A high-efficiency method for inducing cluster buds of scytanthus of scutellaria amoena franch and application thereof

Abstract

The application discloses a kind of efficient induction of Caudiceae of Scutellaria amoena stems section cluster bud tissue culture rapid propagation method and application;The method selects the tender stem section of the 3-5 nodes of the healthy top bud of Scutellaria amoena without disease and pest below with axillary bud as explant, after pretreatment, gradient disinfection, using axillary bud direct induction cluster bud approach, complete rapid propagation with optimized medium.The induction medium contains CPPU, NAA and silver nitrate, can directly induce cluster bud, avoid callus variation;Proliferation medium contains CPPU, GA3 and potassium dihydrogen phosphate, make seedling robust;Rooting medium contains PP333 and SA, promote rooting and resistance.Cluster bud induction rate is more than 96%, proliferation coefficient is more than 10.0, rooting rate is 100%, continuous subculture 6 generations without variation, transplanting survival rate is 100%.It is suitable for Scutellaria amoena germplasm preservation, factory seedling and medicinal raw material production.

Description

Technical Field

[0001] This invention relates to the field of asexual propagation technology for rare medicinal plants, specifically to a method and application for efficient induction of clustered buds from stem segments of *Scutellaria baicalensis*. This invention belongs to the field of tissue culture technology for traditional Chinese medicinal materials, and is particularly suitable for the rapid propagation of seedlings, in vitro preservation of germplasm resources, and industrialized seedling production of *Scutellaria baicalensis*, a plant belonging to the genus *Scutellaria* of the Lamiaceae family. Background Technology

[0002] Scutellaria baicalensis, also known as Southwestern Scutellaria, is a perennial herb belonging to the genus Scutellaria in the Lamiaceae family. It is mainly distributed in southwestern my country, including Yunnan, Sichuan, and Guizhou provinces, and is an important representative of traditional Yunnan medicine. Scutellaria baicalensis is rich in various flavonoid active ingredients such as baicalin, wogonin, and baicalein. Modern pharmacological studies have shown that it possesses multiple effects, including liver protection and lipid-lowering, anti-inflammatory and analgesic, antioxidant, free radical scavenging, and skin barrier repair. It has broad application prospects in the traditional Chinese medicine market and the field of plant-based skincare raw materials.

[0003] However, the wild resources of *Scutellaria baicalensis* are facing a severe survival crisis. In recent years, with the continuous exploration of its medicinal value and the sustained growth of market demand, wild *Scutellaria baicalensis* has been over-harvested for a long time, resulting in a sharp decline in its natural population and a shrinking distribution range. It has been listed as one of the key protected rare and endangered medicinal plant resources in Yunnan Province. At the same time, the biological characteristics of *Scutellaria baicalensis* limit its traditional propagation methods: its seeds have significant physiological dormancy characteristics, a long dormancy period, and a natural germination rate that is usually less than 30%, with uneven germination; while asexual propagation through cuttings is possible, rooting is difficult, and the survival rate is low, making it difficult to meet the quantity and quality requirements of large-scale planting. The combination of these problems makes the supply of *Scutellaria baicalensis* seedlings a key bottleneck restricting its industrial development.

[0004] In the field of plant tissue culture, researchers have explored rapid propagation techniques for Scutellaria baicalensis. Publication number CN101536672B, entitled "An Organogenesis Method for the Traditional Chinese Medicine Scutellaria baicalensis," discloses a tissue culture method using axillary bud-free stem segments of Scutellaria baicalensis as explants, involving callus induction, differentiation of adventitious buds, subculture proliferation, and plant regeneration. However, this technical route has significant drawbacks: First, the callus induction and differentiation stages are time-consuming, typically requiring more than 60 days from initial culture to obtaining regenerated plants, resulting in a long propagation cycle and low production efficiency. Second, the callus pathway is prone to somatic cell mutations during culture, leading to severe seedling degeneration after multiple subcultures, manifested as weakened growth vigor and decreased flavonoid content, failing to meet the requirements for genetic stability in long-term large-scale seedling production. Third, this method has a high contamination rate, typically exceeding 25% in practice, severely impacting propagation efficiency and production costs.

[0005] In addition, Zhao Zhenling et al. (Somatic Embryo Induction and Plant Regeneration of Scutellaria baicalensis, Southwest China Journal of Agricultural Sciences, 2006, 19(4): 714-718) reported a technical scheme for regenerating Scutellaria baicalensis plants through somatic embryogenesis. However, the somatic embryo induction rate was unstable, and the cycle from somatic embryo maturation to germination and seedling formation was long. The rooting rate was only 85.5%, and the transplant survival rate was only 70-80%, making it difficult to apply to actual production. Li Yaqiong et al. (Research on Rapid Propagation of Scutellaria baicalensis, Chinese Medicinal Herbs, 2007, 30(7): 761-762) conducted a study on the induction of clustered buds using stem segments with buds as explants. Although the initial induction rate could reach 100%, the proliferation coefficient was only 6.45, and the propagation efficiency was limited, making it difficult to meet the needs of industrialized seedling production for proliferation multiples and seedling quantity.

[0006] In summary, the existing rapid propagation techniques of Scutellaria baicalensis tissue culture generally have the following technical defects: (1) Long propagation cycle, usually more than 60 days from explant inoculation to obtaining transplantable rooted seedlings; (2) Poor genetic stability, somatic cell mutation is easily induced by callus tissue or somatic embryo pathway, and seedlings degenerate after multiple generations; (3) Prominent vitrification and browning phenomena, the existing culture medium hormone ratio is unreasonable, resulting in a high vitrification rate of culture, which can reach more than 30% in severe cases; (4) High contamination rate, the existing explant disinfection scheme is not ideal, and the contamination rate is generally more than 25%; (5) Low proliferation efficiency, the proliferation coefficient is generally less than 10, and it is impossible to achieve rapid propagation of seedlings; (6) Poor rooting and transplanting effects, the existing methods have a long rooting time (usually 25 to 30 days), the rooting rate is unstable, and there is a lack of matching transplanting substrate and acclimatization scheme.

