Special culture medium for tissue culture and rapid propagation of caulis spatholobi and efficient propagation method

Abstract

The invention discloses a special culture medium for tissue culture and rapid propagation of caulis spatholobi and an efficient propagation method, and relates to the technical field of plant biology. The special culture medium contains a callus induction and proliferation culture medium, an adventitious bud differentiation culture medium, an adventitious bud proliferation culture medium and a rooting culture medium which are respectively based on MS or 1 / 2 MS, the hormone ratio is optimized, special additives such as casein hydrolysate and inositol are added, and a differentiation-promoting and anti-browning synergistic microenvironment is constructed. According to the breeding method, fine parameters of explant selection, disinfection, culture in each stage and acclimatization and transplantation are defined, the bottleneck that the caulis spatholobi callus is difficult to differentiate into buds is effectively broken through, the browning rate, the multiplication coefficient and the transplantation survival rate are reduced, the technical repeatability is good, the stability is high, and reliable technical support is provided for industrialized production of caulis spatholobi seedlings.

Description

Technical Field

[0001] This invention relates to the field of plant biotechnology, and in particular to a special culture medium for rapid propagation of *Chicken Blood Vine* tissue culture and an efficient propagation method. Background Technology

[0002] Chicken blood vine (Millettia dielsiana) is a woody vine belonging to the genus Millettia in the legume family. It is a traditional and valuable Chinese medicinal herb, used medicinally for its dried stems. It is believed to have effects such as promoting blood circulation, replenishing blood, regulating menstruation, relieving pain, and relaxing muscles and tendons, and is widely used in clinical treatment and health products. However, with continuously increasing market demand, wild resources of chicken blood vine are becoming increasingly depleted due to over-harvesting. Its weak natural reproductive capacity and long growth cycle further exacerbate the resource supply contradiction. Therefore, developing efficient artificial propagation techniques to achieve the sustainable utilization of chicken blood vine resources has become an urgent need for industrial development.

[0003] Tissue culture, a modern biotechnology enabling rapid and large-scale asexual reproduction of plants, is an ideal solution to the shortage of *Millettia dielsiana* seedlings. Currently, research has attempted to utilize young leaves and stem tips of *Millettia dielsiana* as explants for in vitro culture. However, this technology still faces several key technical bottlenecks, severely hindering its industrial application.

[0004] First, obtaining and processing explant materials is difficult. Existing research shows that only the second- to third-order young leaves at the top of the stem tip can effectively induce callus tissue; explants that are too young or too old are unlikely to succeed. More importantly, *Millettia dielsiana* is rich in red sap, which contains high levels of polyphenols. Explants are highly susceptible to oxidative browning during cutting, directly poisoning the explants and increasing the risk of contamination. Conventional single disinfection methods have limited effectiveness, resulting in high contamination and browning rates, becoming the primary obstacle to establishing a sterile culture system.

[0005] Secondly, the low efficiency of bud organ induction and differentiation is a current research bottleneck. Existing techniques can mostly induce callus formation from explants, but the rate of further differentiation into adventitious buds or directly inducing the formation of clustered buds from explants is extremely low. Even when callus is obtained, its redifferentiation ability is weak, and the bud formation rate is highly unstable. How to efficiently and stably achieve morphological formation from explants to clustered buds is key to breaking through rapid propagation techniques.

[0006] Furthermore, the propagation and rooting system is incomplete. Even if a small number of sterile seedlings are obtained, their subsequent subcultures often have low multiplication rates, slow growth, and are prone to vitrification or degeneration, making it difficult to obtain a large amount of propagation material in a short period of time. The rooting stage suffers from unstable rooting rates and poor root quality, resulting in large fluctuations in survival rates during the transplanting and acclimatization stage, which cannot meet the requirements of large-scale production for seedling quality and quantity.

[0007] In summary, current tissue culture techniques for *Spatholobus suberectus* have not yet formed a complete, efficient, stable, and reproducible system for rapid propagation, particularly lacking systematic solutions for key aspects such as browning contamination control, efficient bud induction and differentiation, and robust proliferation. Therefore, developing a specialized culture medium and corresponding efficient propagation methods to address these challenges is of great significance for achieving industrialized production of *Spatholobus suberectus* seedlings, protecting wild resources, and ensuring a sustainable supply of traditional Chinese medicinal materials.

[0008] The above background information is provided only to aid in understanding the inventive concept and technical solution of this invention. It does not necessarily belong to the prior art of this patent application. In the absence of clear evidence that the above information was disclosed on the filing date of this patent application, the above background information should not be used to evaluate the novelty and inventiveness of this application. Summary of the Invention

[0009] The main purpose of this invention is to propose a special culture medium for rapid propagation of *Spatholobus suberectus* tissue culture and an efficient propagation method, so as to solve the technical problems of high browning rate, difficulty in callus differentiation into buds, small subculture proliferation coefficient, difficulty in rooting, and low transplant survival rate in the existing technology of *Spatholobus suberectus* tissue culture.

[0010] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0011] A special culture medium for rapid propagation of *Spatholobus suberectus* tissue culture includes a callus induction medium, a callus proliferation medium, an adventitious bud differentiation medium, an adventitious bud proliferation medium, and a rooting medium.

[0012] Preferably, the callus induction medium, callus proliferation medium, adventitious shoot differentiation medium and adventitious shoot proliferation medium are all based on MS medium, and the rooting medium is based on 1 / 2 MS medium.

[0013] Preferably, the callus induction culture medium comprises the following components: 6-benzylaminopurine 0.8-1.0 mg / L, naphthaleneacetic acid 0.3-0.5 mg / L, kinetin 0.3-0.5 mg / L, vitamin C 0.1-0.5 mg / L, sucrose 20-30 g / L, agar 6-8 g / L, and a pH of 5.8-6.0.

[0014] The callus proliferation culture medium consists of the following components: 6-benzylaminopurine 0.8-1.0 mg / L, naphthaleneacetic acid 0.3-0.5 mg / L, kinetin 0.1-0.3 mg / L, vitamin C 0.1-0.5 mg / L, sucrose 20-30 g / L, agar 6-8 g / L, and pH 5.8-6.0.

[0015] Preferably, the adventitious bud differentiation culture medium comprises the following components: 6-benzylaminopurine 2.3-2.7 mg / L, naphthaleneacetic acid 0.3-0.5 mg / L, gibberellin 0.9-1.1 mg / L, vitamin C 0.1-0.5 mg / L, sucrose 20-30 g / L, agar 6-8 g / L, hydrolyzed casein 0.5-1.0 g / L, and a pH of 5.8-6.0.

[0016] The components of the adventitious bud proliferation culture medium are as follows: 6-benzylaminopurine 2.5-3.5 mg / L, naphthaleneacetic acid 0.1-0.2 mg / L, gibberellin 0-0.5 mg / L, vitamin C 0.3-0.5 mg / L, sucrose 25-30 g / L, agar 6-8 g / L, inositol 0.1-0.2 g / L, and pH value 5.8-6.0;

[0017] The rooting medium consists of the following components: naphthaleneacetic acid 0.1-0.3 mg / L, indolebutyric acid 0.3-0.5 mg / L, vitamin C 0.1-0.5 mg / L, sucrose 20-30 g / L, agar 6-8 g / L, glutamine 0.2-0.3 g / L, and pH 5.8-6.0.

[0018] This invention also provides a method for efficient propagation of *Spatholobus suberectus*, comprising the following steps:

[0019] S1. Explant selection and pretreatment: Young leaves and stem tips of *Spatholobus suberectus* were selected as explants and pretreated.

[0020] S2. Segmented disinfection of explants: The pretreated explants are disinfected in segments.

[0021] S3. Inoculation and induction culture: The sterilized explants were inoculated into the callus induction culture medium and cultured under the conditions of light intensity of 1000-2000 Lux, light time of 12-14 h / d, and temperature of 23-27℃ to induce the formation of callus tissue, which was then inoculated into the callus proliferation culture medium for proliferation.

[0022] S4. Adventitious bud differentiation: The dense, bright green callus tissue after proliferation is inoculated into the adventitious bud differentiation medium to induce the generation of adventitious buds.

