. Therefore, it is urgent to construct an asexual reproduction system for *Bupleurum yantaiense*, which is of vital importance to meeting market demand and improving the existing inefficient and poorly systematic cultivation techniques. Summary of the Invention

[0004] In view of the above situation and to overcome the shortcomings of the existing technology, this invention aims to propose a method for the in vitro rapid propagation of young shoots of Bupleurum chinense by constructing and optimizing a scientific and reasonable asexual propagation system for Bupleurum chinense, so as to solve the current difficulties in the propagation of Bupleurum chinense, realize its rapid propagation, and meet the market demand for Bupleurum chinense.

[0005] A method for rapid in vitro propagation of Bupleurum chinense seedlings from Yantai is achieved through the following steps: 1) Surface sterilization of explants: Select young buds at the base of Bupleurum chinense stems as explants. First, rinse with clean water for 2 hours to ensure that surface impurities are fully removed. Then, place the cleaned explants on a sterile operating table and disinfect the surface with 75% alcohol for 30 seconds. Rinse with sterile water 3-5 times to remove residual alcohol. After that, disinfect the surface with 0.1% HgCl2 (mercuric chloride) for 5-15 minutes and rinse with sterile water 5-7 times to thoroughly remove residual HgCl2. 2) Induction of shoot clusters: Explants of young shoots from the base of *Bupleurum chinense* stems, after surface sterilization, were carefully cleaned of the attached root bark and stem bark on a sterile operating table, and the outermost leaves were peeled off. The treated explants were then placed in a high-temperature sterilized shoot cluster induction medium. Each bottle contained 30 ml of medium and 4 *Bupleurum chinense* stem base explants. The culture bottles were placed in a culture room at a temperature of 20-26℃, a light intensity of 10000 lx, and a photoperiod of 12 h / d for 14 days to induce shoot clusters. The shoot cluster induction medium consisted of 1.0 mg 6-BA, 0.2 mg IBA, 0.5 mg ZT, 0.2 mg NAA, 30 g sucrose, and 6 g agar per 1000 ml of 1 / 2 MS medium, and the pH was adjusted to 5.7-5.9. 3) Proliferation and subculturing of clustered shoots: Explant shoot blocks (tissue chimeras) induced with clustered shoots were carefully transferred into sterilized proliferation medium. Each bottle of medium had a volume of 30 ml, and each bottle contained 4 clustered shoot blocks. The culture was carried out in a culture room at a temperature of 20-26℃, a light intensity of 10000 lx, and a light duration of 12 h / d for 15 days to produce subcultured clustered shoots. The proliferation medium formula was as follows: 0.5-2.0 mg of 6-BA, 0.1-0.5 mg of NAA, 30 g of sucrose, and 6 g of agar were added to every 1000 ml of MS medium, and the pH was adjusted to 5.8-6.0. 4) Rooting of subcultured shoot clusters: Subcultured shoot clusters were inoculated into rooting medium for shoot clusters. Each bottle contained 30 ml of medium and 4 shoot clusters. The culture was carried out in a culture room at a controlled temperature of 20-26℃, a light intensity of 10000 lx, and a photoperiod of 12 h / d for 30 days to produce rooted seedlings. The rooting medium formula was as follows: per 1000 ml of 1 / 2 MS medium, add 0.1-0.5 mg NAA, 0.1-1.0 mg IBA, 15 g sucrose, and 6 g agar, and adjust the pH to 5.7-5.9. 5) Hardening off rooted seedlings: After rooting in the rooting medium for 30 days, the rooted seedlings are transferred from the culture room to a shaded hardening-off greenhouse. The temperature in the hardening-off greenhouse should be controlled at 18-25℃, and shade netting should be used to maintain a relative humidity of 60-70%. After hardening off for 4-5 days with the bottle caps open, the rooted seedlings are taken out, the agar on the roots is washed off, and then they are disinfected with a 1000-fold diluted solution of carbendazim (active ingredient and content: 50%) for 10 minutes. Then they are planted in the cultivation substrate and cultivated for 15-25 days. The rooted seedlings will grow new buds, achieving rapid propagation of seedlings. The formula of the cultivation substrate is peat: perlite = 2:1 by weight. Then they can be transplanted into the natural environment for cultivation.