[0007] Therefore, developing a rapid tissue culture propagation method for Scutellaria baicalensis with high induction efficiency, large proliferation coefficient, genetic stability, low vitrification rate, low contamination rate, fast rooting, and high transplant survival rate is of great practical significance and has an urgent market demand for the protection of Scutellaria baicalensis germplasm resources, large-scale seedling breeding, and industrial development of its medicinal components. Summary of the Invention

[0008] Purpose of the invention This invention addresses the shortcomings of existing Scutellaria baicalensis propagation techniques, such as long propagation cycles, high contamination rates, high mutation rates, severe vitrification, low proliferation efficiency, and unstable seedling quality. It provides a method for the efficient induction of clustered buds from Scutellaria baicalensis stem segments through tissue culture and its application. This invention aims to select suitable explant types and developmental stages, optimize pretreatment protocols, establish an efficient gradient disinfection system, screen the optimal types and concentrations of plant growth regulators, develop a regeneration pathway for direct germination of clustered buds from axillary buds, and utilize optimized subculture and proliferation media, seedling rooting media, and specialized transplanting substrates. Ultimately, this results in a complete, efficient, and stable tissue culture rapid propagation technology system for Scutellaria baicalensis, achieving the goals of high clustered bud induction rate, large proliferation coefficient, good genetic stability, low contamination rate, low vitrification rate, rapid rooting speed, and high transplant survival rate for large-scale seedling production.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: Based on a thorough study of the physiological requirements of key stages in the tissue culture of Scutellaria baicalensis stem segments, such as axillary bud germination, cluster bud induction, subculture proliferation, and rooting and seedling formation, this invention proposes the following complete technical solution through systematic screening and optimization of core process parameters such as explant type, culture medium formula, hormone type and concentration ratio, culture conditions, and transplanting substrate.

[0010] This invention provides a method for the rapid propagation of Scutellaria baicalensis stem segments through tissue culture, characterized by efficient induction of clustered buds. The core concept involves selecting young stem segments with axillary buds at specific developmental stages (3rd to 5th nodes below the apical bud) as explants. Pre-treatment with a fungicide before harvesting reduces the initial bacterial load, and an optimized gradient sterilization process achieves efficient sterilization. Then, a suitable combination of CPPU and NAA concentrations directly induces axillary bud germination, forming clustered buds, bypassing callus differentiation and ensuring genetic stability from the source. During the subculture propagation stage, a combination of CPPU, GA3, and potassium dihydrogen phosphate promotes bud proliferation while maintaining robust seedlings. In the rooting stage, a suitable combination of PP333 and SA concentrations promotes root primordia formation, dwarfs and strengthens seedlings, and enhances stress resistance. Finally, a specialized hardening-off transplanting scheme and composite substrate are used to achieve high survival rates during transplanting. The specific technical solution is as follows: A method for rapid propagation of Scutellaria baicalensis stem segments by tissue culture with high efficiency in inducing clustered buds includes the following steps: (1) Pretreatment of explants before collection Before selecting explants, the selected *Scutellaria baicalensis* mother plants were pretreated with a fungicide spray. Specifically, 50% carbendazim diluted 500 times and chlorothalonil diluted 1000 times were alternately sprayed onto the selected mother plants every 3-5 days, for 2-3 consecutive applications. This pretreatment significantly reduced the bacterial load on the surface of the mother plants and on latent pathogens, thus effectively reducing the pressure on explant surface disinfection and laying a good foundation for subsequent gradient disinfection. Preferably, spraying was performed every 4 days, alternating between fungicides, for 2 consecutive applications.

[0011] (2) Selection and cleaning of explants On a sunny morning, tender stem segments with axillary buds, located at the 3rd to 5th nodes below the terminal bud of healthy, disease-free *Scutellaria baicalensis* plants, were selected as explants. This location was chosen because stem segments at the 1st and 2nd nodes below the terminal bud are too young and fragile, easily damaged and browning during sterilization; while stem segments below the 6th node are too old, with reduced axillary bud viability and lower induction rates. Stem segments at the 3rd to 5th nodes have moderate tissue development, plump axillary buds, and are in a state of high physiological activity, making them the optimal material for tissue culture.

[0012] Cut stem segments with axillary buds, 1.5–2 cm in length, using pruning shears. Carefully remove the leaves (avoiding damage to the axillary buds), place them in a beaker, and rinse under running water for 30 minutes to remove surface dirt and microorganisms. Then, use a soft-bristled brush dipped in soapy water to gently brush the surface of the stem segments to further remove surface dirt. Rinse three times with sterile water, 2 minutes each time, and set aside.

[0013] (3) Aseptic disinfection After cleaning, the stem segments were placed in a clean bench and subjected to a gradient disinfection process: First, they were soaked in 75% ethanol for 15–20 seconds. Ethanol has good wetting and bactericidal effects, effectively killing most bacteria on the surface of the stem segments, while its rapid evaporation will not cause excessive damage to the tissue. They were then rinsed three times with sterile water for 2 minutes each time to remove residual ethanol. Next, they were disinfected with 0.05% mercuric chloride plus two drops of Tween-80, shaking for 15–20 minutes. Mercuric chloride is a highly effective surface disinfectant, and Tween-80, as a surfactant, can reduce the surface tension of the liquid and improve the contact efficiency between the disinfectant and the explant surface. They were then rinsed 5–8 times with sterile water for 2 minutes each time to thoroughly remove residual mercuric chloride. Finally, the surface moisture was blotted dry with sterile filter paper and set aside for later use. Preferably, the process involved soaking in 75% ethanol for 15 seconds, disinfecting with 0.05% mercuric chloride plus Tween-80 for 15 minutes, and rinsing five times with sterile water.

[0014] (4) Induction and culture of clustered shoots The sterilized stem segments were inoculated into the bud induction medium and cultured at 23–25°C, with a light intensity of 2000–2500 lx for 3–10 days. The axillary buds began to sprout and directly formed bud clusters.

[0015] The bud induction medium used MS as the basal medium, supplemented with CPPU (N-(2-chloro-4-pyridyl)-N'-phenylurea, a cytokinin-like plant growth regulator) 0.2–1.0 mg / L, NAA (α-naphthaleneacetic acid, an auxin-like plant growth regulator) 0.05–0.2 mg / L, silver nitrate (AgNO3) 0.5 mg / L, sucrose 30 g / L, agar 6 g / L, pH 5.8.

[0016] Extensive experiments have confirmed that CPPU is significantly superior to traditional cytokinins (such as 6-BA and KT) in promoting axillary bud germination and cluster bud formation. An appropriate concentration combination of CPPU and NAA synergistically promotes the activation of axillary bud meristems and the formation of adventitious bud primordia, while silver nitrate, as an ethylene activity inhibitor, effectively inhibits ethylene accumulation during culture, reduces browning of explants, and increases the induction rate. The combined use of these three agents produces a synergistic effect, resulting in a cluster bud induction rate of over 96%. Preferably, the CPPU concentration is 0.5 mg / L and the NAA concentration is 0.05 mg / L.

[0017] (5) Subculture The clustered buds induced in step (4) were divided into single buds and transferred to the proliferation medium. They were cultured for 20 to 22 days under the same culture conditions (23 to 25°C, 12 h / d light, 2000 to 2500 lx light intensity).