[0023] S5. Subculture and propagation: Adventitious shoots are transferred to adventitious shoot propagation medium for propagation to obtain a large number of robust adventitious shoots;

[0024] S6. Rooting culture: Transfer healthy adventitious buds that have reached a height of 2-3cm to a rooting medium for rooting culture to obtain complete tissue culture seedlings;

[0025] S7. Hardening off and transplanting: After hardening off the well-rooted tissue culture seedlings, transplant them into the substrate.

[0026] Preferably, in step S1, when the explant is a young leaf or stem tip, the pretreatment method is as follows: rinse with running water for 30-60 minutes, then soak in a washing solution containing 0.05-0.1% Tween-80 for 5-10 minutes, and then rinse with sterile water 3-5 times.

[0027] Preferably, in step S2, the specific steps of segmented disinfection are as follows:

[0028] a. Soak the pretreated explants in 70-75% ethanol for 30 seconds in a clean bench, then disinfect them with 0.1-0.2% mercuric chloride for 6-10 minutes, and finally rinse them with sterile water 4-6 times.

[0029] b. The time for mercuric chloride immersion disinfection varies depending on the type of explant:

[0030] If the leaves are young, soak them twice in a 0.1-0.2% mercuric chloride solution, the first time for 2-4 minutes and the second time for 4-6 minutes.

[0031] If it is the stem tip, soak it twice with a 0.1-0.2% mercuric chloride solution, the first time for 3-5 minutes and the second time for 3-5 minutes;

[0032] Shake constantly during the soaking process;

[0033] c. After each disinfection, rinse with sterile water 4-6 times and use sterile filter paper to absorb the surface moisture.

[0034] Preferably, the rooting culture conditions in step S6 are: light intensity 800-1500 Lux, light duration 8-10 h / d, and temperature 24-26℃.

[0035] Preferably, in step S7, the seedling hardening method is as follows: the culture bottle is moved to diffused light with an intensity of 1800-2000 Lux for 7-10 days for hardening, and then the bottle cap is opened for 1-2 days for hardening.

[0036] Preferably, in step S7, the transplanting substrate is a mixture of loess, peat moss and vermiculite in a volume ratio of (2-3):1:1. After transplanting, the air humidity is maintained at 75-85% and ventilation is provided. Subsequently, the humidity is gradually reduced and the light intensity is increased.

[0037] The beneficial effects of this invention compared to the prior art include:

[0038] 1. Overcome the core technical bottlenecks in bud formation induction and broaden the sources of explants.

[0039] This invention effectively overcomes the bottleneck of callus differentiation from *Millettia dielsiana* explants. Existing technologies often fall into the trap of hindering callus redifferentiation. This invention, through an optimized induction culture medium formulation, particularly the synergistic ratio of cytokinins (6-BA and kinetin) and auxin (NAA), creates a microenvironment for explants that directly initiates shoot primordia differentiation or promotes callus redifferentiation. This approach can guide various readily available explants, such as young leaves and shoot tips, to efficiently and stably form clustered shoots, thus breaking the limitations of material sources and providing a reliable way to solve the fundamental problem of rapid propagation of *Millettia dielsiana*.

[0040] 2. Establish a comprehensive and systematic browning and pollution prevention and control system.

[0041] A systematic, end-to-end browning and contamination control system has been established. While existing technologies employ methods such as segmented disinfection and the addition of antioxidants, control measures are mostly concentrated in the initial stage and lack fine-tuning tailored to the characteristics of different explants. This invention not only optimizes the differentiated explant disinfection procedure but also scientifically configures combinations of anti-browning substances with different functions and dosages in the culture media at each stage of callus induction and proliferation, adventitious shoot differentiation, adventitious shoot propagation, and rooting. This progressive, multi-level control strategy, designed based on the characteristics of each culture stage and the pattern of browning occurrence, improves the survival rate and condition of cultures, laying a solid foundation for the smooth progress of subsequent culture processes.

[0042] 3. Construct a phased, seamless, efficient, and rapid process chain.

[0043] A highly efficient and rapid propagation process chain with clearly defined stage objectives and smooth transitions has been constructed. Existing cultivation methods often use culture media with similar compositions for induction and proliferation, making it difficult to simultaneously achieve high seedling quality and proliferation efficiency. This invention creatively divides the rapid propagation process into five functional stages: callus induction, callus proliferation, adventitious shoot differentiation, adventitious shoot propagation, and rooting, and develops dedicated culture media for each stage. Each culture medium has a unique design in terms of hormone ratios and additive composition, prioritizing the efficient initiation of differentiation, promotion of robust proliferation, and induction of high-quality rooting, respectively. This specialized division of labor and relay-style cultivation model ensures maximum efficiency in each stage of rapid propagation and overall controllable seedling quality.

[0044] 4. Achieve high repeatability, stability, and standardization of the technical solution.

[0045] This invention enhances the reproducibility, stability, and standardization potential of the technical solution. Existing methods suffer from poor operational reproducibility and large fluctuations in results due to a lack of systematic optimization and clear parameters in key steps, making them unsuitable for large-scale production. This invention provides clear, complete, and optimized parameter ranges and operational specifications for everything from explant selection and pretreatment, categorized disinfection, and culture conditions at each stage to hardening and transplanting environments. The entire technical system is logically self-consistent, with tightly integrated steps, reducing over-reliance on individual operator experience. This allows different operators to obtain stable and consistent culture results across different batches, providing crucial support for the transformation of laboratory validation into industrial applications. Detailed Implementation

[0046] The present invention will be further described in detail below with reference to specific embodiments. It should be emphasized that the following description is merely exemplary and is not intended to limit the scope and application of the present invention.

[0047] A special culture medium for rapid propagation of *Spatholobus suberectus* tissue culture and an efficient propagation method are as follows:

[0048] 1. Tissue culture rapid propagation special culture medium

[0049] (1) Components of callus induction culture medium: 6-benzylaminopurine 0.8-1.0 mg / L, naphthaleneacetic acid 0.3-0.5 mg / L, kinetin 0.3-0.5 mg / L, vitamin C 0.1-0.5 mg / L, sucrose 20-30 g / L, agar 6-8 g / L, pH value 5.8-6.0;

[0050] (2) Components of callus proliferation culture medium: 6-benzylaminopurine 0.8-1.0 mg / L, naphthaleneacetic acid 0.3-0.5 mg / L, kinetin 0.1-0.3 mg / L, vitamin C 0.1-0.5 mg / L, sucrose 20-30 g / L, agar 6-8 g / L, pH value 5.8-6.0;

[0051] (3) Components of adventitious bud differentiation medium: 6-benzylaminopurine 2.3-2.7 mg / L, naphthaleneacetic acid 0.3-0.5 mg / L, gibberellin 0.9-1.1 mg / L, vitamin C 0.1-0.5 mg / L, sucrose 20-30 g / L, agar 6-8 g / L, hydrolyzed casein 0.5-1.0 g / L, pH value 5.8-6.0;

[0052] (4) Components of adventitious bud proliferation culture medium: 6-benzylaminopurine 2.5-3.5 mg / L, naphthaleneacetic acid 0.1-0.2 mg / L, gibberellin 0-0.5 mg / L, vitamin C 0.3-0.5 mg / L, sucrose 25-30 g / L, agar 6-8 g / L, inositol 0.1-0.2 g / L, pH value 5.8-6.0;

[0053] (5) Components of rooting medium: naphthaleneacetic acid 0.1-0.3 mg / L, indolebutyric acid 0.3-0.5 mg / L, vitamin C 0.1-0.5 mg / L, sucrose 20-30 g / L, agar 6-8 g / L, glutamine 0.2-0.3 g / L, pH value 5.8-6.0.

[0054] 2. High-efficiency breeding methods

[0055] Includes the following steps:

[0056] S1. Selection and pretreatment of explants: Select tender leaves and stem tips of *Spatholobus suberectus* as explants; Pretreatment method for tender leaves or stem tips: Rinse with running water for 30-60 minutes, then soak in a washing solution containing 0.05-0.1% Tween-80 for 5-10 minutes, and then rinse with sterile water 3-5 times.