[0006] The beneficial effects of this invention are: 1) High-quality and efficient propagation system: The in vitro rapid propagation system of Bupleurum chinense provided by this invention utilizes the young buds at the base of the Bupleurum chinense stems for bud induction and propagation, and then the induced clustered buds are rooted and planted, successfully establishing a high-quality and efficient in vitro rapid propagation system. This system can effectively solve the problem of difficult propagation of Bupleurum chinense and provide a reliable technical guarantee for its large-scale propagation.

[0007] 2) Unaffected by the natural environment: This method has the significant characteristic of being unaffected by the natural environment. Traditional plant propagation methods are often limited by natural factors such as season and climate, while the in vitro rapid propagation technology of this invention can be carried out in an artificially controlled environment, ensuring the stability and controllability of the propagation process and greatly improving propagation efficiency.

[0008] 3) Meeting Market Demand: Given the extreme scarcity of Bupleurum chinense germplasm resources in Yantai, which cannot meet the enormous market demand, the method of this invention can be used for the large-scale and rapid propagation of young shoots at the base of Bupleurum chinense stems. This technology enables the harvesting of a large number of sterile seedlings in a short time, providing a scientific and convenient path for the efficient cultivation of Bupleurum chinense in Yantai, and has extremely high application value. It not only alleviates the market demand pressure for Bupleurum chinense in Yantai, but also provides solid raw material support for the development of related pharmaceutical industries. Detailed Implementation

[0009] The present invention will be described in detail below with reference to embodiments and specific circumstances.

[0010] To make the objectives, technical solutions, and advantages of this invention clearer, the following detailed description of the invention is provided in conjunction with embodiments, but this is not intended to limit the scope of the invention. The embodiments provided below can serve as a reference and guide for those skilled in the art to further improve the invention, and do not constitute a limitation on the invention in any way. Any product that is the same as or similar to this invention, formed under the guidance of this invention or by combining features of this invention with other prior art, falls within the protection scope of this invention.

[0011] The present invention can be described in the following embodiments.

[0012] Example 1: A method for rapid in vitro propagation of Bupleurum chinense buds from Yantai, comprising the following steps: 1) Surface sterilization of explants: Using the young shoots at the base of the stem of Bupleurum chinense as explants, rinse with clean water for 2 hours, disinfect the surface with 75% alcohol for 30 seconds on a sterile operating table, rinse with sterile water 4 times, disinfect with 0.1% HgCl2 (mercuric chloride) for 10 minutes, and rinse with sterile water 6 times to thoroughly remove HgCl2; 2) Induction of clustered shoots: Explants of young shoots from the base of *Bupleurum chinense* stems, which had been surface-sterilized, were placed on a sterile operating table with the attached root bark and stem bark removed, the outermost leaves peeled off, and placed in a high-temperature sterilized clustered shoot induction medium. Each bottle of medium contained 30 ml, and each bottle contained 4 explants from the base of *Bupleurum chinense* stems. The culture was carried out in a culture room at a temperature of 23℃, a light intensity of 10000 lx, and a light duration of 12 h / d for 14 days to induce clustered shoots. The medium used in this process was prepared by adding 1 mg of 6-BA, 0.2 mg of IBA, 0.5 mg of ZT, 0.2 mg of NAA, 30 g of sucrose, 6 g of agar, and a pH of 5.8 to every 1000 ml of MS medium. 3) Proliferation and subculturing of clustered shoots: The explanted shoot blocks induced with clustered shoots were transferred into a sterilized clustered shoot proliferation medium. Each bottle contained 30 ml of medium and 4 clustered shoot blocks. The culture was carried out for 15 days at a temperature of 23℃, a light intensity of 10000 lx, and a light duration of 12 h / d. The proliferation medium was prepared by adding 1.0 mg of 6-BA, 0.1 mg of NAA, 30 g of sucrose, 6 g of agar, and a pH of 5.8 to 1000 ml of MS medium. 4) Rooting of subcultured shoot clusters: Subcultured shoot clusters were inoculated into rooting medium for shoot clusters, 30 ml per bottle, with 4 shoot clusters per bottle. The culture was carried out for 30 days at a temperature of 23 ℃ and a light intensity of 10000 lx, with a photoperiod of 12 h / d. Rooted seedlings were obtained. The rooting medium consisted of 1000 ml of 1 / 2 MS medium with the addition of 0.5 mg IBA, 0.1 mg NAA, 15 g sucrose, and 6 g agar, with a pH of 5.8. 5) Hardening off rooted tissue culture seedlings: After 30 days of rooting culture in the rooting medium, the rooted seedlings are transferred from the culture room to a shaded hardening-off greenhouse. The temperature in the hardening-off greenhouse is 18-25℃, shaded with a shade net, and the relative humidity is 65%. After hardening off for 4-5 days with the bottle caps open, the seedlings are taken out, the roots are washed clean of agar, and then disinfected with a 1000-fold diluted solution of 50% carbendazim active ingredient. The solution is then planted into the cultivation substrate, and new shoots will emerge successively in 15-25 days.