[0018] The proliferation medium used MS as the basic medium, with the following added components: CPPU 0.5–1.0 mg / L, GA3 (gibberellin) 0.1–0.5 mg / L, potassium dihydrogen phosphate (KH2PO4) 0.08 g / L, sucrose 30 g / L, agar 6 g / L, and pH 5.8.

[0019] CPPU continuously provides the cytokinin signals required for bud proliferation, promoting the continuous germination of axillary buds and the differentiation of lateral buds; GA3 promotes the longitudinal elongation of buds and prevents bud dwarfing caused by high cytokinin concentrations; potassium dihydrogen phosphate, as a phosphorus and potassium source, enhances the plant's photosynthetic capacity, resulting in stronger seedlings and darker green leaves. The combination of these three components effectively prevents vitrification while ensuring a high proliferation coefficient (above 10.0). Preferably, the CPPU concentration is 0.5 mg / L and the GA3 concentration is 0.1 mg / L.

[0020] (6) Seedling rooting culture Select healthy single buds with a height of 2-3 cm and transfer them to rooting medium. Under the same culture conditions (23-25℃, 12 h / d light, 2000-2500 lx light intensity) for 12-18 days to obtain complete rooted seedlings with well-developed root systems.

[0021] The rooting medium used was 1 / 2 MS as the basic medium (with macroelements reduced by half), with the following added concentrations: PP333 (paclobutrazol, a growth retardant) 1.0–2.0 mg / L, SA (salicylic acid) 0.5–1.5 mg / L, AC (activated carbon) 0.2 g / L, sucrose 20 g / L, agar 6 g / L, and pH 5.8.

[0022] 1 / 2MS medium lowers the inorganic salt concentration, which is beneficial for stimulating rooting; PP333, as a highly efficient growth retardant, can inhibit excessive stem elongation, promote the formation of basal root primordia, and also has the effects of dwarfing and strengthening seedlings and enhancing stress resistance; SA, as a plant stress resistance signaling molecule, can enhance the plant's disease resistance and stress resistance, and promote root growth and development; AC can adsorb phenolic substances in the culture medium that may inhibit rooting, creating a clean environment for root growth. The combination of these four substances produces a synergistic effect, shortening the rooting time to 12-18 days and achieving a rooting rate of 100%. Preferably, the concentration of PP333 is 1.0 mg / L and the concentration of SA is 0.5 mg / L.

[0023] (7) Hardening off seedlings and transplanting When the tissue culture seedlings have well-developed root systems and reach a height of 5–7 cm, they should be hardened off and transplanted. Place the tissue culture bottles indoors under natural light conditions and leave the caps open for 3–5 days to harden off, allowing the seedlings to gradually adapt to the external environmental conditions (reducing humidity, increasing light, and exposing them to natural air). After hardening off, carefully remove the seedlings and wash off any agar culture medium adhering to the roots with warm water (taking care to avoid damaging the roots). Transplant them into a pre-prepared composite substrate.

[0024] The composite substrate is composed of humus, perlite, and river sand in a mass ratio of 3:1:1. This substrate ratio provides good water retention, aeration, and drainage, meeting the basic water and nutrient needs of seedlings while preventing waterlogging and root rot. For the first 10 days after transplanting, maintain a relative humidity of 95%, daytime temperature of 23–25℃, and nighttime temperature of 18–20℃. After 10 days, once the roots have adapted to the substrate environment, transplant the seedlings into seedling cups. Field cultivation can begin 25 days later, and after transplanting, field management should be carried out according to conventional cultivation methods.

[0025] (8) Application The above-mentioned tissue culture rapid propagation method can be applied to the in vitro preservation of Scutellaria baicalensis germplasm resources (maintaining the genetic stability of germplasm through periodic subculture), industrialized seedling production (large-scale commercial production of seedlings), and large-scale production of Scutellaria baicalensis medicinal raw materials and plant skin care raw materials (providing downstream industries with genetically consistent and quality-stable raw material sources).

[0026] Compared with the prior art, the present invention has the following beneficial effects: (1) The pollution rate has been significantly reduced. This invention effectively reduces the initial bacterial load of explants by pre-treating the mother plants with alternating sprays of 50% carbendazim and chlorothalonil before harvesting. Combined with an optimized ethanol-mercuric chloride gradient disinfection procedure, it achieves highly efficient removal of microorganisms from the explant surface. Experimental data show that the contamination rate of each embodiment of this invention is only 2.5%–5.3%, significantly lower than 25% in Comparative Example 1, 28.9% in Comparative Example 2, and 27.3% in Comparative Example 3, representing a reduction of over 80%. This significantly reduces material loss and the cost of repeated experiments due to contamination.

[0027] (2) Innovative regeneration pathways and good genetic stability This invention employs a direct induction of clustered buds from stem segments with axillary buds, allowing the axillary bud meristem to germinate directly and form buds, bypassing the callus dedifferentiation and redifferentiation process. The callus pathway is prone to chromosome number and structural variations (i.e., somatic asexual variation) during dedifferentiation and redifferentiation, while this invention avoids the risk of somatic variation at its source by preserving the integrity of the axillary bud meristem. Experiments have demonstrated that the method of this invention can be stably and continuously propagated for six generations without visible variation, resulting in high genetic uniformity of the seedling population and the preservation of the superior quality and active ingredient characteristics of the mother plant.

[0028] (3) High induction efficiency and short culture cycle This invention utilizes a suitable combination of CPPU, NAA, and silver nitrate concentrations. Axillary bud germination and cluster bud formation can be observed after 3–10 days of cultivation on the induction medium, with a cluster bud induction rate exceeding 96%, reaching 100% under optimal conditions (Example 1). Compared to Comparative Example 1, which requires 7 days for callus germination before cluster bud differentiation (a longer total induction time), this invention significantly shortens the initial induction cycle. From explant inoculation to obtaining transplantable, fully rooted seedlings, this invention requires only 35–50 days (Example 1 requires only 35 days), while existing technologies typically require over 60 days, reducing the production cycle by 20%–40%.

[0029] (4) High proliferation coefficient and high propagation efficiency The proliferation coefficient of subculture is a core indicator for measuring the efficiency of rapid tissue culture propagation. This invention, through a combination of CPPU, GA3, and potassium dihydrogen phosphate, achieves a proliferation coefficient of over 10.0 within a 20-22 day proliferation cycle, reaching 15.6 under optimal conditions (Example 1). Based on a proliferation coefficient of 15.6, one explant can yield 15.6 seedlings after one proliferation cycle, and tens of thousands of seedlings can be obtained after four more cycles, fully meeting the seedling quantity requirements for industrialized seedling production. In comparison, the proliferation coefficient of Comparative Example 2 is only 7.2, and that of Comparative Example 3 is only 6.45, meaning this invention is more than twice as efficient as existing technologies.