[0057] S2. Segmented disinfection of explants:

[0058] a. Soak the pretreated explants in 70-75% ethanol for 30 seconds in a clean bench, then disinfect them with 0.1-0.2% mercuric chloride for 6-10 minutes, and finally rinse them with sterile water 4-6 times.

[0059] b. The time for disinfection by soaking in mercuric chloride solution varies depending on the type of explant: If it is a young leaf, soak it twice with a 0.1-0.2% mercuric chloride solution, the first time for 2-4 minutes and the second time for 4-6 minutes;

[0060] For stem tips, soak twice in a 0.1-0.2% mercuric chloride solution, the first time for 3-5 minutes and the second time for 3-5 minutes; shake constantly during the soaking process.

[0061] c. After each disinfection, rinse with sterile water 4-6 times and dry the surface with sterile filter paper.

[0062] S3. Inoculation and induction culture: The sterilized explants were inoculated into the above-mentioned callus induction culture medium and cultured under the conditions of light intensity of 1000-2000 Lux, light time of 12-14 h / d, and temperature of 23-27℃ to induce the formation of callus. Then, they were inoculated into the callus proliferation culture medium for proliferation.

[0063] S4. Adventitious bud differentiation: The dense, bright green callus tissue after proliferation is inoculated into the adventitious bud differentiation medium to induce the generation of adventitious buds.

[0064] S5. Subculture and propagation: Adventitious buds are transferred to adventitious bud propagation medium for propagation to obtain a large number of robust adventitious buds;

[0065] S6. Rooting culture: Transfer healthy adventitious buds that have reached a height of 2-3cm to the rooting culture medium. The culture conditions are light intensity of 800-1500 Lux, light duration of 8-10h / d, and temperature of 24-26℃ to obtain complete tissue culture seedlings.

[0066] S7. Hardening off and transplanting: Harden off the well-rooted tissue culture seedlings by placing the culture bottles under diffused light with an intensity of 1800-2000 Lux for 7-10 days, then open the bottle caps for 1-2 days. Transplant the seedlings into a substrate, which is a mixture of loess, peat moss and vermiculite in a volume ratio of (2-3):1:1. After transplanting, maintain an air humidity of 75-85% and provide ventilation, then gradually reduce the humidity and increase the light intensity.

[0067] Technical principle of the invention:

[0068] The core technical principle of this invention lies in following the physiological laws of *Spatholobus suberectus* in vitro culture, deconstructing the rapid propagation process of tissue culture into three functionally independent stages: "induced differentiation," "robust proliferation," and "rooting and acclimatization." Furthermore, it designs specialized culture medium formulations and corresponding culture methods with complementary functions and synergistic effects, addressing the core contradictions and biological objectives of each stage. This approach is not a simple superposition or concentration adjustment of existing tissue culture components, but rather an organic whole where various elements, under specific ratios, produce functional coupling and enhanced effects. This systematically solves a series of interconnected technical challenges from stable dedifferentiation and efficient redifferentiation of explants to large-scale seedling production.

[0069] 1. Induction Stage: Constructing a budding microsystem that coordinates "initiation signals" and "environmental protection".

[0070] The goal of this stage is to overcome the dormancy or dedifferentiation tendency of explants, promote callus formation, and efficiently redifferentiate into adventitious shoots. The induction medium of this invention achieves this goal through the synergistic effect of the following ingredients:

[0071] Cytokinin combination (6-BA and kinetin): serving as the core "morphogenesis signal." 6-BA plays a major role, strongly promoting cell division and the initiation of shoot primordia; kinetin helps to break apical dominance and promote the activation of lateral meristems. The two produce a synergistic effect at specific concentration ratios, increasing the number of shoot formation sites.

[0072] Low concentrations of auxin (NAA): acting as a "balance regulatory signal." At low concentrations of 0.1-0.3 mg / L, NAA can synergistically induce cell dedifferentiation with cytokinin, providing a cellular basis for redifferentiation, while also preventing abnormal structures caused by excessive cytokinin activity and guiding the differentiation direction to bud organs in an orderly manner.

[0073] Stress resistance and stabilization system: This system creates a stable internal environment for the differentiation process. Vitamin C, as the main antioxidant, directly removes toxins such as quinones produced by the oxidation of phenolic substances released from explant wounds.

[0074] 2. Proliferation Stage: Achieving coordinated regulation of "quantitative expansion" and "quality maintenance".

[0075] The goal at this stage is to efficiently propagate the initial seedlings and maintain their robustness. The design of the proliferation medium reflects a strategic shift from "initiating differentiation" to "stable propagation":

[0076] Moderately adjusted hormone concentration: By slightly reducing the kinetin concentration on the basis of callus induction medium, the physiological state of the culture is switched from a strong "differentiation initiation" mode to a stable "lateral bud germination and elongation" mode. This ratio can maintain sufficient cell division activity to promote the formation of clustered shoots, while effectively preventing bud thinning, vitrification, or premature aging of regenerative capacity caused by excessive hormone levels.

[0077] The retained auxiliary components: Vitamin C concentration is retained but appropriately reduced. Its role has changed from dealing with the initial severe stress to combating the oxidative stress and nutritional stress accumulated in long-term subculture, thereby maintaining the continuous vitality and robustness of the seedlings. This is the key to obtaining "a large number of robust adventitious buds" rather than weak and etiolated seedlings.

[0078] Increased sucrose concentration: Increasing sucrose concentration provides a more abundant carbon source and energy for rapid and continuous cell division and proliferation, supporting efficient biomass accumulation.

[0079] 3. Synergy of Methodologies: Process-oriented design ensures stable achievement of technical results.

[0080] The parameter design for each step in the entire breeding method serves to fully realize the efficacy of the aforementioned culture medium:

[0081] Differentiated disinfection pretreatment: Based on the different characteristics of different parts of the chicken blood vine, mild and strong disinfection strategies were adopted respectively. While minimizing the contamination rate, the viability of the explants was protected, thus preserving the foundation for subsequent response to induction signals in a special culture medium.

[0082] Stage transition and environmental adaptation: The relay use of culture media in the three stages of induction, proliferation, and rooting is precisely matched with the corresponding light and temperature parameters. For example, low light intensity and short days are used in the rooting stage, which is conducive to the accumulation of endogenous auxin, promotes the formation of root primordia, and synergizes with the exogenous auxin (IBA, NAA) in the rooting medium. The gradient design of light and humidity during the hardening-off process helps tissue culture seedlings smoothly complete the physiological transition from heterotrophic to autotrophic.

[0083] To make the present invention more fully disclosed, more specific embodiments are described below.

[0084] Example 1:

[0085] A special culture medium for rapid propagation of *Spatholobus suberectus* tissue culture and an efficient propagation method are as follows:

[0086] 1. Tissue culture rapid propagation special culture medium

[0087] (1) Components of callus induction culture medium: 0.9 mg / L of 6-benzylaminopurine, 0.4 mg / L of naphthaleneacetic acid, 0.4 mg / L of kinetin, 0.3 mg / L of vitamin C, 25 g / L of sucrose, 7 g / L of agar, and pH adjusted to 5.9;

[0088] (2) Components of callus proliferation culture medium: 1.0 mg / L of 6-benzylaminopurine, 0.5 mg / L of naphthaleneacetic acid, 0.2 mg / L of kinetin, 0.3 mg / L of vitamin C, 25 g / L of sucrose, 7 g / L of agar, and pH adjusted to 5.9;

[0089] (3) Components of adventitious shoot differentiation medium: 6-benzylaminopurine 2.5 mg / L, naphthaleneacetic acid 0.4 mg / L, gibberellin 1.0 mg / L, vitamin C 0.3 mg / L, sucrose 25 g / L, agar 7 g / L, hydrolyzed casein 0.8 g / L, pH adjusted to 5.9;

[0090] (4) Components of adventitious bud proliferation medium: 6-benzylaminopurine 3.0 mg / L, naphthaleneacetic acid 0.15 mg / L, gibberellin 0.3 mg / L, vitamin C 0.4 mg / L, sucrose 28 g / L, agar 7 g / L, inositol 0.15 g / L, pH adjusted to 5.9;

[0091] (5) Components of rooting medium: naphthaleneacetic acid 0.2 mg / L, indolebutyric acid 0.4 mg / L, vitamin C 0.3 mg / L, sucrose 25 g / L, agar 7 g / L, glutamine 0.25 g / L, pH adjusted to 5.9.

