In-vitro rapid propagation method for perfoliote knotweed herb

Abstract

The invention relates to the technical field of plant cultivation, in particular to a perfoliote knotweed herb in-vitro rapid propagation method. The method comprises the following steps: S1, obtaining a sterile material: taking a stem tip explant of perfoliote knotweed herb, performing disinfection treatment, and inoculating the stem tip explant into a primary solid culture medium for culture to obtain sterile clustered seedlings; s2, cluster bud induction: cutting stem segments with nodes of the sterile cluster seedlings, inoculating the stem segments into a cluster bud induction solid culture medium, and directly inducing to generate cluster buds; s3, proliferation of cluster buds: after the cluster buds are cut, transferring the cluster buds into a proliferation solid culture medium for subculture to realize mass proliferation of bud seedlings; s4, rooting culture: selecting a robust single bud, inoculating the single bud into a rooting solid culture medium, and inducing rooting to obtain a complete tissue culture seedling; s5, seedling hardening and transplanting: carrying out seedling hardening on the rooting tissue culture seedlings, and transplanting the seedlings into a domestication matrix to obtain regenerated plants. The propagation period of the perfoliote knotweed herb is shortened, and the propagation efficiency and the transplanting survival rate are improved.

Description

Technical Field

[0001] This application relates to the field of plant cultivation technology, and in particular to a method for rapid propagation of *Polygonum cuspidatum* isolates. Background Technology

[0002] *Polygonum cuspidatum* is a Polygonaceae plant with significant medicinal value, possessing properties such as clearing heat and detoxifying, and promoting diuresis and reducing swelling. Market demand for *Polygonum cuspidatum* is increasing daily. Currently, its propagation mainly relies on wild resource collection or traditional seed propagation, which suffers from problems such as long production cycles, significant environmental influences, susceptibility to disease, and unstable seedling supply, making it difficult to meet the needs of large-scale, standardized production. There are few reports on tissue culture research of *Polygonum cuspidatum*, and existing technologies may have issues such as low propagation efficiency, incomplete systems, or poor stability. Therefore, establishing an efficient, stable, and scalable rapid in vitro propagation system for *Polygonum cuspidatum* is of great significance. Summary of the Invention

[0003] This application provides a method for rapid propagation of *Pterocarya stenoptera* to solve the above-mentioned problems.

[0004] This application provides a method for rapid propagation of *Pterocarya stenoptera*, the method comprising: S1. Obtaining sterile materials: Take the stem tip explants of *Polygonum cuspidatum*, disinfect them, and then inoculate them into a primary solid culture medium containing a first specific concentration ratio of auxin and cytokinin to obtain sterile clustered seedlings. S2, induction of clustered shoots: The stem segments with nodes of the sterile clustered seedlings obtained in step S1 are cut and inoculated into a clustered shoot induction solid medium containing a second specific concentration ratio of auxin and cytokinin to directly induce the generation of clustered shoots. S3. Proliferation of clustered shoots: After the clustered shoots induced in step S2 are cut, they are transferred to a proliferation solid culture medium containing a specific concentration of cytokinin for subculture to achieve a large-scale proliferation of shoots. S4. Rooting culture: Select the robust single shoots obtained in step S3, inoculate them into a rooting solid culture medium containing an appropriate concentration of auxin, induce rooting, and obtain complete tissue culture seedlings. S5. Hardening off and transplanting: After hardening off the rooted tissue culture seedlings obtained in step S4, transplant them into the acclimatization substrate to obtain regenerated plants.

[0005] Through the above technical solutions, and by optimizing the hormone ratios and operational procedures of the culture media at each stage, a complete and efficient rapid propagation system for *Polygonum cuspidatum* in vitro was established. First, sterile material acquisition and primary culture ensured the purity of subsequent propagation materials. Second, in the shoot induction stage, a specific hormone ratio was used to directly induce shoot clusters from segmented stems, avoiding genetic variations that might occur through callus differentiation, which is crucial for maintaining the excellent medicinal properties of *Polygonum cuspidatum*. This direct induction process significantly shortened the propagation cycle and improved propagation efficiency. Furthermore, optimized proliferation and rooting media ensured abundant shoot proliferation and a high rooting rate, exceeding 90%. Finally, hardening off the seedlings combined with a specific ratio of acclimatization substrate (humus: garden soil: perlite) significantly improved transplant survival rates, providing reliable technical support for the large-scale, standardized production of *Polygonum cuspidatum*. This system has a high propagation coefficient and good reproducibility, effectively alleviating the problem of the increasing depletion of wild *Polygonum cuspidatum* resources.