[0013] Example 2, a method for rapid in vitro propagation of Bupleurum chinense seedlings from Yantai, is implemented by the following steps: 1) Surface sterilization of explants: Using the young shoots at the base of the stem of Bupleurum chinense as explants, rinse with running water for 2 hours, disinfect the surface with 75% alcohol for 30 seconds, rinse with sterile water 3-5 times, then disinfect the surface with 0.1% HgCl2 (mercuric chloride) for 5 minutes, and then rinse with sterile water 5-7 times. 2) Induction and subculturing of clustered shoots: Explants of young shoots from the base of *Bupleurum chinense* stems, after surface sterilization, were placed on a sterile operating table. The attached root bark and stem bark were removed, and the outermost leaves were peeled off. The explants were then placed in a 30ml bottle of sterilized clustered shoot induction medium, containing four explants from the base of *Bupleurum chinense* stems per bottle. The culture was carried out at 23℃, 10000lx light intensity, and 12h / d for 14 days to induce clustered shoots. The explants that induced clustered shoots were then inoculated back into the same medium and cultured for 15 days. The growth and development of the subcultured clustered shoots were then assessed. This medium was prepared by adding 1mg 6-BA, 0.2mg IBA, 0.5mg ZT, 0.2mg NAA, 30g sucrose, 6g agar, and maintaining a pH of 5.7-5.9 to every 1000ml of 1 / 2 MS medium. 3) Rooting of clump shoots: The subcultured clump shoots were inoculated into the rooting medium for clump shoots, 30 ml per bottle, with 4 clump shoot blocks in each bottle. They were cultured in a culture room at a temperature of 20-26℃ and a light intensity of 10000 lx for 12 h / d for 30 days to form rooted seedlings. The rooting medium was composed of 0.1 mg IBA, 0.1 mg NAA, 15 g sucrose, and 6 g agar per 1000 ml of 1 / 2 MS medium, with a pH of 5.8. 4) Hardening off rooted tissue culture seedlings: After 30 days of rooting culture in the rooting medium, transfer the rooted seedlings from the culture room to a shaded hardening-off greenhouse. The temperature in the hardening-off greenhouse should be 18-25℃, shaded with a shade net, and the relative humidity 60-70%. After hardening off for 4-5 days with the bottle caps open, remove the seedlings, wash off the agar from the roots, and disinfect them for 10 minutes with a 50% solution of carbendazim diluted 1000 times with clean water. Then, plant them in the cultivation substrate. New shoots will appear after 15-25 days.