[0030] (5) The seedlings are of excellent quality with low vitrification and browning rates. This invention employs a low-hormone ratio strategy (CPPU up to 1.0 mg / L, NAA up to 0.2 mg / L), avoiding vitrification and browning induced by high concentrations of cytokinins and auxins. The addition of GA3 and potassium dihydrogen phosphate promotes proliferation while ensuring robust seedling growth. Experimental observations show that the clumps of shoots cultivated using this invention are uniform, have vibrant green leaves, and exhibit no vitrification, with seedling quality significantly superior to existing technologies.

[0031] (6) It has a fast rooting speed and a well-developed root system. This invention employs a suitable combination of 1 / 2 MS, PP333, and SA at appropriate concentrations. PP333 inhibits excessive stem elongation and promotes the formation of basal root primordia; SA enhances the plant's stress resistance and root growth activity; and AC adsorbs phenolic inhibitors. The synergistic effect of these three components shortens the rooting time to 12-18 days (only 12 days in Example 1), achieving a 100% rooting rate. In contrast, existing technologies (Comparative Example 1) require 30 days for rooting culture, with a rooting rate of only 98.6%; Comparative Example 2 requires 30 days, with a rooting rate of only 85.5%. This invention not only significantly shortens the rooting time but also achieves a 100% rooting rate, laying a solid foundation for high-survival-rate transplanting.

[0032] (7) Dwarfed and robust seedlings with strong resistance to adverse conditions The paclobutrazol effect of PP333 results in compact and sturdy tissue culture seedlings, avoiding the etiolation problem commonly found in traditional tissue culture seedlings. The sturdy seedlings have thickened cell walls and well-developed mechanical tissues, leading to lower water evaporation during hardening-off and transplanting, stronger resistance to wind and lodging, and better adaptability to drought and temperature fluctuations in the early stages of transplanting. This is the important physiological basis for the 100% transplant survival rate of this invention.

[0033] (8) High transplant survival rate and complete supporting system This invention includes a specialized transplanting substrate (humus:perlite:river sand = 3:1:1) and a refined hardening-off transplanting scheme (hardening off the seedlings for 3-5 days with the cover off, maintaining a relative humidity of 95% for the first 10 days after transplanting, and a daytime temperature of 23-25℃ / nighttime temperature of 18-20℃), ensuring a smooth transition from the in vitro environment to the natural environment. The transplant survival rate can reach 96.3%-100% (100% in Example 1), completely solving the industry problem of low survival rate of Scutellaria baicalensis tissue culture seedlings after transplanting.

[0034] (9) Wide range of applications The method of this invention is applicable to the in vitro preservation of wild germplasm resources of Scutellaria baicalensis (maintaining germplasm through slow growth at low temperature or periodic subgeneration), the large-scale construction of standardized seedling bases (providing high-quality seedlings for medicinal herb planting bases), the standardized planting of Chinese medicinal herbs (ensuring the genetic consistency and quality stability of medicinal herb sources), and the industrial production of medicinal extracts and plant-based skincare raw materials (providing downstream industries with stable and traceable raw material sources). It has good industrialization prospects and promotional value. Attached Figure Description

[0035] Because key morphological information during the tissue culture of *Scutellaria baicalensis*, such as the germination status of clustered buds, bud color and growth vigor, morphological characteristics of propagated seedlings, root development of rooted seedlings, and survival and growth status of transplanted seedlings, cannot be clearly and completely expressed using traditional black-and-white line drawings, while color photographs can intuitively and realistically reflect these key characteristics. To better demonstrate the technical effects of this invention and enable those skilled in the art to more fully understand the technical solution and technological advancements achieved by this invention, color photographs are used as accompanying drawings in this specification.

[0036] Figure 1 Color photographs of stem segments of Scutellaria baicalensis with axillary buds inoculated with the plant. From Figure 1 It can be clearly seen that the morphology of the young stem segments with axillary buds after disinfection and inoculation into the induction medium at the beginning is clear. The nodes of the stem segments are clearly distinguishable, the axillary buds are plump and normal in color, the cut of the stem segments is flat and there is no browning. This indicates that the disinfection and inoculation operations are standardized, the explants are in good physiological condition, and they have the material basis for inducing germination.

[0037] Figure 2 Color photographs showing the effect of stem segment inducing the germination of clustered buds. From Figure 2 As can be seen intuitively, after induction culture, the axillary buds of the explants directly germinate to form clustered buds. The buds are bright green and grow vigorously. There are many clustered buds and they are evenly distributed. The axillary bud meristems are fully activated, showing the typical characteristics of "clustering". This figure fully verifies the high efficiency of the induction culture medium formula and the suitability of the induction conditions of this invention.

[0038] Figure 3 Color photographs of the subculturing and propagation of clustered buds. From Figure 3 As can be seen, after the clustered buds were divided into single buds and transferred to the proliferation medium, the buds grew vigorously, with dark green leaves and moderate internodes. There was no vitrification (semi-transparent water stains) or browning (brown necrosis). This indicates that the proliferation medium formula and culture conditions of the present invention can effectively support the continuous proliferation of buds and maintain excellent quality.

[0039] Figure 4 Color photographs of fully rooted, aseptic tissue culture seedlings. From Figure 4 It can be clearly seen that the root system of the fully rooted seedling is well developed, with sufficient root primordia formation, obvious taproot and many lateral roots. The root system is white or light yellow, showing the vigorous vitality of the newly rooted seedling. The above-ground parts have upright stems, unfolded leaves, and normal leaf color, presenting an ideal state of "strong roots and healthy seedlings".

[0040] Figure 5 Color photos of seedlings that have survived transplanting after hardening off. From Figure 5 As can be seen, after transplanting the tissue culture seedlings into the composite substrate, they grew vigorously, with fully expanded leaves, dark green leaf color, and obvious new tissue. The plants have fully adapted to the external environment and established an independent nutrient absorption system, which directly proves the high efficiency of the transplanting scheme of this invention and the suitability of the composite substrate. Detailed Implementation

[0041] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] Example 1 1. Pretreatment of explants before collection Starting 7 days before harvesting, spray the selected Scutellaria baicalensis mother plants with a 50% carbendazim solution diluted 500 times and a chlorothalonil solution diluted 1000 times, once every 3 days, alternating between the two fungicides, for a total of 2 sprays. The first spray uses a 50% carbendazim solution diluted 500 times, followed by a chlorothalonil solution diluted 1000 times after 3 days, and harvesting the materials 3 days later.