[0092] 2. High-efficiency breeding methods

[0093] Includes the following steps:

[0094] S1. Explant selection and pretreatment: Select the top 2nd-3rd grade tender leaves and stem tips of healthy chicken blood vine plants as explants; Tender leaf pretreatment: Rinse with running water for 45 minutes, then soak in a washing solution containing 0.08% Tween-80 for 8 minutes, and then rinse 4 times with sterile water.

[0095] S2. Segmented disinfection of explants: a. After pretreatment, the explants were immersed in 72% ethanol for 45 seconds in a clean bench, then disinfected with 0.15% mercuric chloride solution for 9 minutes, and rinsed three times with sterile water; b. Young leaves were immersed twice with 0.15% mercuric chloride solution, the first time for 3 minutes and the second time for 5 minutes; the stem tips were immersed twice with 0.15% mercuric chloride solution, the first time for 4 minutes and the second time for 5 minutes, shaking continuously during the immersion process; c. After each disinfection, the explants were rinsed five times with sterile water and the surface moisture was absorbed with sterile filter paper.

[0096] S3. Inoculation and induction culture: The sterilized explants were inoculated into the above callus induction culture medium and cultured under the conditions of light intensity of 1500 Lux, light time of 13 h / d and temperature of 25℃ for 3 weeks. After inducing the formation of callus, the explants were then inoculated into the callus proliferation culture medium for proliferation.

[0097] S4. Adventitious bud differentiation: The dense, bright green callus tissue after proliferation is inoculated into the adventitious bud differentiation medium to induce the generation of adventitious buds.

[0098] S5. Subculture and propagation: The adventitious buds are transferred to the adventitious bud propagation medium for propagation. The subculture and propagation cycle is 3.5 weeks to obtain a large number of robust adventitious buds.

[0099] S6. Rooting culture: Transplant robust adventitious buds that have reached a height of 2.5cm into rooting medium. The culture conditions are light intensity of 1200 Lux, light duration of 9h / d, and temperature of 25℃. Complete tissue culture seedlings are obtained after 4 weeks of culture.

[0100] S7. Hardening off and transplanting: Transplant well-rooted tissue culture seedlings to diffused light with an intensity of 1800 Lux for 9 days to harden off, then open the bottle cap for 2 days. The transplanting substrate is a mixture of loess, peat moss and vermiculite in a volume ratio of 2.5:1:1. After transplanting, maintain an air humidity of 80% and ventilate 3 times a day for 30 minutes each time. Then, in a gradient of 2 days, decrease the humidity by 6% and increase the light intensity by 250 Lux each time until the humidity drops to 65% and the light intensity reaches 1900 Lux.

[0101] Example 1 Results: The callus induction rate was 92.5%, the callus proliferation coefficient was 3.8, the adventitious bud differentiation rate was 89.6%, the rooting rate was 92.3%, the transplant survival rate was 88.7%, and the tissue culture seedlings grew vigorously without obvious browning or contamination.

[0102] Example 2:

[0103] A special culture medium for rapid propagation of *Spatholobus suberectus* tissue culture and an efficient propagation method are as follows:

[0104] 1. Special culture medium for rapid tissue culture propagation:

[0105] (1) Components of callus induction culture medium: 0.8 mg / L of 6-benzylaminopurine, 0.3 mg / L of naphthaleneacetic acid, 0.3 mg / L of kinetin, 0.2 mg / L of vitamin C, 22 g / L of sucrose, 6.5 g / L of agar, and pH adjusted to 5.8;

[0106] (2) Components of callus proliferation culture medium: 0.8 mg / L of 6-benzylaminopurine, 0.3 mg / L of naphthaleneacetic acid, 0.1 mg / L of kinetin, 0.2 mg / L of vitamin C, 22 g / L of sucrose, 6.5 g / L of agar, and pH adjusted to 5.8;

[0107] (3) Components of adventitious shoot differentiation medium: 6-benzylaminopurine 2.3 mg / L, naphthaleneacetic acid 0.4 mg / L, gibberellin 0.9 mg / L, vitamin C 0.2 mg / L, sucrose 22 g / L, agar 6.5 g / L, hydrolyzed casein 0.5 g / L, pH adjusted to 5.8;

[0108] (4) Components of adventitious bud proliferation medium: 6-benzylaminopurine 2.5 mg / L, naphthaleneacetic acid 0.1 mg / L, gibberellin 0 mg / L, vitamin C 0.3 mg / L, sucrose 25 g / L, agar 6.5 g / L, inositol 0.1 g / L, pH adjusted to 5.8;

[0109] (5) Components of rooting medium: naphthaleneacetic acid 0.1 mg / L, indolebutyric acid 0.3 mg / L, vitamin C 0.2 mg / L, sucrose 22 g / L, agar 6.5 g / L, glutamine 0.2 g / L, pH adjusted to 5.8.

[0110] 2. High-efficiency breeding methods

[0111] S1. Selection and pretreatment of explants: Select the second and third grade tender leaves and stem tips of disease-free *Spatholobus suberectus* plants as explants; Pretreatment method for tender leaves at the top of stem tips: rinse with running water for 30 minutes, then soak in a washing solution containing 0.05% Tween-80 for 5 minutes, and then rinse 3 times with sterile water.

[0112] S2. Segmented disinfection of explants: a. Immerse the pretreated explants in 70% ethanol for 30 seconds in a clean bench, disinfect with 0.1% mercuric chloride solution for 10 minutes, and finally rinse with sterile water 5 times; b. Immerse the tender top leaves twice with 0.1% mercuric chloride solution, the first time for 3 minutes and the second time for 5 minutes; immerse the stem tips twice with 0.1% mercuric chloride solution, the first time for 4 minutes and the second time for 5 minutes; shake continuously during the immersion process; c. Rinse with sterile water 4 times and gently absorb the surface moisture with sterile filter paper;

[0113] S3. Inoculation and induction culture: Light intensity 1200 Lux, light time 14 h / d, temperature 23℃, culture for 5 weeks, induce callus formation and then inoculate into callus proliferation medium for proliferation.

[0114] S4. Adventitious bud differentiation: The dense, bright green callus tissue after proliferation is inoculated into the adventitious bud differentiation medium to induce the generation of adventitious buds.

[0115] S5. Subculture and propagation: The adventitious buds are transferred to the adventitious bud propagation medium for propagation. The subculture and propagation cycle is 4 weeks to obtain a large number of robust adventitious buds.

[0116] S6. Rooting culture: Transplant robust adventitious buds that have reached a height of 2.5cm into rooting medium. The culture conditions are light intensity of 900 Lux, light duration of 10h / d, and temperature of 24℃. Tissue culture seedlings are obtained after 5 weeks of culture.

[0117] S7. Hardening off and transplanting: Transplant well-rooted tissue culture seedlings to diffused light with an intensity of 1900 Lux for 8 days, then open the bottle cap for 1 day of hardening off. The volume ratio of transplanting substrate is 2:1:1. After transplanting, the humidity is 75%. Ventilate twice a day for 25 minutes each time. The gradient adjustment is to reduce the humidity by 5% and increase the light intensity by 200 Lux every 2 days until the humidity drops to 65% and the light intensity reaches 1900 Lux.

[0118] Example 2 Results: The callus induction rate was 85.3%, the callus proliferation coefficient was 3.2, the adventitious bud differentiation rate was 82.1%, the rooting rate was 87.6%, the transplant survival rate was 83.2%, the tissue culture seedlings grew stably, and a small number of leaves showed slight browning.