[0014] Example 3, a method for rapid in vitro propagation of Bupleurum chinense seedlings from Yantai, is implemented by the following steps: 1) Surface sterilization of explants: Using the young shoots at the base of the stem of Bupleurum chinense as explants, rinse with running water for 2 hours, disinfect the surface with 75% alcohol for 30 seconds, rinse with sterile water 3-5 times, then disinfect the surface with 0.1% HgCl2 (mercuric chloride) for 15 minutes, and then rinse with sterile water 6 times. 2) Induction of clustered shoots: Explants of young shoots from the base of *Bupleurum chinense* stems, which had been surface-sterilized, were placed on a sterile operating table with the attached root bark and stem bark removed, the outermost leaves peeled off, and placed in a sterilized clustered shoot induction medium. Each bottle of medium contained 30 ml, and each bottle contained 4 explants from the base of *Bupleurum chinense* stems. The culture was carried out in a culture room at a temperature of 23 ± 3℃, a light intensity of 10000 lx, and a light duration of 12 h / d for 14 days to induce clustered shoots. The medium was prepared by adding 1 mg of 6-BA, 0.2 mg of IBA, 0.5 mg of ZT, 0.2 mg of NAA, 15 g of sucrose, 6 g of agar, and a pH of 5.8 to 1000 ml of 1 / 2 MS medium. 3) Proliferation and subculturing of clustered shoots: The explants induced with clustered shoots were transferred to a sterilized clustered shoot proliferation medium, 30 ml per bottle, with 4 clustered shoots per bottle. The plants were cultured for 15 days at 23℃, 10000 lx light intensity, and 12 h / d to produce subcultured clustered shoots. The proliferation medium consisted of 1.0 mg 6-BA, 0.5 mg NAA, 30 g sucrose, 6 g agar, and a pH of 5.8 per 1000 ml MS medium. 4) Rooting of subcultured shoot clusters: Subcultured shoot clusters were inoculated into rooting medium for shoot clusters, 30 ml per bottle, with 4 shoot clusters per bottle. The culture was carried out in a culture room at a temperature of 23 ± 3℃ and a light intensity of 10000 lx for 12 h / d for 30 days to produce rooted seedlings. The rooting medium consisted of 0.3 mg NAA, 0.5 mg IBA, 30 g sucrose, and 6 g agar per 1000 ml of 1 / 2 MS medium, with a pH of 5.8. 5) Hardening off rooted tissue culture seedlings: After 30 days of rooting culture in the rooting medium, transfer the rooted seedlings from the culture room to a shaded hardening-off greenhouse. The temperature in the hardening-off greenhouse should be 18-25℃, shaded with a shade net, and the relative humidity 60-70%. After hardening off for 4-5 days with the bottle caps open, remove the seedlings, wash off the agar from the roots, and disinfect them for 10 minutes with a 1000-fold dilution of 50% carbendazim. Then, plant them in the cultivation substrate. New shoots will emerge successively in 15-25 days.

[0015] The method of this invention is simple, easy to operate, and has good cultivation results. It establishes an efficient asexual propagation system for Bupleurum chinense in Yantai, providing technical support for meeting market demand for Bupleurum chinense in Yantai. Experiments have yielded very good and beneficial technical results, and the relevant experimental data are as follows: Unless otherwise specified, the experimental methods in the following examples are conventional operating methods, and the materials and reagents used in the examples are commercially available unless otherwise specified.

[0016] 1. Test materials In this invention, the winter stem basal shoots of Bupleurum chinense from Yantai were selected as experimental materials, and healthy stem basal shoots free from pests and diseases were chosen as explants. Winter stem basal shoots were chosen because they are in a relatively dormant state at this time, with stable cell activity, which is more conducive to in vitro culture and can improve the success rate of culture. 2. Instruments and reagents Reagents: Agar powder, sucrose, MS medium, 1 / 2 MS medium, naphthaleneacetic acid (NAA), indolebutyric acid (IBA), zeatin (ZT), and 6-benzylaminopurine (6-BA). These reagents are all commonly used basic reagents in plant tissue culture. Among them, MS medium and 1 / 2 MS medium are the basic nutrient supply systems, while various hormones are used to regulate the division, differentiation, and growth of plant cells. Instruments: ultra-clean workbench, incubator, autoclave, light culture rack, etc., are used to ensure a sterile environment and suitable conditions for explant processing, culture medium sterilization, and culture process.