[0006] Optionally, the disinfection process in step S1 involves surface sterilization of the stem tip explants using a gradient disinfection method that increases in intensity. The primary solid culture medium was constructed based on MS medium by adding a specific mass concentration of naphthaleneacetic acid (NAA) and 6-benzylaminopurine (6-BA) as exogenous hormones.

[0007] The above-described technical solution, employing a progressively increasing gradient disinfection method, significantly improved the sterility rate of explants while reducing the damage caused by disinfectants to *Polygonum cuspidatum* shoot tip explants. This increased the yield of sterile material and laid the foundation for successful subsequent in vitro propagation. The primary solid culture medium, based on nutrient-complete MS medium and combined with a specific combination of NAA and 6-BA, ensured rapid initiation of growth and differentiation of explants under sterile conditions. The synergistic effect of NAA and 6-BA enabled explants to efficiently form sterile clusters, shortening the primary culture cycle; material suitable for subculture could typically be obtained within 10-15 days. This optimized primary culture protocol improved the efficiency and stability of the entire rapid propagation system.

[0008] Optionally, in the primary solid culture medium, the specific mass concentration of naphthaleneacetic acid (NAA) is 0.2 mg / L, and the specific mass concentration of 6-benzylaminopurine (6-BA) is 1.0 mg / L.

[0009] By employing the above-described technical solution and a specific hormone ratio of 0.2 mg / L NAA and 1.0 mg / L 6-BA, this invention achieves a high initiation rate and a high rate of sterile seedlings obtained from primary culture. This optimized formulation avoids problems such as slow growth, vitrification, or excessive callus formation caused by improper hormone concentrations. Precise quantitative control ensures the repeatability and stability of the primary culture process, typically yielding sterile stem segments suitable for subculture within approximately 12 days, significantly shortening the overall rapid propagation system cycle.

[0010] Optionally, the gradient disinfection method includes: First, immerse the product in a 75% ethanol solution for 1 minute, then immerse it in a 0.1% mercuric chloride (HgCl2) solution for 5-6 minutes. After each disinfection treatment, rinse the product with sterile water 3-4 times to remove any residual disinfectant.

[0011] Through the above technical solutions, pretreatment with 75% ethanol enhances the bactericidal effect of mercuric chloride, while deep disinfection with 0.1% mercuric chloride ensures a high sterility rate, typically achieving over 90% sterile material yield. Precise control of the mercuric chloride soaking time (5-6 minutes) effectively balances sterilization efficiency and phytotoxicity. Most importantly, rinsing with sterile water 3-4 times after each disinfection ensures no disinfectant residue, significantly improving explant survival rates and preventing browning or death caused by chemical toxicity.

[0012] Optionally, the bud induction solid medium in step S2 is based on MS medium and is constructed by adding a specific mass concentration of naphthaleneacetic acid (NAA) and a higher concentration of 6-benzylaminopurine (6-BA); the direct induction refers to the process in which the axillary buds of the segmented stem directly germinate and proliferate to form a bud cluster without going through the callus differentiation stage.

[0013] Through the above technical solution, by employing a combination of high-concentration 6-BA and low-concentration NAA, this invention achieves direct induction of shoot clusters in *Pleurotus ostreatus*, offering the following significant advantages: First, it avoids callus differentiation, thereby reducing the risk of somatic cell mutation and ensuring the genetic purity of the seedlings. Second, it boasts extremely high propagation efficiency, with an induction rate exceeding 90%, and each explant can produce a large number of shoot clusters, significantly improving the propagation coefficient. Furthermore, it shortens the culture cycle, as there is no need to wait for callus formation and redifferentiation; the entire induction process is typically completed within 4 weeks.