[0119] Example 3:

[0120] A special culture medium for rapid propagation of *Spatholobus suberectus* tissue culture and an efficient propagation method are as follows:

[0121] 1. Special culture medium for rapid tissue culture propagation:

[0122] (1) Components of callus induction culture medium: 0.9 mg / L of 6-benzylaminopurine, 0.5 mg / L of naphthaleneacetic acid, 0.5 mg / L of kinetin, 0.5 mg / L of vitamin C, 30 g / L of sucrose, 7.5 g / L of agar, and pH adjusted to 6.0;

[0123] (2) Components of callus proliferation culture medium: 1.0 mg / L of 6-benzylaminopurine, 0.5 mg / L of naphthaleneacetic acid, 0.2 mg / L of kinetin, 0.5 mg / L of vitamin C, 30 g / L of sucrose, 7.5 g / L of agar, and pH adjusted to 6.0;

[0124] (3) Components of adventitious shoot differentiation medium: 6-benzylaminopurine 2.5 mg / L, naphthaleneacetic acid 0.4 mg / L, gibberellin 1.1 mg / L, vitamin C 0.5 mg / L, sucrose 30 g / L, agar 7.5 g / L, hydrolyzed casein 1.0 g / L, pH adjusted to 6.0;

[0125] (4) Components of adventitious bud proliferation medium: 6-benzylaminopurine 3.5 mg / L, naphthaleneacetic acid 0.2 mg / L, gibberellin 0.5 mg / L, vitamin C 0.5 mg / L, sucrose 30 g / L, agar 7.5 g / L, inositol 0.2 g / L, pH adjusted to 6.0;

[0126] (5) Components of rooting medium: naphthaleneacetic acid 0.3 mg / L, indolebutyric acid 0.4 mg / L, vitamin C 0.5 mg / L, sucrose 30 g / L, agar 7.5 g / L, glutamine 0.3 g / L, pH adjusted to 6.0.

[0127] 2. High-efficiency breeding methods

[0128] S1. Selection and pretreatment of explants: Select the second-to-third grade tender leaves and stem tips from the top of superior *Spatholobus suberectus* plants; among them, the tender leaves at the top of the stem tips are pretreated by rinsing with running water for 50 minutes, then soaking in a washing solution containing 0.09% Tween-80 for 9 minutes, and then rinsing with sterile water 5 times.

[0129] S2. Segmented disinfection of explants: a. Immerse the pretreated explants in 74% ethanol (v / v) for 55 seconds in a clean bench, then disinfect with 0.2% mercuric chloride solution for 8 minutes, and finally rinse 5 times with sterile water; b. Immerse the tender top leaves twice with 0.2% mercuric chloride solution, the first time for 3 minutes and the second time for 5 minutes; immerse the stem tips twice with 0.2% mercuric chloride solution, the first time for 4 minutes and the second time for 5 minutes; shake continuously during the immersion process; c. Rinse 5 times with sterile water and blot dry the surface moisture with sterile filter paper;

[0130] S3. Inoculation and induction culture: Light intensity 1800 Lux, light time 12 h / d, temperature 27℃, culture for 4 weeks, induce callus formation and then inoculate into callus proliferation medium for proliferation.

[0131] S4. Adventitious bud differentiation: The dense, bright green callus tissue after proliferation is inoculated into the adventitious bud differentiation medium to induce the generation of adventitious buds.

[0132] S5. Subculture and propagation: The adventitious buds are transferred to the adventitious bud propagation medium for propagation. The subculture and propagation cycle is 3 weeks to obtain a large number of robust adventitious buds.

[0133] S6. Rooting culture: Transplant robust adventitious buds that have reached a height of 2.5cm into rooting medium. The culture conditions are light intensity of 1400 Lux, light duration of 8h / d, and temperature of 26℃. Tissue culture seedlings are obtained after 3.5 weeks of culture.

[0134] S7. Hardening off and transplanting: Transplant well-rooted tissue culture seedlings to diffused light with an intensity of 2000 Lux for 7 days to harden off, then open the bottle cap for 2 days to harden off. The volume ratio of transplanting substrate is 3:1:1. After transplanting, the humidity is 81%. Ventilate 4 times a day for 35 minutes each time. The gradient adjustment is to reduce the humidity by 8% and increase the light intensity by 300 Lux every 2 days until the humidity drops to 65% and the light intensity reaches 1900 Lux.

[0135] Example 3 Results: The callus induction rate was 98.3%, the callus proliferation coefficient was 5.2, the adventitious bud differentiation rate was 97.8%, the rooting rate was 96.5%, the transplant survival rate was 93.7%, the tissue culture seedlings reached a height of 5.8 cm, the buds were robust, the leaves were bright green, the root system was well developed, and there were no browning, pollution or deformities. The tissue culture seedlings performed best overall.

[0136] Example 4:

[0137] A special culture medium for rapid propagation of *Spatholobus suberectus* tissue culture and an efficient propagation method are as follows:

[0138] 1. Special culture medium for rapid tissue culture propagation:

[0139] (1) Components of callus induction culture medium: 0.9 mg / L of 6-benzylaminopurine, 0.4 mg / L of naphthaleneacetic acid, 0.4 mg / L of kinetin, 0.3 mg / L of vitamin C, 25 g / L of sucrose, 6.2 g / L of agar, and pH adjusted to 5.9;

[0140] (2) Components of callus proliferation culture medium: 0.9 mg / L of 6-benzylaminopurine, 0.4 mg / L of naphthaleneacetic acid, 0.2 mg / L of kinetin, 0.3 mg / L of vitamin C, 25 g / L of sucrose, 6.2 g / L of agar, and pH adjusted to 5.9;

[0141] (3) Components of adventitious bud differentiation medium: 6-benzylaminopurine 2.4 mg / L, naphthaleneacetic acid 0.4 mg / L, gibberellin 1.0 mg / L, vitamin C 0.3 mg / L, sucrose 25 g / L, agar 6.2 g / L, hydrolyzed casein 0.7 g / L, pH adjusted to 5.9;

[0142] (4) Components of adventitious bud proliferation medium: 3.2 mg / L of 6-benzylaminopurine, 0.18 mg / L of naphthaleneacetic acid, 0.4 mg / L of gibberellin, 0.4 mg / L of vitamin C, 27 g / L of sucrose, 6.2 g / L of agar, 0.18 g / L of inositol, and pH adjusted to 5.9;

[0143] (5) Components of rooting medium: naphthaleneacetic acid 0.25 mg / L, indolebutyric acid 0.45 mg / L, vitamin C 0.3 mg / L, sucrose 25 g / L, agar 6.2 g / L, glutamine 0.28 g / L, pH adjusted to 5.9.

[0144] 2. High-efficiency breeding methods

[0145] S1. Selection and pretreatment of explants: Select the second- to third-level tender leaves and stem tips from the top of healthy current-year plants of *Spatholobus suberectus*. Pretreatment method for tender leaves at the top: rinse with running water for 60 minutes, then soak in a washing solution containing 0.1% Tween-80 for 10 minutes, and then rinse 5 times with sterile water.

[0146] S2. Segmented disinfection of explants: a. Immerse the pretreated explants in 75% ethanol for 60 seconds in a clean bench, then disinfect with 0.18% mercuric chloride solution for 9 minutes, and finally rinse 5 times with sterile water; b. Immerse the tender top leaves twice with 0.18% mercuric chloride solution, the first time for 3 minutes and the second time for 5 minutes; immerse the stem tips twice with 0.18% mercuric chloride solution, the first time for 4 minutes and the second time for 5 minutes; shake gently and continuously during the immersion process; c. Rinse 6 times with sterile water and blot dry the surface moisture with sterile filter paper;

[0147] S3. Inoculation and induction culture: The sterilized explants were inoculated into the above callus induction culture medium and cultured under the conditions of light intensity of 1900 Lux, light time of 12.5 h / d and temperature of 26℃. Direct induction culture was carried out for 4.5 weeks. After callus formation, the explants were inoculated into the callus proliferation culture medium for proliferation.

[0148] S4. Adventitious bud differentiation: The dense, bright green callus tissue after proliferation is inoculated into the adventitious bud differentiation medium to induce the generation of adventitious buds.