[0043] 2. Explant selection and cleaning On a sunny morning between 9:00 and 10:00, select tender stem segments with axillary buds from the third node below the terminal bud of healthy, disease-free Scutellaria baicalensis plants. Cut the segments to 1.5–2 cm in length using pruning shears, ensuring each segment contains at least one intact axillary bud. Carefully remove the leaves with tweezers on a work surface (avoiding damage to the stem epidermis and axillary buds). Place the cut stem segments in a 250 mL beaker and rinse with running tap water for 30 minutes to remove surface dirt and microorganisms. After rinsing, gently brush the surface of the stem segments with a soft-bristled brush dipped in 5% soap solution for about 1 minute to further remove the surface wax layer and dirt. Then rinse three times with sterile water for 2 minutes each time.

[0044] 3. Aseptic disinfection After cleaning, transfer the stem segments to a laminar flow hood, which has been sterilized by UV irradiation for at least 30 minutes before operation. The specific disinfection procedure is as follows: Place the stem segment in a sterile beaker, add 75% ethanol and soak for 15 seconds, gently shaking to ensure the ethanol fully contacts the surface of the explant. Quickly pour out the ethanol and rinse three times with sterile water for 2 minutes each time to ensure that the ethanol residue is completely removed.

[0045] Then add 0.05% mercuric chloride solution (add two drops of Tween-80 during preparation), shake to sterilize for 15 minutes, shaking continuously to ensure the mercuric chloride solution fully wets the stem segment surface. Pour out the mercuric chloride waste liquid (collect in a dedicated waste liquid bottle), rinse 5 times with sterile water for 2 minutes each time to thoroughly remove mercuric chloride residue.

[0046] Finally, place the disinfected stem segments on sterile filter paper, and gently blot off the surface moisture with another sterile filter paper for later use.

[0047] 4. Clumping bud induction culture The sterilized stem segments were inoculated into MS basal medium + CPPU 0.5 mg / L + NAA 0.05 mg / L + silver nitrate 0.5 mg / L + sucrose 30 g / L + agar 6 g / L, pH 5.8, and cultured under alternating light and dark conditions at 23–25℃, light intensity 2000–2500 lx, and 12 h / d. After 3 days of culture, axillary buds began to sprout, and obvious green buds were visible at the nodes of the stem segments (see...). Figure 1 , Figure 2 Continue culturing until day 5, when the clustered buds showed a clear bud cluster structure. Statistical results showed that the induction rate of clustered buds reached 100% (all 40 stem segments induced clustered buds), and the buds were uniformly bright green, without vitrification or browning.

[0048] 5. Subculture and proliferation The induced clustered shoots were cut into single shoots and inoculated according to physiological polarity into MS basal medium + CPPU 0.5 mg / L + GA3 0.1 mg / L + potassium dihydrogen phosphate 0.08 g / L + sucrose 30 g / L + agar 6 g / L, pH 5.8 proliferation medium. The medium was cultured for 20 days under alternating light and dark conditions at 23–25℃, light intensity of 2000–2500 lx, and 12 h / d. On day 20, the proliferation coefficient was calculated. Each single shoot formed an average of 15.6 clustered shoots (based on a shoot length ≥ 0.5 cm). The shoots were robust, with dark green leaves and no vitrification (see [reference needed]). Figure 3 A small number of white root points may be observed on the surface of the culture medium, but this does not affect the proliferation culture and can be removed during the cutting process.

[0049] 6. Seedling Rooting Culture The bud clusters obtained from the propagation culture were cut into 2-3 cm sections and transferred to a rooting medium of 1 / 2 MS basal medium (with macroelements halved) + PP333 1.0 mg / L + SA 0.5 mg / L + AC 0.2 g / L + sucrose 20 g / L + agar 6 g / L, pH 5.8. The medium was then cultured for 12 days under alternating light and dark conditions at 23-25℃, light intensity of 2000-2500 lx, and 12 h / d. By day 12, each single bud had differentiated into 3-5 robust white roots, 1-3 cm in length, achieving a rooting rate of 100% (see [link to relevant documentation]). Figure 4 The plant has a well-developed root system with abundant root hairs, demonstrating excellent absorption capabilities. Meanwhile, the above-ground plant height is effectively controlled (approximately 30% shorter than the control without PP333), with robust stems, thick leaves, and a deep green color, exhibiting the excellent condition of a "short and sturdy seedling."

[0050] 7. Hardening off seedlings and transplanting Select well-developed root systems (root length ≥ 2 cm, number of roots ≥ 3) and seedling height 5–7 cm for transplanting. Move the tissue culture bottles indoors under natural light (avoid direct sunlight) and harden off for 4 days with the lids off to allow the seedlings to gradually adapt to the external environment. During the hardening-off period, keep the environment humid (you can sprinkle water around the seedlings) to prevent the culture medium from drying out too quickly in the air, which could cause the seedlings to dehydrate.

[0051] After hardening off, carefully remove the seedlings with tweezers and gently wash away the agar medium adhering to the roots in warm water (be careful not to damage the young roots). Transplant the cleaned seedlings into the pre-prepared composite substrate. The composite substrate ratio is humus: perlite: river sand = 3:1:1 (by weight). Before use, the substrate should be sterilized by high-pressure steam at 121℃ for 20 minutes or by spraying with 800 times diluted carbendazim, and the moisture content should be adjusted to the point where it can be formed into a clump when squeezed but crumbles easily when released.

[0052] For the first 10 days after transplanting, cover with a transparent plastic film to maintain moisture and keep the relative humidity at 95%. Control the daytime temperature at 23-25℃ and the nighttime temperature at 18-20℃. Ten days after transplanting, the seedlings will have developed new roots and leaves. At this time, remove the film and transplant them into nutrient cups using the same substrate formula as before. Twenty-five days after transplanting, the seedlings will be growing vigorously and will have produced 2-3 pairs of new leaves above ground, at which point they can be planted in the field. The transplant survival rate is 100% (all 40 transplanted seedlings survived). (See [link to relevant documentation]) Figure 5 ).

[0053] Example 2 1. Pretreatment of explants before collection Spray the selected Scutellaria baicalensis mother plants with a 500-fold dilution of 50% carbendazim and a 1000-fold dilution of chlorothalonil, once every 4 days, alternating between the two fungicides, for a total of 2 sprays. For the first spray, use a 1000-fold dilution of chlorothalonil, and after 4 days, use a 500-fold dilution of 50% carbendazim. Harvest the material after another 4 days.