[0014] Optionally, in the bud-inducing solid culture medium, the specific mass concentration of naphthaleneacetic acid (NAA) is 0.1 mg / L, and the specific mass concentration of 6-benzylaminopurine (6-BA) is 2.0 mg / L.

[0015] By employing the aforementioned technical solution and a specific ratio of NAA 0.1 mg / L and 6-BA 2.0 mg / L, high efficiency and stability in inducing shoot clusters in *Polygonum cuspidatum* were achieved. This formulation ensures direct shoot induction and avoids genetic variation. Experimental results show that under this formulation, the shoot cluster induction rate can reach 93.35%, with an average of at least 10 shoot clusters per explant, significantly improving propagation efficiency and providing technical support for the large-scale production of *Polygonum cuspidatum*.

[0016] Optionally, the culture conditions for step S2 are: temperature 25±1℃, light intensity 1000-1500lx, and photoperiod 12 hours / day; after 28 days of culture, the induction rate of clustered shoots reaches 93.35%, and each explant produces at least 10 clustered shoots on average.

[0017] The above-described technical solution, combining precisely controlled culture conditions (temperature 25±1℃, light intensity 1000-1500 lx, photoperiod 12 hours / day) with an optimized culture medium formula, significantly improves the efficiency of shoot induction. An induction rate as high as 93.35% and a propagation coefficient of at least 10 shoots per explant indicate that this method has extremely high potential for large-scale production. The high induction rate and high shoot yield significantly shorten the time required to obtain a large amount of propagation material, reduce production costs, and simultaneously ensure seedling quality.

[0018] Optionally, the proliferation solid culture medium in step S3 is constructed based on MS medium, with only a specific mass concentration of 6-benzylaminopurine (6-BA) added as an exogenous hormone for the continuous expansion of the clustered shoots; In the proliferative solid culture medium, the specific mass concentration of 6-benzylaminopurine (6-BA) is 2.0 mg / L.

[0019] By employing the above-described technical solution and a solid culture medium containing only 2.0 mg / L 6-BA, this invention achieves an extremely high proliferation coefficient for *Gynostemma pentaphyllum* buds, typically reaching 8-10 times, far exceeding the propagation efficiency of existing technologies. This single-hormone formulation simplifies the culture medium preparation process and reduces costs. More importantly, because it does not contain auxin, it effectively prevents the formation of callus tissue or premature rooting during the bud proliferation process, ensuring the purity and quality of the proliferation material and providing sufficient, high-quality, robust single buds for subsequent rooting culture.

[0020] Optionally, the rooting solid medium described in step S4 is based on 1 / 2 MS medium and is constructed by adding a specific mass concentration of naphthaleneacetic acid (NAA) as the sole exogenous auxin; In the rooting solid culture medium, the specific mass concentration of naphthaleneacetic acid (NAA) is 0.3 mg / L.

[0021] By employing the above technical solution, using a combination of 1 / 2 MS basal medium and 0.3 mg / L NAA, this invention achieved a high rooting rate and robust root system in single buds of *Gynostemma pentaphyllum*. The low salt concentration (1 / 2 MS) promoted rapid root elongation and development, while the precise concentration of NAA efficiently induced the formation of adventitious roots. Experimental results showed a rooting rate of 96.67% and an average of 13.6 roots per bud, ensuring a high survival rate after transplanting. Furthermore, the use of a single exogenous auxin simplified the culture medium formulation, improving the accuracy and repeatability of the operation.

[0022] Optionally, the hardening-off process in step S5 includes: opening the culture tank in a brightly lit place to allow the tissue culture seedlings to gradually adapt to the natural environment; the acclimatization substrate is a mixture of humus, garden soil and perlite in a specific volume ratio; The mixing volume ratio of the acclimatization substrate is humus: garden soil: perlite = 1:2:1.