[0149] S5. Subculture and propagation: The adventitious buds are transferred to the adventitious bud propagation medium for propagation. The subculture and propagation cycle is 3.2 weeks to obtain a large number of robust adventitious buds.

[0150] S6. Rooting culture: Healthy adventitious buds with a height of 2.5cm were transferred to rooting medium. The culture conditions were: light intensity 1300Lux, light duration 8.5h / d, temperature 25.5℃, and tissue culture seedlings were obtained after 3.8 weeks of culture.

[0151] S7. Hardening off and transplanting: Transplant well-rooted tissue culture seedlings to diffused light with an intensity of 1800 Lux for 8 days, then open the bottle cap for 1.5 days of hardening off. The volume ratio of transplanting substrate is 2.2:1:1. After transplanting, maintain humidity at 78%, ventilate 3 times a day for 28 minutes each time, and gradually adjust the humidity by decreasing by 6% and increasing the light intensity by 220 Lux every 2 days until the humidity drops to 65% and the light intensity reaches 1900 Lux.

[0152] Example 4 Results: The callus induction rate was 90.2%, the callus proliferation coefficient was 3.7, the adventitious bud differentiation rate was 86.7%, the rooting rate was 89.8%, the transplant survival rate was 85.4%, the tissue culture seedlings grew well, and there was no serious pollution.

[0153] Example 5:

[0154] A special culture medium for rapid propagation of *Spatholobus suberectus* tissue culture and an efficient propagation method are as follows:

[0155] 1. Special culture medium for rapid tissue culture propagation:

[0156] (1) Components of callus induction culture medium: 0.85 mg / L of 6-benzylaminopurine, 0.35 mg / L of naphthaleneacetic acid, 0.35 mg / L of kinetin, 0.25 mg / L of vitamin C, 27 g / L of sucrose, 7.2 g / L of agar, and pH adjusted to 5.85;

[0157] (2) Components of callus proliferation culture medium: 0.85 mg / L of 6-benzylaminopurine, 0.35 mg / L of naphthaleneacetic acid, 0.15 mg / L of kinetin, 0.25 mg / L of vitamin C, 27 g / L of sucrose, 7.2 g / L of agar, and pH adjusted to 5.85;

[0158] (3) Components of adventitious bud differentiation medium: 6-benzylaminopurine 2.6 mg / L, naphthaleneacetic acid 0.4 mg / L, gibberellin 1.0 mg / L, vitamin C 0.25 mg / L, sucrose 27 g / L, agar 7.2 g / L, hydrolyzed casein 0.6 g / L, pH adjusted to 5.85;

[0159] (4) Components of adventitious bud proliferation medium: 6-benzylaminopurine 2.8 mg / L, naphthaleneacetic acid 0.12 mg / L, gibberellin 0.2 mg / L, vitamin C 0.35 mg / L, sucrose 26 g / L, agar 7.2 g / L, inositol 0.12 g / L, pH adjusted to 5.85;

[0160] (5) Components of rooting medium: naphthaleneacetic acid 0.15 mg / L, indolebutyric acid 0.35 mg / L, vitamin C 0.25 mg / L, sucrose 27 g / L, agar 7.2 g / L, glutamine 0.22 g / L, pH adjusted to 5.85.

[0161] 2. High-efficiency breeding methods

[0162] S1. Selection and pretreatment of explants: Select the 2nd-3rd grade young leaves and stem tips of healthy plants of *Spatholobus suberectus*. Pretreatment method for young leaves at the top: rinse with running water for 40 minutes, then soak in a washing solution containing 0.07% Tween-80 for 7 minutes, and then rinse 4 times with sterile water.

[0163] S2. Segmented disinfection of explants: a. Immerse the pretreated explants in 73% ethanol (v / v) for 50 seconds in a clean bench, then disinfect with 0.12% mercuric chloride solution for 9.5 minutes, and finally rinse 5 times with sterile water; b. Immerse the tender leaves at the top of the stem tip in 0.12% mercuric chloride solution twice, for 3 minutes the first time and 5 minutes the second time; immerse the stem tip in 0.12% mercuric chloride solution twice, for 4 minutes the first time and 5 minutes the second time; shake continuously during the immersion process; c. Rinse 5 times with sterile water and blot dry the surface moisture with sterile filter paper;

[0164] S3. Inoculation and induction culture: The sterilized explants were inoculated into the above callus induction culture medium and cultured under the conditions of light intensity of 1000 Lux, light time of 13.5 h / d and temperature of 24℃ for 5.5 weeks. After inducing the formation of callus, the explants were then inoculated into the callus proliferation culture medium for proliferation.

[0165] S4. Adventitious bud differentiation: The dense, bright green callus tissue after proliferation is inoculated into the adventitious bud differentiation medium to induce the generation of adventitious buds.

[0166] S4. Subculture and propagation: Adventitious buds are transferred to adventitious bud propagation medium for propagation. The subculture and propagation cycle is 3.8 weeks to obtain a large number of robust adventitious buds.

[0167] S5. Rooting culture: Healthy adventitious buds with a height of 2.5cm were transferred to rooting medium. The culture conditions were 850 Lux light intensity, 9.5 h / d light duration, and 24.5℃ temperature. Tissue culture seedlings were obtained after 4.2 weeks of culture.

[0168] S6. Hardening off and transplanting: Transplant well-rooted tissue culture seedlings to diffused light with an intensity of 1900 Lux for 8 days, and harden off for 1 day with the bottle caps off; the volume ratio of transplanting substrate is 2.8:1:1, and the humidity is 82% after transplanting. Ventilate twice a day for 32 minutes each time, and adjust the gradient by decreasing the humidity by 7% and increasing the light intensity by 280 Lux every 2 days until the humidity drops to 65% and the light intensity reaches 1900 Lux.

[0169] Example 5 Results: The callus induction rate was 87.6%, the callus proliferation coefficient was 3.4, the adventitious bud differentiation rate was 84.3%, the rooting rate was 90.2%, the transplant survival rate was 86.1%, and the tissue culture seedlings had bright green leaves and low browning rate.

[0170] Except for the parts that are specified to be modified or missing, the following comparative examples are completely consistent with Example 1 in terms of culture medium components, propagation steps and culture conditions.

[0171] Comparative Example 1: Kinetin-deficient proliferation medium

[0172] 1. Tissue culture rapid propagation special medium: The proliferation medium lacks the "kinetin" component, but the rest is the same as in Example 3;

[0173] 2. High-efficiency breeding method: completely consistent with Example 3.

[0174] Comparative Example 1: The callus induction rate was 93.2%, which was not significantly different from Example 3; the callus proliferation coefficient was only 2.1, which was significantly lower than 4.3 in Example 3; the adventitious bud differentiation rate was 88.7%, and some adventitious buds showed yellowing; the final rooting rate was 83.8%, and the transplant survival rate was 79.3%. The core problem was that the lack of kinetin led to a decrease in bud proliferation capacity and a weakening of seedling resistance.

[0175] Comparative Example 2: Modification of hormone ratio in induction medium

[0176] 1. Tissue culture rapid propagation special culture medium: The concentration of 6-benzylaminopurine in the induction medium was changed to 0.8 mg / L and the concentration of naphthaleneacetic acid was changed to 0.5 mg / L, the rest was the same as in Example 3;

[0177] 2. High-efficiency breeding method: completely consistent with Example 3.

[0178] Comparative Example 2: The callus induction rate was 95.1%, the callus proliferation coefficient was 4.2, the adventitious shoot differentiation rate was 93.1%, the subsequent subculture proliferation coefficient was 2.7, the rooting rate was 74.5%, and the transplant survival rate was 72.4%. The core problem was the imbalance of hormone ratio (too low cytokinin concentration and too high auxin concentration), which led to the differentiation of explants into callus tissue and inhibited the formation of clustered shoots.

[0179] Comparative Example 3: Modification of light intensity and duration for seedling hardening

[0180] 1. Tissue culture rapid propagation special culture medium: completely consistent with Example 3;

[0181] 2. High-efficiency propagation method: Only the hardening conditions in step S7 are modified to "move to diffused light with an intensity of 800 Lux for 2 days, and then open the bottle cap for 3 days" (insufficient light intensity and too short hardening time in the early stage). The remaining steps and conditions are the same as in Example 3.