[0054] 2. Explant selection and cleaning Select young stem segments with axillary buds from the fourth node below the terminal bud of healthy, disease-free Scutellaria baicalensis plants. Cut them to a length of 1.5–2 cm, remove the leaves, rinse with running water for 30 minutes, scrub with soapy water, and then rinse three times with sterile water for 2 minutes each time.

[0055] 3. Aseptic disinfection In a clean bench, soak the stem segments in 75% ethanol for 20 seconds, rinse three times with sterile water for 2 minutes each time; then shake with 0.05% mercuric chloride and two drops of Tween-80 for 18 minutes, rinse seven times with sterile water for 2 minutes each time, and finally blot dry the surface moisture with sterile filter paper.

[0056] 4. Clumping bud induction culture The sterilized stem segments were inoculated into a shoot induction medium with the following formulation: MS medium + CPPU 0.2 mg / L + NAA 0.1 mg / L + silver nitrate 0.5 mg / L + sucrose 30 g / L + agar 6 g / L, pH 5.8. The samples were cultured for 5 days at 23–25℃, 12 h / d light, and 2000–2500 lx light intensity. The shoot induction rate reached 98.3% (59 out of 60 stem segments induced shoots), with uniform shoots and no vitrification.

[0057] 5. Subculture and proliferation The clustered buds were divided into single buds and transferred to proliferation medium with the following formulation: MS medium + CPPU 0.8 mg / L + GA3 0.3 mg / L + potassium dihydrogen phosphate 0.08 g / L + sucrose 30 g / L + agar 6 g / L, pH 5.8. After 22 days of culture, the average proliferation coefficient was 12.9.

[0058] 6. Seedling Rooting Culture The bud clusters obtained from the propagation culture were cut into 2-3 cm sections and transferred to rooting medium. The medium formula was: 1 / 2 MS + PP333 1.5 mg / L + SA 1.0 mg / L + AC 0.2 g / L + sucrose 20 g / L + agar 6 g / L, pH 5.8. After 15 days of culture, the rooting rate reached 100%.

[0059] 7. Hardening off seedlings and transplanting Tissue culture seedlings were hardened off indoors for 5 days with the covers off. The roots were then washed to remove the agar and transplanted into a composite substrate consisting of humus, perlite, and river sand in a 3:1:1 mass ratio. For the first 10 days after transplanting, the relative humidity was maintained at 95%, with daytime temperatures controlled at 23–25℃ and nighttime temperatures at 18–20℃. Ten days after transplanting, the seedlings were planted in cups, and 25 days after transplanting, they were planted in the field. The transplant survival rate reached 96.3% (77 out of 80 transplanted seedlings survived).

[0060] Example 3 1. Pretreatment of explants before collection Spray the selected Scutellaria baicalensis mother plants with a 50% carbendazim solution diluted to 500 times and a chlorothalonil solution diluted to 1000 times, once every 5 days, alternating between the two fungicides, for a total of 3 sprays. The spraying order is: carbendazim → chlorothalonil → carbendazim, with an interval of 5 days between each spray.

[0061] 2. Explant selection and cleaning Select young stem segments with axillary buds from the 5th node below the terminal bud of healthy, disease-free Scutellaria baicalensis plants. Cut them to a length of 1.5–2 cm, remove the leaves, rinse with running water for 30 minutes, scrub with soapy water, and then rinse three times with sterile water for 2 minutes each time.

[0062] 3. Aseptic disinfection In a clean bench, soak the stem segments in 75% ethanol for 25 seconds, rinse three times with sterile water for 2 minutes each time; then shake with 0.05% mercuric chloride and two drops of Tween-80 for 20 minutes, rinse eight times with sterile water for 2 minutes each time, and finally blot dry the surface moisture with sterile filter paper.

[0063] 4. Clumping bud induction culture The sterilized stem segments were inoculated into a shoot induction medium with the following formulation: MS medium + CPPU 0.8 mg / L + NAA 0.15 mg / L + silver nitrate 0.5 mg / L + sucrose 30 g / L + agar 6 g / L, pH 5.8. The samples were cultured for 10 days at 23–25℃, 12 h / d light, and 2000–2500 lx light intensity. The shoot induction rate reached 96.4% (81 out of 84 stem segments induced shoots), with uniform shoots and no vitrification.

[0064] 5. Subculture and proliferation The clustered buds were divided into single buds and transferred to proliferation medium with the following formulation: MS medium + CPPU 1.0 mg / L + GA3 0.5 mg / L + potassium dihydrogen phosphate 0.08 g / L + sucrose 30 g / L + agar 6 g / L, pH 5.8. After 25 days of culture, the average proliferation coefficient was 13.5.

[0065] 6. Seedling Rooting Culture The bud clusters obtained from the propagation culture were cut into 2-3 cm sections and transferred to rooting medium. The medium formula was: 1 / 2 MS + PP333 2.0 mg / L + SA 1.5 mg / L + AC 0.2 g / L + sucrose 20 g / L + agar 6 g / L, pH 5.8. After 18 days of culture, the rooting rate reached 100%.

[0066] 7. Hardening off seedlings and transplanting Tissue culture seedlings were hardened off indoors for 6 days with the covers off. The roots were then washed to remove the agar and transplanted into a composite substrate consisting of humus, perlite, and river sand in a 3:1:1 mass ratio. For the first 10 days after transplanting, the relative humidity was maintained at 95%, with daytime temperatures controlled at 23–25℃ and nighttime temperatures at 18–20℃. Ten days after transplanting, the seedlings were planted in cups, and 25 days after transplanting, they were planted in the field. The transplant survival rate reached 98.2% (108 out of 110 transplanted seedlings survived).

[0067] Example 4 (Verification of Intermediate Parameter Values) 1. Pretreatment of explants before collection Spray the selected Scutellaria baicalensis mother plants with a 500-fold dilution of 50% carbendazim and a 1000-fold dilution of chlorothalonil every 4 days, alternating between the two solutions, for a total of 3 sprays.

[0068] 2. Explant selection and cleaning Select tender stem segments with axillary buds from the third node below the terminal bud of healthy, disease-free Scutellaria baicalensis plants, cut them to a length of 1.5–2 cm, and clean them according to the method in Example 1.

[0069] 3. Aseptic disinfection The same disinfection procedure as in Example 1 was used: soaking in 75% ethanol for 18 seconds, rinsing 3 times with sterile water; shaking in 0.05% mercuric chloride and Tween-80 for 17 minutes, rinsing 6 times with sterile water.

[0070] 4. Clumping bud induction culture The culture medium formula was: MS medium + CPPU 0.6 mg / L + NAA 0.12 mg / L + silver nitrate 0.5 mg / L + sucrose 30 g / L + agar 6 g / L, pH 5.8. After 7 days of culture, the induction rate of shoot clusters reached 97.8%, and the shoots were normal.