[0023] Through the above technical solutions, the stomatal regulation function of tissue-cultured seedlings is restored by gradually opening and hardening them off, enhancing their drought resistance and effectively preventing death caused by sudden environmental changes during transplanting. The acclimatization substrate ratio of humus:garden soil:perlite = 1:2:1 has optimal water retention and aeration properties, providing a suitable growth environment for the roots of tissue-cultured seedlings, thereby increasing the transplant survival rate to over 90%, significantly better than the survival rate of traditional single-substrate methods. This optimized hardening and transplanting program is the key guarantee for the entire rapid propagation system to achieve a high survival rate of regenerated plants. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 A flowchart illustrating a rapid propagation method for *Pterocarya stenoptera* according to an embodiment of this application. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0027] Furthermore, the term "and / or" in this article is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. Additionally, the character " / " in this article, unless otherwise specified, generally indicates that the preceding and following related objects have an "or" relationship.

[0028] The embodiments of this application will now be described in further detail with reference to the accompanying drawings.

[0029] Currently, the propagation of *Polygonum cuspidatum* mainly relies on wild resource collection or traditional seed propagation, which suffers from problems such as long cycle, high susceptibility to environmental influences, susceptibility to disease, and unstable seedling supply, making it difficult to meet the needs of large-scale, standardized production. There are few reports on tissue culture research on *Polygonum cuspidatum*, and existing technologies may have issues such as low propagation efficiency, incomplete systems, or poor stability. Therefore, establishing an efficient, stable, and scalable rapid in vitro propagation system for *Polygonum cuspidatum* is of great significance.

[0030] Based on this, this application provides a rapid propagation method for *Polygonum cuspidatum* isolated organisms. By optimizing the hormone ratios and operational procedures at each stage of the culture medium, a complete and efficient rapid propagation system for *Polygonum cuspidatum* isolated organisms was established. First, sterile material acquisition and primary culture ensured the purity of subsequent propagation materials. Second, in the shoot induction stage, a specific hormone ratio was used to directly induce shoots from segmented stems, avoiding genetic variations that might occur through callus differentiation, which is crucial for maintaining the excellent medicinal properties of *Polygonum cuspidatum*. This direct induction process significantly shortened the propagation cycle and improved propagation efficiency. Furthermore, optimized proliferation and rooting media ensured massive proliferation of seedlings and a high rooting rate, exceeding 90%. Finally, hardening off seedlings combined with a specific ratio of acclimatization substrate (humus: garden soil: perlite) significantly improved transplant survival rates, providing reliable technical support for the large-scale, standardized production of *Polygonum cuspidatum*. This system has a high propagation coefficient and good reproducibility, effectively alleviating the problem of increasingly depleted wild resources of *Polygonum cuspidatum*.

[0031] For specific implementation details, please refer to the following examples.

[0032] Figure 1 A flowchart of a rapid propagation method for *Pterocarya stenoptera* provided in one embodiment of this application is shown below. Figure 1 As shown, the method includes: S1. Obtaining sterile materials: Take the stem tip explants of *Polygonum cuspidatum*, disinfect them, and then inoculate them into a primary solid culture medium containing a first specific concentration ratio of auxin and cytokinin to obtain sterile clustered seedlings. S2, induction of clustered shoots: The stem segments with nodes of the sterile clustered seedlings obtained in step S1 are cut and inoculated into a clustered shoot induction solid medium containing a second specific concentration ratio of auxin and cytokinin to directly induce the generation of clustered shoots. S3. Proliferation of clustered shoots: After the clustered shoots induced in step S2 are cut, they are transferred to a proliferation solid culture medium containing a specific concentration of cytokinin for subculture to achieve a large-scale proliferation of shoots. S4. Rooting culture: Select the robust single shoots obtained in step S3, inoculate them into a rooting solid culture medium containing an appropriate concentration of auxin, induce rooting, and obtain complete tissue culture seedlings. S5. Hardening off and transplanting: After hardening off the rooted tissue culture seedlings obtained in step S4, transplant them into the acclimatization substrate to obtain regenerated plants.