[0017] 3. Explant disinfection Mercuric chloride treatment at three time points (5, 10, and 15 minutes) with 0.1% HgCl2 significantly affected the germination rate of *Bupleurum chinense*. The contamination rate gradually decreased with increasing 0.1% HgCl2 treatment time. However, mercuric chloride is toxic to plant larvae, and prolonged treatment can negatively impact bud germination. The study found that the contamination rate was lowest at 15 minutes and the germination rate was highest at 10 minutes. Based on the significant differences, 10 minutes of mercuric chloride disinfection was selected as the optimal disinfection time.

[0018]

[0019] Data are presented as mean ± standard deviation. The results of the analysis of variance are expressed as a, b, c, d to indicate significant differences between different groups (P < 0.05). 4. Effects of different hormone combinations on the proliferation of shoots from Bupleurum chinense in Yantai Different types and concentrations of hormones had varying effects on the proliferation and growth of adventitious buds of *Bupleurum chinense* var. *yantaiense*. Based on MS + 6 g / L agar + 30 g / L sucrose, different concentrations of 6-BA + NAA combinations were established, with other components and treatment methods remaining constant. Each group contained 30 buds, with 3 replicates per group. The study found that the combined addition of 6-BA and NAA significantly affected the proliferation of adventitious buds of *Bupleurum chinense* var. *yantaiense*. Statistical analysis of experimental data at different gradients:

[0020] Data are presented as mean ± standard deviation. The results of the analysis of variance are expressed as a, b, c, d to indicate significant differences between different groups (P < 0.05). The data above show that the optimal concentration of 6-BA for inducing the proliferation of Bupleurum chinense shoots is 1.0 mg / L and NAA is 0.1 mg / L. Under this combination, about 8 adventitious shoots can be induced in 15 days, resulting in optimal plant growth, moderate leaf and shoot size, and less callus tissue. This combination can be used as a proliferation culture medium for Bupleurum chinense from Yantai. 4. Effects of different concentrations of IBA on the rooting of Bupleurum chinense in Yantai The effects of different concentrations of IBA and NAA on the rooting of Bupleurum chinense were studied using 1 / 2 MS medium as the basal medium. Each treatment group consisted of 30 plants, with 3 replicates per group. The rooting rate gradually decreased with increasing IBA and NAA concentrations. Comparative analysis of plant growth and rooting characteristics under different treatments revealed that treatment K showed the best rooting rate and average number of roots, and the plants were healthier compared to other treatments. The specific experimental data are as follows:

[0021] Data are presented as mean ± standard deviation. The results of the analysis of variance are expressed as a, b, c, d to indicate significant differences between different groups (P < 0.05). Adding 0.5 mg / L IBA and 0.1 mg / L NAA to 1 / 2 MS medium resulted in the best rooting effect for sterile Bupleurum chinense seedlings from Yantai, with a rooting rate of 87% and a root count of up to 10.64 roots after 20 days. 5. Hardening medium for Bupleurum chinense seedlings in Yantai Rooted seedlings that have been rooted in the rooting medium for 30 days are transferred from the culture room to a shaded hardening-off greenhouse. The temperature in the hardening-off greenhouse should be maintained at 18-25℃, with shade netting used to maintain a relative humidity of 60-70%. After hardening off for 4-5 days with the bottle caps open, the seedlings are removed, the agar on the roots is washed off, and then disinfected with a 1000-fold diluted solution of carbendazim (active ingredient and content: 50%) for 10 minutes. They are then planted in the cultivation substrate, and new shoots will emerge after 15-25 days. The cultivation substrate is a peat moss:perlite ratio of 2:1. This ratio provides good aeration and water retention, creating a suitable environment for the growth of rooted seedlings and improving the survival rate of hardening-off seedlings. Based on the comprehensive experimental data, different treatment methods had a significant impact on the rapid in vitro propagation effect of Bupleurum chinense from Yantai.

[0022] Example 1 Experimental Data 1) Explant contamination rate: A total of 30 explants were treated, and 3 explants were contaminated, with an explant contamination rate of 10.0%.

[0023] 2) Clump bud germination rate: 26 explants successfully germinated clump buds, and the clump bud induction rate was 86.67%.