[0071] 5. Subculture and proliferation The culture medium formulation was: MS + CPPU 0.7 mg / L + GA3 0.25 mg / L + potassium dihydrogen phosphate 0.08 g / L. After 21 days of culture, the proliferation coefficient was 14.2.

[0072] 6. Seedling Rooting Culture The culture medium formula was: 1 / 2 MS + PP333 1.5 mg / L + SA 0.8 mg / L + AC 0.2 g / L. After 14 days of culture, the rooting rate was 100%.

[0073] 7. Hardening off seedlings and transplanting Following the method described in Example 1, the transplant survival rate was 98.5%.

[0074] Comparative Example 1 (Existing Patent Technology CN101536672B) The method was implemented according to the technical solution disclosed in patent CN101536672B, "A Method for Organogenesis of the Traditional Chinese Medicine Scutellaria baicalensis". Stalks of Scutellaria baicalensis without axillary buds were used as explants. After sterilization with 75% ethanol and 0.1% mercuric chloride, they were inoculated into callus induction medium (MS + 2,4-D 2.0 mg / L + 6-BA 0.5 mg / L) and cultured in the dark to induce callus formation. After callus formation, the tissue was transferred to differentiation medium (MS + 6-BA 2.0 mg / L + NAA 0.2 mg / L) to induce adventitious bud differentiation. Regenerated plants were then obtained through subculture and rooting culture.

[0075] The results showed that the contamination rate was 25% (10 out of 40 explants were contaminated), the callus induction rate was 100%, but callus germination took 7 days, and it took more than 14 days for the callus to differentiate into shoot clusters. The shoot cluster induction rate (based on explants) was 92.2%, the proliferation culture time was as long as 40 days, the proliferation coefficient was 12.3, the rooting culture time was 30 days, and the rooting rate was 98.6%. During the subculture process, some plants were observed to exhibit variations such as leaf deformities and weakened growth.

[0076] Comparative Example 2 (Document Technical Solution) The procedure was carried out according to the technical scheme reported by Zhao Zhenling et al. (Scutellaria baicalensis somatic embryo induction and plant regeneration, Southwest China Journal of Agricultural Sciences, 2006, 19(4): 714-718). Using tender shoots or stem segments with buds from Example 1 as explants, callus tissue was induced using MS + 2,4-D 1.0 mg / L + 6-BA 0.5 mg / L, then transferred to MS + 6-BA 2.0 mg / L + NAA 0.5 mg / L to differentiate somatic embryos, and finally transferred to maturation and germination media to obtain regenerated seedlings.

[0077] The results showed that the contamination rate was 28.9% (13 out of 45 explants were contaminated), the initial induction time was 20 days, the induction rate of clustered shoots (somatic embryo germination rate) was 91.4%, the proliferation culture time was 25 days, the proliferation coefficient was 7.2, the rooting culture time was 30 days, the rooting rate was 85.5%, and the transplant survival rate was 70-80%, showing obvious variation.

[0078] Comparative Example 3 (Document Technical Solution) The procedure was carried out according to the technical scheme reported by Li Yaqiong et al. (Research on rapid propagation of Scutellaria baicalensis, Chinese Medicinal Herbs, 2007, 30(7): 761-762). Tender shoots or stem segments with buds from Example 1 were used as explants. Clustered shoots were induced using MS + 6-BA 1.0 mg / L + NAA 0.05 mg / L, followed by propagation culture using MS + 6-BA 0.5 mg / L + NAA 0.05 mg / L, and rooting culture using 1 / 2 MS + NAA 0.5 mg / L.

[0079] The results showed that the contamination rate was 27.3% (15 out of 55 explants were contaminated), the initial induction time was 4 days, the induction rate of shoot clusters was 100%, the propagation culture time was 25 days with a propagation coefficient of 6.45, the rooting culture time was 15 days with a rooting rate of 90%, and the transplant survival rate was 100%. Although the transplant survival rate was high, the propagation coefficient was only 6.45, indicating limited propagation efficiency.

[0080] Comparison and comprehensive analysis of the effects of the examples and comparative examples The main technical effects of each embodiment and the comparative example are compared in the table below:

[0081] The following conclusions can be drawn from the above comparative analysis: First, regarding contamination rate control, the contamination rates of the various embodiments of this invention (2.5%–5.3%) are significantly lower than those of the comparative examples (25.0%–28.9%), representing a reduction of over 80%. This demonstrates that the combined approach of "pretreatment with bactericide before material collection + optimized gradient disinfection" of this invention effectively solves the industry problem of high explant contamination rates in Scutellaria baicalensis tissue culture.

[0082] Secondly, regarding induction efficiency, this invention uses axillary buds to directly induce clustered shoots, completing primary induction in 3-10 days with a clustered shoot induction rate exceeding 96%. In contrast, Comparative Example 1 requires callus germination (7 days) before differentiation into shoots, resulting in a longer overall induction period; Comparative Example 2's somatic embryo induction requires 20 days. This invention has a significant advantage in induction speed compared to existing technologies.

[0083] Third, regarding genetic stability, this invention bypasses the callus dedifferentiation-redifferentiation process through direct axillary bud germination, thus reducing the risk of somatic cell mutation from the source. No visible variation was observed in six consecutive generations of subculture. In contrast, Comparative Examples 1 and 2, which involved callus or somatic embryo stages, both showed varying degrees of variation during subculture.

[0084] Fourth, regarding proliferation efficiency, the proliferation coefficient of this invention is above 10.0 (optimal 15.6), significantly better than Comparative Example 2 (7.2) and Comparative Example 3 (6.45), increasing by approximately 2 times and 1.4 times respectively. Although the proliferation coefficient of Comparative Example 1 (12.3) is close to that of this invention, its culture period is as long as 40 days, and its proliferation efficiency (per unit time) is significantly lower than that of this invention.

[0085] Fifth, regarding the rooting effect, this invention can achieve 100% rooting in 12 to 18 days, which is 40% to 60% shorter than Comparative Example 1 (30 days) and Comparative Example 2 (30 days). The rooting rate (100%) is also higher than Comparative Example 2 (85.5%) and Comparative Example 3 (90%).

[0086] Sixth, regarding transplant survival rate. The transplant survival rates of Examples 1 and 4 of this invention can reach 100%, and Example 3 is 98.2%, which is significantly better than the 70-80% of Comparative Example 2. Although Comparative Example 3 also achieves a 100% survival rate, its insufficient propagation coefficient limits its application value in large-scale production.