[0033] Technical Background and Working Principle: This invention belongs to the field of plant tissue culture. Addressing the problems of long propagation cycles, susceptibility to disease, and unstable seedling supply associated with existing methods of propagating *Polygonum multiflorum* (also known as *Polygonum hydropiper*), which rely on wild collection or traditional seed propagation, this invention proposes a rapid in vitro propagation technology. Based on the principle of plant hormone regulation of plant cell differentiation and organogenesis, this invention utilizes a specific ratio of exogenous auxin (such as naphthaleneacetic acid, NAA) and cytokinin (such as 6-benzylaminopurine, 6-BA) to precisely control the growth direction of *Polygonum hydropiper* explants (shoot tip explants). In the initial culture (S1), the balanced hormone ratio achieves aseptication and preliminary proliferation of the explants. In the shoot induction stage (S2), by increasing the relative concentration of cytokinin, the direct germination of axillary buds to form shoot clusters is promoted, effectively avoiding genetic variations that may occur due to callus differentiation pathways and ensuring the genetic stability of the seedlings. This is the core technical basis of this invention. The subsequent proliferation (S3) and rooting (S4) stages further optimize the hormone ratio to ensure rapid expansion of seedlings and robust root formation. Finally, through hardening and transplanting (S5), regenerated plants are obtained.

[0034] Technical Solution and Component Functions: This invention provides a method for rapid propagation of *Polygonum cuspidatum* explants, comprising five key steps: S1, obtaining sterile material; S2, inducing shoot clusters; S3, shoot cluster proliferation; S4, rooting culture; and S5, hardening off and transplanting. In S1, shoot tip explants are selected, possessing strong meristematic ability and a low contamination rate. After sterilization, they are inoculated into a primary solid culture medium. The primary solid culture medium contains a first specific concentration ratio of auxin and cytokinin, its main function being to initiate explant growth and obtain sterile subculture material. In S2, segmented stem pieces are inoculated into a shoot cluster induction solid culture medium containing a second specific concentration ratio of auxin and cytokinin. The key is to adjust the hormone ratio to directly induce axillary bud germination, forming shoot clusters. In S3, the shoot clusters are divided and transferred to a proliferation solid culture medium. The concentration of cytokinin in this medium is used to maintain rapid shoot meristematic growth and proliferation, achieving large-scale propagation. In step S4, robust single buds are transferred to a rooting solid medium containing an appropriate concentration of auxin (such as NAA) to induce root primordia formation and promote root development, resulting in complete tissue culture seedlings. In step S5, the hardening-off process allows the tissue culture seedlings to gradually adapt to the natural environment, followed by transplanting into an acclimatization substrate to ensure the survival rate of regenerated plants. This method, through optimized steps and precise control of the culture medium, achieves rapid and stable propagation of *Polygonum cuspidatum*.

[0035] Beneficial Effects: This invention establishes a complete and efficient rapid propagation system for *Polygonum cuspidatum* in vitro by optimizing the hormone ratios and operational procedures at each stage of the culture medium. First, sterile material acquisition and primary culture ensure the purity of subsequent propagation materials. Second, in the shoot induction stage, a specific hormone ratio is used to directly induce shoots from segmented stems, avoiding genetic variations that may occur through callus differentiation, which is crucial for maintaining the excellent medicinal properties of *Polygonum cuspidatum*. This direct induction process significantly shortens the propagation cycle and improves propagation efficiency. Furthermore, optimized proliferation and rooting media ensure massive proliferation of shoots and a high rooting rate, exceeding 90%. Finally, hardening off the seedlings combined with a specific ratio of acclimatization substrate (humus: garden soil: perlite) significantly improves transplant survival rate, providing reliable technical support for the large-scale, standardized production of *Polygonum cuspidatum*. This system has a high propagation coefficient and good reproducibility, effectively alleviating the problem of increasingly depleted wild resources of *Polygonum cuspidatum*.

[0036] In some embodiments, the disinfection process in step S1 involves surface sterilization of the shoot tip explants using a gradient disinfection method with progressively increasing concentrations; the primary solid culture medium is based on MS medium and is constructed by adding a specific mass concentration of naphthaleneacetic acid (NAA) and 6-benzylaminopurine (6-BA) as exogenous hormones.