[0182] Comparative Example 3: The callus induction rate was 95.1%, the callus proliferation coefficient was 4.2, and the adventitious bud differentiation rate was 93.1%; the final rooting rate was 89.5% (similar to Example 3), but the transplant survival rate was only 64.8%. The core problem was that the hardening conditions were not suitable, resulting in insufficient resistance of the seedlings and difficulty in adapting to the external environment after transplanting.

[0183] Single-factor experiment:

[0184] Single-factor experimental design principle: Only one core process parameter is changed in each experiment, and the remaining parameters are fixed at the baseline value of Example 3. Five levels are set for each parameter, and the difference between adjacent levels is equal. Core response indicators (such as shoot induction rate, rooting rate, etc.) are detected. Three parallel replicates are set for each group. The average value of the results is taken and one decimal place is retained to accurately screen the optimal parameter range.

[0185] Experiment 1: Single-factor screening of 6-benzylaminopurine (6-BA) concentration in induction medium

[0186] Variable parameters: 6-BA concentration (mg / L), level settings: 0.80, 0.85, 0.90, 0.95, 1.00; other parameters and conditions are the same as in Example 3. Detection index: bud induction rate (%). Experimental results are shown in Table 1:

[0187]

[0188] Conclusion and Analysis: When the concentration of 6-BA was 0.90 mg / L, it formed the optimal synergistic effect with kinetin and naphthaleneacetic acid, achieving a shoot induction rate of 98.1%, significantly higher than other concentration groups. Below 0.90 mg / L, insufficient cytokinin signaling resulted in a lower induction rate; above 0.90 mg / L, the hormonal balance was disrupted, leading to a slight decrease in the induction rate. This experiment determined that the optimal concentration of 6-BA in the induction medium was 0.90 mg / L.

[0189] Experiment 2: Single-factor screening of kinetin (KT) concentration in proliferation medium

[0190] Variable parameters: KT concentration (mg / L), level settings: 0.10, 0.15, 0.20, 0.25, 0.30; other parameters and conditions are the same as in Example 3. Detection index: subgeneration proliferation coefficient. Experimental results are shown in Table 2:

[0191]

[0192] Conclusions and analysis: When the KT concentration is 0.20 mg / L, it exhibits the strongest synergistic effect with 6-benzylaminopurine, achieving a subculture proliferation coefficient of 5.2, and producing robust buds without vitrification. Below 0.20 mg / L, the bud proliferation signal is insufficient, resulting in a lower coefficient. Above 0.20 mg / L, excessive bud differentiation is likely, leading to a decrease in the coefficient. This experiment determined the optimal KT concentration in the proliferation medium to be 0.20 mg / L.

[0193] Experiment 3: Single-factor screening of 6-benzylaminopurine (6-BA) concentration in adventitious shoot differentiation medium

[0194] Variable parameters: 6-BA concentration (mg / L), level settings: 2.3, 2.4, 2.5, 2.6, 2.7; other parameters and conditions are the same as in Example 3. Detection index: Adventitious bud differentiation rate (%). Experimental results are shown in Table 3:

[0195]

[0196] Conclusion and Analysis: When the concentration of 6-BA is 2.5 mg / L, the synergistic induction effect with gibberellin and naphthaleneacetic acid is optimal, achieving an adventitious shoot differentiation rate of 97.6% and thorough callus differentiation. Below 2.5 mg / L, the differentiation signal is insufficient, and the rate value is low; above 2.5 mg / L, the shoots are prone to being weak, and the differentiation rate decreases. This experiment determined that the optimal concentration of 6-BA in the adventitious shoot differentiation medium is 2.5 mg / L.

[0197] Experiment 4: Single-factor screening of indolebutyric acid (IBA) concentration in rooting medium

[0198] Variable parameters: IBA concentration (mg / L), level settings: 0.30, 0.35, 0.40, 0.45, 0.50; other parameters and conditions are the same as in Example 3. Detection indicators: rooting rate (%), average root length (cm). Experimental results are shown in Table 4:

[0199]

[0200] When the IBA concentration was 0.4 mg / L, its synergistic effect with naphthaleneacetic acid and glutamine was optimal, achieving a rooting rate of 96.3%, an average of 9.8 roots per root, and an average root length of 3.9 cm, resulting in a robust and well-developed root system. At concentrations below 0.4 mg / L, rooting signals were insufficient, and the indicators were low; at concentrations above 0.4 mg / L, root growth was uneven, and the indicators slightly decreased. This experiment determined that the optimal IBA concentration in the rooting medium was 0.4 mg / L.

[0201] Experiment 5: Single-factor screening of initial air humidity after transplanting

[0202] Variable parameters: Initial air humidity after transplanting (%), horizontal settings: 75, 77, 79, 81, 85; other parameters and conditions are the same as in Example 3. Detection indicators: Transplant survival rate (%), seedling establishment time (d). Experimental results are shown in Table 5:

[0203]

[0204] Conclusion and Analysis: When the initial air humidity is 81%, the transpiration and water absorption of tissue culture seedlings reach equilibrium, resulting in a transplant survival rate of 93.5% and a recovery time of only 6.1 days. When the humidity is below 81%, the seedlings are prone to water loss and wilting, leading to a lower survival rate. When the humidity is above 81%, mold growth is likely, further reducing the survival rate. This experiment determined that the optimal initial air humidity after transplanting is 81%.

[0205] Performance index testing:

[0206] To fully verify the superiority of the technical solution of the present invention, Examples 1-5 and Comparative Examples 1-5 were selected for performance index testing. The test indexes included the induction rate of clustered shoots, the subculture proliferation coefficient, the rooting rate, the transplant survival rate, and the height of tissue culture seedlings. Each group was repeated 3 times, and the results were averaged and retained to one decimal place.

[0207] 1. Detection object and parameter settings

[0208] Examples 1-5: carried out according to the culture medium components and propagation method disclosed in this invention, with parameters selected within the scope of the claims;

[0209] Comparative Example 1: Kinetin was missing from the proliferation medium; otherwise, it was the same as in Example 3.

[0210] Comparative Example 2: The concentration of 6-BA in the induction medium was changed to 0.8 mg / L and the concentration of naphthaleneacetic acid was changed to 0.5 mg / L, and the rest was the same as in Example 3;

[0211] Comparative Example 3: The light intensity and time for hardening off seedlings were modified (the seedlings were hardened off for 2 days under diffused light with an intensity of 800 Lux, and then the bottle caps were opened for hardening off for 3 days), and the rest was the same as in Example 3.

[0212] 2. Test Results

[0213] The performance index test results are shown in Table 6:

[0214]

[0215] Data comparison and theoretical analysis:

[0216] 1. Data Comparison and Analysis

[0217] (1) Mean values ​​of the examples vs. the comparative examples: The mean callus induction rate of examples 1-5 was 90.8%, the mean callus proliferation coefficient was 4.1, the mean adventitious bud differentiation rate was 88.1%, the mean rooting rate was 91.3%, the mean transplant survival rate was 87.8%, and the mean plant height of tissue culture seedlings was 4.5 cm; the mean values ​​of the above indicators of comparative examples 1-3 were 92.6%, 3.0%, 73.5%, 3.1%, 82.6%, 72.2%, and 3.9 cm, respectively. Except for the callus induction rate being slightly lower than that of the comparative examples (because the induction-related parameters of comparative examples 1 and 3 were not changed), all other indicators of the examples were significantly better than those of the comparative examples, with the callus proliferation coefficient increasing by 36.7%, the adventitious bud differentiation rate increasing by 20.0%, the rooting rate increasing by 10.5%, the transplant survival rate increasing by 21.6%, and the plant height increasing by 15.4%.

[0218] (2) Verification of the impact of key parameters: The adventitious shoot differentiation rate of Comparative Example 2 (with modified induction hormone ratio) decreased by 59.3% compared with Example 3, which confirms the key role of hormone balance in the induction stage on the callus differentiation potential; the callus proliferation coefficient of Comparative Example 1 (without proliferative kinin) decreased by 3.1 compared with Example 3; the transplant survival rate of Comparative Example 3 (with modified hardening conditions) decreased by 28.9% compared with Example 3, indicating that a suitable hardening procedure is crucial to ensuring the final survival.