[0024] 3) Bud proliferation multiple: After 15 days, the average bud proliferation multiple was 8.67 times.

[0025] 4) Rooting rate: Of the 30 bud clusters inoculated with the rooting medium, 28 successfully rooted, with a rooting rate of 93.33% and an average of 10 roots per bud.

[0026] 5) Seedling survival rate: A total of 50 seedlings were hardened, and 47 of them survived, resulting in a seedling survival rate of 94.0%.

[0027] Example 2 Experimental Data 1) Explant contamination rate: Of the 30 explants treated, 13 were contaminated, resulting in an explant contamination rate of 43.3%.

[0028] 2) Clump bud germination rate: 23 explants successfully germinated clump buds, and the clump bud induction rate was 76.67%.

[0029] 3) Bud proliferation multiple: After 15 days, the average bud proliferation multiple was 8 times, but 46.7% of the tissue culture seedlings vitrified.

[0030] 4) Rooting rate: Of the 30 bud clusters inoculated with the rooting medium, 22 successfully rooted, with a rooting rate of 73.0% and an average number of roots of 7.33.

[0031] 5) Seedling survival rate: A total of 50 seedlings were hardened, and 43 of them survived, resulting in a seedling survival rate of 86%.

[0032] Example 3 Experimental Data 1) Explant contamination rate: 30 explants were treated, and 4 explants were contaminated, with an explant contamination rate of 13.3%.

[0033] 2) Sprouting rate of clustered shoots: 25 explants successfully sprouted clustered shoots, and the induction rate of clustered shoots was 83.33%.

[0034] 3) Bud proliferation multiple: After 15 days, the average bud proliferation multiple was 4 times.

[0035] 5) Rooting rate: Of the 30 shoot clusters inoculated with the rooting medium, 18 successfully rooted, with a rooting rate of 60% and an average number of roots of 3.75.

[0036] 6) Seedling survival rate: A total of 50 seedlings were hardened, and 36 of them survived, resulting in a seedling survival rate of 72%. Comparative analysis

[0037] Based on the experimental data from the comprehensive experiment and the examples, different treatment methods have a significant impact on the rapid propagation effect of Bupleurum chinense in vitro. Explant contamination rate: Example 2 showed the highest contamination rate at 43.3%, while Example 1 showed the lowest at 10%. This indicates that disinfection with 0.1% HgCl2 for 10 and 15 minutes, combined with 75% alcohol for 30 seconds, is more effective in controlling explant contamination; while disinfection with 0.1% HgCl2 for 5 minutes is relatively less effective. The germination rate of clustered shoots was highest in Example 1 (85%) and lowest in Example 2 (83.33%). The results indicate that HgCl2 has a good bactericidal effect, but excessive use should be avoided to prevent adverse effects on the germination rate of clustered shoots. Bud proliferation rate: Example 1 showed the highest rate, averaging 8.67 times, while Example 3 showed the lowest rate, averaging 4 times. Different concentrations of hormones can promote bud proliferation to some extent, but the concentration must be carefully controlled to avoid adverse effects on subsequent growth. Rooting rate: Example 1 showed the highest rate at 93.33%, while Example 3 showed the lowest rate at 60%. The hormone concentrations of IBA and NAA showed a promoting effect on rooting of Bupleurum yantaiense at low concentrations and an inhibiting effect at high concentrations, indicating that these two hormones play an important role in promoting the rooting of Bupleurum yantaiense bud clusters. Seedling survival rate: Example 1 had the highest survival rate at 94%, while Example 3 had the lowest at 72%. The survival rate of seedlings after hardening off is closely related to the rooting effect in the early stage and the quality of the rooted seedlings. A higher rooting rate and stronger rooted seedlings result in a relatively higher survival rate after hardening off.