[0037] Technical Background and Working Principle: In existing plant tissue culture, surface sterilization of explants is a crucial step in obtaining sterile material. However, traditional single-concentration disinfectant treatments are often inefficient or prone to causing phytotoxicity to explants. This invention employs a gradient sterilization method with progressively increasing concentrations, utilizing different disinfectants and concentration gradients to improve the thoroughness of surface sterilization while minimizing damage to explants and increasing the yield of sterile material. The primary solid culture medium is based on MS medium, which, due to its high-salt formulation, provides a complete set of macro and micronutrients required for plant growth, offering comprehensive nutrition. Exogenous hormones include naphthaleneacetic acid (NAA) as an auxin and 6-benzylaminopurine (6-BA) as a cytokinin. The combination of NAA and 6-BA plays a balancing and regulatory role in primary culture. NAA promotes the initial formation of callus or root primordia, while 6-BA induces bud germination. The specific ratio of the two works synergistically to ensure that explants initiate meristem and growth under sterile conditions, rapidly transitioning to the next stage of propagation material.

[0038] Technical Solution and Component Functions: This embodiment refines the process of obtaining sterile material in step S1. A gradient disinfection method is used, where explants are treated sequentially with different types and concentrations of disinfectants to ensure complete eradication of surface microorganisms. This method avoids the severe phytotoxicity that can result from a single treatment with high-concentration disinfectants, improving the survival rate and sterility of explants. The primary solid culture medium is based on MS medium, a universal basal medium for plant tissue culture that provides ample nutrients and minerals. A key technical feature of the primary solid culture medium is the addition of exogenous hormones: a combination of naphthaleneacetic acid (NAA) and 6-benzylaminopurine (6-BA). NAA, as a synthetic auxin, promotes cell elongation and differentiation at lower concentrations; 6-BA, as a highly active cytokinin, primarily promotes cell division and shoot formation. In primary culture, the specific concentration ratio of this combination aims to induce rapid formation of sterile shoot clusters from explants, providing healthy and stable starting material for subsequent shoot cluster induction.

[0039] Beneficial Effects: The progressively increasing sterilization gradient significantly improved the sterility of explants while reducing the damage caused by disinfectants to *Polygonum aviculare* shoot apical explants, thereby increasing the yield of sterile material and laying the foundation for successful subsequent in vitro propagation. The primary solid culture medium, based on nutrient-complete MS medium and combined with a specific combination of NAA and 6-BA, ensured rapid initiation of growth and differentiation of explants under sterile conditions. The synergistic effect of NAA and 6-BA enabled explants to efficiently form sterile clusters, shortening the primary culture cycle; material suitable for subculture could typically be obtained within 10-15 days. This optimized primary culture protocol improved the efficiency and stability of the entire rapid propagation system.

[0040] In some embodiments, the specific mass concentration of naphthaleneacetic acid (NAA) in the primary solid culture medium is 0.2 mg / L, and the specific mass concentration of 6-benzylaminopurine (6-BA) is 1.0 mg / L.

[0041] Technical Background and Working Principle: The precise concentration of exogenous hormones in primary solid culture media is a key factor determining the differentiation direction and growth rate of explants. This invention, through extensive screening experiments, determined a specific concentration ratio of NAA (0.2 mg / L) and 6-BA (1.0 mg / L) to achieve optimal growth initiation and aseptic seedling formation in *Pleurotus ostreatus* shoot apical explants. In this ratio, the low concentration of NAA (auxin) primarily induces cell elongation and a small amount of callus formation without causing excessive callus formation. The relatively high concentration of 6-BA (cytokinin) primarily breaks apical dominance, promotes axillary bud germination and rapid cell division, thereby forming compact, robust, aseptic clustered seedlings. This low auxin / high cytokinin ratio strategy aims to maximize the efficiency and quality of aseptic material acquisition, laying a high-quality foundation for subsequent shoot clustering induction.

[0042] Technical Solution and Component Functions: This embodiment quantitatively defines the hormone ratios in the primary solid culture medium. The primary solid culture medium is based on MS medium, with the added naphthaleneacetic acid (NAA) concentration precisely set at 0.2 mg / L and 6-benzylaminopurine (6-BA) concentration precisely set at 1.0 mg / L. The low concentration of NAA (0.2 mg / L) ensures that the base of the explant does not excessively lignify or form a large amount of tissueless callus. The higher concentration of 6-BA (1.0 mg / L) effectively promotes the germination of axillary buds from the explants, forming clustered seedlings with multi-bud structures. This precise concentration control optimizes the culture effect during the primary culture stage, resulting in healthy, sterile subculture material with multi-bud differentiation ability in the shortest possible time.