[0219] 2. Theoretical Analysis

[0220] (1) Prominent substantive features: This invention breaks through the pain points of traditional chicken blood vine tissue culture technology. Through single-factor experiments, it accurately screens the optimal values ​​of key parameters. The culture medium at each stage achieves the synergistic effect of hormone balance regulation, anti-browning and stress resistance enhancement, effectively guiding the explant to gradually complete morphogenesis, reflecting a profound understanding of the physiological characteristics of chicken blood vine tissue culture.

[0221] (2) Progress: This invention systematically solves the core problems of the prior art by optimizing the culture medium components and process parameters. The adventitious bud differentiation rate of the best example 3 is 97.8% and the transplant survival rate is 93.7%, which are 33.2% and 30.0% higher than the average of the comparative examples, respectively. The adventitious bud differentiation rate of the comparative example 2 is 152.1% higher than that of the comparative example 3, and the transplant survival rate of the comparative example 3 is 44.6% higher, achieving a leapfrog improvement. At the same time, the clarification of process parameters and the standardization of the process improve the repeatability and operability of the technology, which is conducive to industrial application and has economic and technical value.

[0222] The above description, in conjunction with preferred embodiments, provides a further detailed explanation of the present invention and should not be construed as limiting the specific implementation of the invention to these descriptions. For those skilled in the art, various substitutions or modifications can be made to these described embodiments without departing from the inventive concept, and all such substitutions or modifications should be considered within the scope of protection of the present invention.

[0223] Although the invention and its advantages have been described in detail, it should be understood that various changes, substitutions, and modifications can be made without departing from the spirit and scope of the invention. Furthermore, the scope of the invention is not limited to the specific embodiments of the processes, methods, and steps described in the specification. From the disclosure of this invention, those skilled in the art will readily utilize existing or future processes, methods, steps that substantially perform the same function or achieve the same results as the corresponding embodiments described herein. Therefore, the appended claims are intended to cover such processes, methods, steps.

Claims

1. A special culture medium for rapid propagation of *Spatholobus suberectus* through tissue culture, characterized in that... This includes callus induction medium, callus proliferation medium, adventitious shoot differentiation medium, adventitious shoot proliferation medium, and rooting medium.

2. The special culture medium for rapid propagation of *Spatholobus suberectus* tissue culture according to claim 1, characterized in that, The callus induction medium, callus proliferation medium, adventitious shoot differentiation medium, and adventitious shoot proliferation medium are all based on MS medium, and the rooting medium is based on 1 / 2 MS medium.

3. The special culture medium for rapid propagation of *Spatholobus suberectus* tissue culture according to claim 1, characterized in that, The callus induction culture medium consists of the following components: 6-benzylaminopurine 0.8-1.0 mg / L, naphthaleneacetic acid 0.3-0.5 mg / L, kinetin 0.3-0.5 mg / L, vitamin C 0.1-0.5 mg / L, sucrose 20-30 g / L, agar 6-8 g / L, and pH 5.8-6.

0. The callus proliferation culture medium consists of the following components: 6-benzylaminopurine 0.8-1.0 mg / L, naphthaleneacetic acid 0.3-0.5 mg / L, kinetin 0.1-0.3 mg / L, vitamin C 0.1-0.5 mg / L, sucrose 20-30 g / L, agar 6-8 g / L, and pH 5.8-6.

0.

4. The special culture medium for rapid propagation of *Spatholobus suberectus* tissue culture according to claim 1, characterized in that, The components of the adventitious bud differentiation culture medium are as follows: 6-benzylaminopurine 2.3-2.7 mg / L, naphthaleneacetic acid 0.3-0.5 mg / L, gibberellin 0.9-1.1 mg / L, vitamin C 0.1-0.5 mg / L, sucrose 20-30 g / L, agar 6-8 g / L, hydrolyzed casein 0.5-1.0 g / L, and pH value 5.8-6.0; The components of the adventitious bud proliferation culture medium are as follows: 6-benzylaminopurine 2.5-3.5 mg / L, naphthaleneacetic acid 0.1-0.2 mg / L, gibberellin 0-0.5 mg / L, vitamin C 0.3-0.5 mg / L, sucrose 25-30 g / L, agar 6-8 g / L, inositol 0.1-0.2 g / L, and pH value 5.8-6.0; The rooting medium consists of the following components: naphthaleneacetic acid 0.1-0.3 mg / L, indolebutyric acid 0.3-0.5 mg / L, vitamin C 0.1-0.5 mg / L, sucrose 20-30 g / L, agar 6-8 g / L, glutamine 0.2-0.3 g / L, and pH 5.8-6.

0.

5. A method for efficient propagation of *Spatholobus suberectus*, characterized in that, Includes the following steps: S1. Explant selection and pretreatment: Young leaves and stem tips of *Spatholobus suberectus* were selected as explants and pretreated. S2. Segmented disinfection of explants: The pretreated explants are disinfected in segments. S3. Inoculation and induction culture: The sterilized explants were inoculated into the callus induction culture medium and cultured under the conditions of light intensity of 1000-2000 Lux, light time of 12-14 h / d, and temperature of 23-27℃ to induce the formation of callus tissue, which was then inoculated into the callus proliferation culture medium for proliferation. S4. Adventitious bud differentiation: The dense, bright green callus tissue after proliferation is inoculated into the adventitious bud differentiation medium to induce the generation of adventitious buds. S5. Subculture and propagation: Adventitious shoots are transferred to adventitious shoot propagation medium for propagation to obtain a large number of robust adventitious shoots; S6. Rooting culture: Transfer healthy adventitious buds that have reached a height of 2-3cm to a rooting medium for rooting culture to obtain complete tissue culture seedlings; S7. Hardening off and transplanting: After hardening off the well-rooted tissue culture seedlings, transplant them into the substrate.

6. The method for efficient propagation of *Spatholobus suberectus* according to claim 5, characterized in that, In step S1, the pretreatment method is as follows: rinse with running water for 30-60 minutes, then soak in a washing solution containing 0.05-0.1% Tween-80 for 5-10 minutes, and then rinse with sterile water 3-5 times.

7. The method for efficient propagation of *Spatholobus suberectus* according to claim 5, characterized in that, In step S2, the specific steps of segmented disinfection are as follows: a. Soak the pretreated explants in 70-75% ethanol for 30 seconds in a clean bench, then disinfect them with 0.1-0.2% mercuric chloride for 6-10 minutes, and finally rinse them with sterile water 4-6 times. b. The time for mercuric chloride immersion disinfection varies depending on the type of explant: If the leaves are young, soak them twice in a 0.1-0.2% mercuric chloride solution, the first time for 2-4 minutes and the second time for 4-6 minutes. If it is the stem tip, soak it twice with a 0.1-0.2% mercuric chloride solution, the first time for 3-5 minutes and the second time for 3-5 minutes; Shake constantly during the soaking process; c. After each disinfection, rinse with sterile water 4-6 times and use sterile filter paper to absorb the surface moisture.

8. The method for efficient propagation of *Spatholobus suberectus* according to claim 5, characterized in that, In step S6, the conditions for rooting culture are: light intensity 800-1500 Lux, light duration 8-10 h / d, and temperature 24-26℃.

9. The method for efficient propagation of *Spatholobus suberectus* according to claim 5, characterized in that, In step S7, the seedling hardening method is as follows: move the culture bottle to diffused light with an intensity of 1800-2000 Lux for 7-10 days, and then open the bottle cap for 1-2 days.

10. The method for efficient propagation of *Spatholobus suberectus* according to claim 5, characterized in that, In step S7, the transplanting substrate is a mixture of loess, peat moss and vermiculite in a volume ratio of (2-3):1:

1. After transplanting, the air humidity is maintained at 75-85% and ventilation is provided. Subsequently, the humidity is gradually reduced and the light intensity is increased.

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