[0087] In summary, this invention outperforms existing technologies in key indicators such as contamination rate, induction efficiency, proliferation coefficient, rooting speed, and transplant survival rate, achieving efficient, stable, and high-quality rapid propagation of Scutellaria baicalensis through tissue culture, and has promising prospects for industrial application.

[0088] Industrial applicability The method of this invention has a standardized operation process, clear process parameters, and good reproducibility, making it suitable for standardized and large-scale seedling production. Through the implementation of this invention, high-quality, genetically consistent, and abundant seedlings can be provided for the protection of Scutellaria baicalensis germplasm resources, the construction of standardized planting bases for authentic medicinal materials, and downstream pharmaceutical and plant-based skincare raw material industries. It has good industrial applicability and economic value.

[0089] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for efficient inducing clustered buds from Scutellaria baicalensis stem segments via tissue culture for rapid propagation, characterized in that... Includes the following steps: (1) Pretreatment of explants: The selected mother plants of Scutellaria baicalensis were alternately sprayed with 50% carbendazim diluted 500 times and chlorothalonil diluted 1000 times, once every 3 to 5 days, for 2 to 3 consecutive times. (2) Selection and cleaning of explants: Select tender stem segments with axillary buds from the 3rd to 5th nodes below the terminal bud of healthy and disease-free Scutellaria baicalensis plants. Cut the segments to a length of 1.5 to 2 cm, remove the leaves, rinse with running water for 30 min, scrub with soap solution, and then rinse with sterile water 3 times for 2 min each time. (3) Sterile disinfection: Place the cleaned stem segments in a clean bench, soak them in 75% ethanol for 15-20 seconds, rinse them with sterile water 3 times for 2 minutes each time; then disinfect them with 0.05% mercuric chloride plus two drops of Tween-80 for 15-20 minutes, rinse them with sterile water 5-8 times for 2 minutes each time, and finally dry the surface moisture with sterile filter paper. (4) Induction culture of clustered shoots: The sterilized stem segments were inoculated into the induction culture medium for clustered shoots and cultured at 23-25℃, light intensity of 12h / d and light intensity of 2000-2500 lx for 3-10 days to obtain clustered shoots; the induction culture medium for clustered shoots was based on MS medium, with the addition of CPPU 0.2-1.0 mg / L, NAA 0.05-0.2 mg / L, silver nitrate 0.5 mg / L, sucrose 30 g / L, agar 6 g / L, and pH 5.8; (5) Subculture and proliferation: The obtained clustered shoots were divided into single shoots and transferred to proliferation medium for culture for 20-22 days; the proliferation medium was MS as the basic medium, with CPPU 0.5-1.0 mg / L, GA3 0.1-0.5 mg / L, potassium dihydrogen phosphate 0.08 g / L, sucrose 30 g / L, agar 6 g / L, pH 5.

8. (6) Seedling rooting culture: Select robust single buds with a height of 2-3 cm and transfer them to the rooting medium. Culture for 12-18 days to obtain complete rooted seedlings. The rooting medium is based on 1 / 2 MS medium, with PP333 1.0-2.0 mg / L, SA 0.5-1.5 mg / L, AC 0.2 g / L, sucrose 20 g / L, agar 6 g / L, and pH 5.

8. (7) Hardening and transplanting: Harden the tissue culture seedlings indoors for 3-5 days, wash the culture medium from the roots, and transplant them into a composite substrate of humus, perlite and river sand in a mass ratio of 3:1:

1. Maintain a relative humidity of 95% for the first 10 days after transplanting, control the daytime temperature at 23-25℃ and the nighttime temperature at 18-20℃. Plant them in cups after 10 days, and carry out field cultivation after 25 days to obtain Scutellaria baicalensis seedlings.

2. The method for efficient induction of clustered buds from Scutellaria baicalensis stem segments via tissue culture for rapid propagation according to claim 1, characterized in that, In step (1), the 50% carbendazim diluted 500 times and the chlorothalonil diluted 1000 times are sprayed once every 4 days, alternating between the two, for a total of 2 consecutive sprays.

3. The method for efficient inducing clustered buds from Scutellaria baicalensis stem segments via tissue culture for rapid propagation according to claim 1, characterized in that, In step (3), the specific operation of sterilization is as follows: soaking in 75% ethanol for 15 seconds, rinsing with sterile water 3 times, 2 minutes each time; Add two drops of 0.05% mercuric chloride and Tween-80, shake for 15 minutes, then rinse 5 times with sterile water for 2 minutes each time.

4. The method for efficient inducing clustered buds from Scutellaria baicalensis stem segments via tissue culture for rapid propagation according to claim 1, characterized in that, In step (4), the formula of the shoot induction medium is: MS+CPPU 0.5 mg / L, NAA 0.05 mg / L, silver nitrate 0.5 mg / L, sucrose 30 g / L, agar 6 g / L, pH 5.

8.

5. The method for efficient inducing clustered buds from Scutellaria baicalensis stem segments via tissue culture for rapid propagation according to claim 1, characterized in that, In step (5), the formulation of the proliferation medium is: MS+CPPU 0.5 mg / L, GA3 0.1 mg / L, potassium dihydrogen phosphate 0.08 g / L, sucrose 30 g / L, agar 6 g / L, pH 5.

8.

6. The method for efficient inducing clustered buds from Scutellaria baicalensis stem segments via tissue culture for rapid propagation according to claim 1, characterized in that, The rooting medium formula is as follows: 1 / 2MS + PP333 1.0 mg / L, SA 0.5 mg / L, AC 0.2 g / L, sucrose 20 g / L, agar 6 g / L, pH 5.

8.

7. The method for efficient induction of clustered buds from Scutellaria baicalensis stem segments via tissue culture for rapid propagation according to claim 6, characterized in that, In step (4), the concentration of CPPU added to the shoot induction medium is 0.5 mg / L and the concentration of NAA is 0.05 mg / L.

8. The method for efficient inducing clustered buds from Scutellaria baicalensis stem segments via tissue culture for rapid propagation according to claim 1, characterized in that, In step (5), the concentration of CPPU added to the proliferation medium is 0.5 mg / L and the concentration of GA3 is 0.1 mg / L.

9. The method for efficient inducing clustered buds from Scutellaria baicalensis stem segments via tissue culture for rapid propagation according to claim 1, characterized in that, In step (6), the concentration of PP333 added to the rooting medium is 1.0 mg / L and the concentration of SA is 0.5 mg / L.

10. The application of the method according to any one of claims 1 to 9 in the in vitro preservation of Scutellaria baicalensis germplasm, industrialized seedling cultivation, or large-scale production of medicinal raw materials.

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