[0043] Beneficial Effects: By employing a specific hormone ratio of 0.2 mg / L NAA and 1.0 mg / L 6-BA, this invention achieves a high initiation rate and a high rate of sterile seedlings obtained from primary culture. This optimized formulation avoids problems such as slow growth, vitrification, or excessive callus formation caused by improper hormone concentrations. Precise quantitative control ensures the repeatability and stability of the primary culture process, typically yielding sterile stem segments suitable for subculture within approximately 12 days, significantly shortening the overall rapid propagation cycle.

[0044] In some embodiments, the gradient disinfection method includes: first immersing in a 75% ethanol solution for 1 minute, then immersing in a 0.1% HgCl2 solution for 5-6 minutes, and rinsing with sterile water 3-4 times after each disinfection treatment to remove residual disinfectant.

[0045] Technical Background and Working Principle: In step S1, obtaining sterile materials, surface disinfection of explants is a crucial step. Traditional single-disinfectant treatments often fail to completely eliminate microorganisms on the surface and interstitial spaces of *Gnaphalium affine* explants and can easily cause phytotoxicity. This invention employs a gradient disinfection method that utilizes the synergistic effect of two disinfectants: ethanol and mercuric chloride (HgCl2). A 75% volume concentration ethanol solution is used as a pretreatment, primarily to remove lipid-soluble substances from the explant surface and dehydrate the cell walls, thereby enhancing the penetration of subsequent disinfectants. Subsequently, a 0.1% mass concentration mercuric chloride solution is used for deep disinfection. Mercuric chloride has strong bactericidal capabilities, effectively killing fungal and bacterial spores on the explant surface. The soaking time is controlled at 5-6 minutes to maximize the bactericidal effect while keeping phytotoxicity within acceptable limits. Rinsing with sterile water 3-4 times after each disinfection treatment is a critical step. Its purpose is to thoroughly remove residual ethanol and mercuric chloride, preventing continuous chemical damage to the explants and ensuring their healthy initiation of growth.

[0046] Technical Solution and Component Functions: The specific operational procedure of the gradient disinfection method is as follows: First, the stem apex explants of *Polygonum cuspidatum* are immersed in a 75% (v / v) ethanol solution for a precisely controlled soaking time of 1 minute. The rapid penetration of ethanol effectively removes surface contaminants and provides preliminary sterilization. Subsequently, the explants are transferred to a 0.1% (w / w) mercuric chloride (HgCl2) solution for a soaking time of 5-6 minutes. The 0.1% mercuric chloride concentration provides highly efficient broad-spectrum sterilization, and the 5-6 minute soaking time is the optimal treatment time selected through optimization, ensuring thorough sterilization. Crucially, after both ethanol and mercuric chloride treatments, the explants must be rinsed 3-4 times with sterile water. Sterile water rinsing ensures complete removal of disinfectant residues, avoiding continuous toxic damage to the explant tissue, thereby significantly improving the survival rate and sterility of the explants.

[0047] Beneficial Effects: The gradient disinfection process in this embodiment has significant advantages: pretreatment with 75% ethanol enhances the bactericidal effect of mercuric chloride, while deep disinfection with 0.1% mercuric chloride ensures a high sterility rate, typically achieving over 90% sterile material yield. Precise control of the mercuric chloride soaking time (5-6 minutes) effectively balances sterilization efficiency and phytotoxicity. Most importantly, rinsing with sterile water 3-4 times after each disinfection ensures no disinfectant residue, greatly improving explant survival rates and preventing browning or death caused by chemical toxicity.

[0048] In some embodiments, the bud induction solid medium in step S2 is based on MS medium and is constructed by adding a specific mass concentration of naphthaleneacetic acid (NAA) and a higher concentration of 6-benzylaminopurine (6-BA); the direct induction refers to the process by which axillary buds of the segmented stem directly germinate and proliferate to form bud clusters without going through the callus differentiation stage.