[0038] In summary, the in vitro rapid propagation method for *Bupleurum chinense* seedlings provided by this invention affects the induction, proliferation, rooting, and survival rate of *Bupleurum chinense* seedlings by factors such as the surface sterilization method of the explants, the formulation of the culture medium (including the types and amounts of various hormones, sucrose content, etc.), and the culture conditions. In practical applications, appropriate implementation methods can be selected according to specific circumstances to achieve a relatively optimized overall effect, resulting in a high seedling induction rate, high seedling multiplication rate, and high rooting rate, significantly improving the rapid propagation efficiency of *Bupleurum chinense*. This invention represents a major innovation in the cultivation method of *Bupleurum chinense* seedlings and has significant potential for widespread application.

Claims

1. A method for rapid in vitro propagation of Bupleurum chinense buds from Yantai, characterized in that, Includes the following steps: (1) Surface sterilization of explants: Select the young shoots at the base of the stem of Bupleurum chinense as explants, rinse with clean water for 2 hours, and then disinfect the surface with 75% alcohol for 30 seconds, rinse with sterile water 3-5 times, disinfect with 0.1% HgCl2 for 5-15 minutes on a sterile operating table, and then rinse with sterile water 5-7 times. (2) Induction of shoot clusters: The root bark, stem bark and outermost leaves of the surface-sterilized explants were removed and placed in a high-temperature sterilized shoot cluster induction medium. Four explants were placed in each 30ml bottle of medium and cultured for 14 days at a temperature of 20-26℃, a light intensity of 10000lx and a light duration of 12h / d to induce shoot clusters. The shoot cluster induction medium was prepared by adding 1.0mg of 6-BA, 0.2mg of IBA, 0.5mg of ZT, 0.2mg of NAA, 15g of sucrose and 6g of agar to 1000ml of 1 / 2MS medium, with a pH of 5.7-5.

9. (3) Proliferation and subculturing of clustered shoots: The induced clustered shoot blocks were transferred into a high-temperature sterilized proliferation medium. Four clustered shoot blocks were placed in each 30ml bottle of medium and cultured for 15 days under the conditions of temperature 20-26℃, light intensity 10000lx, and light duration 12h / d to obtain subcultured clustered shoots. The proliferation medium consisted of 0.5-2.0mg of 6-BA, 0.1-0.5mg of NAA, 30g of sucrose, and 6g of agar per 1000ml of MS medium, with a pH of 5.8-6.

0. (4) Rooting of subcultured shoots: Subcultured shoots were inoculated into a sterilized rooting medium. Four shoots were placed in each 30ml bottle of medium. The shoots were cultured for 30 days at a temperature of 20-26℃, a light intensity of 10000lx, and a light duration of 12h / d to obtain rooted seedlings. The rooting medium consisted of 0.1-0.5mg NAA, 0.1-1.0mg IBA, 15g sucrose, and 6g agar per 1000ml of 1 / 2MS medium, with a pH of 5.7-5.

9. (5) Hardening off rooted seedlings: Transfer the rooted seedlings to a shaded hardening-off greenhouse. Harden off the seedlings for 4-5 days with the bottle caps open under the conditions of 18-25℃ and 60-70% relative humidity. After that, take them out, wash off the agar from the roots, disinfect them with a 1000-fold dilution of 50% carbendazim for 10 minutes, and plant them in a cultivation substrate with a peat:perlite ratio of 2:1 by weight. Cultivate for 15-25 days until new shoots grow.

2. The method according to claim 1, characterized in that, In step (1), the disinfection time is 30 seconds for 75% alcohol disinfection and 10 minutes for HgCl2 disinfection.

3. The method according to claim 1, characterized in that, In step (2), the bud induction medium contains 1.0 mg of 6-BA, 0.2 mg of IBA, 0.5 mg of ZT, 0.2 mg of NAA, and 15 g of sucrose.

4. The method according to claim 1, characterized in that, In step (3), the proliferation medium contains 1.0 mg of 6-BA, 0.1 mg of NAA, and 30 g of sucrose.

5. The method according to claim 1, characterized in that, In step (4), the rooting medium contains 0.1 mg of NAA, 0.5 mg of IBA, and 15 g of sucrose.

6. The method according to claim 1, characterized in that, The explants were young buds at the base of the stems of Bupleurum chinense from Yantai during winter.

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