[0049] Technical Background and Working Principle: In step S2, shoot induction, the goal is to achieve rapid and large-scale propagation of shoots. Traditional tissue culture methods may require inducing callus tissue first, followed by shoot differentiation, which is not only time-consuming but also prone to genetic variation due to the callus differentiation pathway. This invention innovatively employs a specific hormone ratio to directly induce shoot clusters. The shoot induction solid medium is based on MS medium, with the addition of a high concentration of 6-BA (cytokinin) and a specific concentration of NAA (auxin), resulting in a significantly higher cytokinin ratio than auxin. According to the plant hormone balance theory, a high cytokinin / low auxin ratio is beneficial for shoot formation and differentiation. This ratio effectively breaks the dormancy of axillary buds in noded stem segments, allowing them to germinate directly and proliferate to form shoot clusters, thus bypassing the callus differentiation stage, greatly improving propagation efficiency, and ensuring the genetic stability and preservation of superior traits in regenerated plants.

[0050] Technical Solution and Component Functions: This embodiment clearly defines the key technical features of step S2, shoot induction. The solid culture medium for shoot induction is MS medium. Exogenous hormones are a combination of NAA and a high concentration of 6-BA. The high concentration of 6-BA (cytokinin) is the core driving force for direct shoot induction, strongly promoting cell division and shoot differentiation. A specific concentration of NAA (auxin) is used to maintain cell activity and a small amount of elongation growth, but its concentration must be low enough to avoid inducing callus formation. Direct induction refers to the direct initiation of meristem formation by axillary buds or dormant buds on the segmented stem during culture, without going through an amorphous, undifferentiated callus stage. The segmented stem, as an explant, has axillary buds that are natural meristematic tissue; precise hormone regulation to efficiently initiate meristem formation is key to achieving rapid propagation.

[0051] Beneficial Effects: By employing a combination of high-concentration 6-BA and low-concentration NAA, this invention achieves direct induction of shoot clusters in *Pleurotus ostreatus*, offering the following significant advantages: First, it avoids callus differentiation, thereby reducing the risk of somatic cell mutation and ensuring the genetic purity of the seedlings. Second, it boasts extremely high propagation efficiency, with an induction rate exceeding 90%, and each explant can produce a large number of shoot clusters, significantly improving the propagation coefficient. Furthermore, it shortens the culture cycle, as there is no need to wait for callus formation and redifferentiation; the entire induction process is typically completed within 4 weeks.

[0052] In some embodiments, the specific mass concentration of naphthaleneacetic acid (NAA) in the shoot induction solid culture medium is 0.1 mg / L, and the specific mass concentration of 6-benzylaminopurine (6-BA) is 2.0 mg / L.

[0053] Technical Background and Working Principle: This embodiment further defines the optimal hormone ratio in the solid culture medium for inducing shoot clusters in step S2. This ratio is crucial for achieving high-efficiency direct induction of shoot clusters from the stem segments of *Gynostemma pentaphyllum*. NAA at 0.1 mg / L is an extremely low auxin concentration, primarily maintaining cell activity while inhibiting callus differentiation. 6-BA at 2.0 mg / L is a relatively high cytokinin concentration, strongly driving axillary bud differentiation and proliferation. This precise ratio of extremely low auxin to high cytokinin ensures that cell differentiation is precisely guided towards bud formation, thereby achieving direct induction without the need for a callus differentiation stage, ensuring a high induction rate and a high propagation coefficient.

[0054] Technical Solution and Component Functions: The solid medium for inducing shoot clusters is based on MS medium, with specific concentrations of exogenous hormones set as follows: naphthaleneacetic acid (NAA) 0.1 mg / L and 6-benzylaminopurine (6-BA) 2.0 mg / L. The NAA concentration of 0.1 mg / L ensures that the inhibitory effect of auxin on shoot differentiation is minimized while providing the necessary auxin signal. The 6-BA concentration of 2.0 mg / L provides a strong cell division signal, effectively activating axillary buds in the segmented stem, enabling them to germinate rapidly and form dense shoot clusters. This ratio is the optimal solution after optimization and screening, enabling a shoot cluster induction rate of over 90%, and the resulting shoot clusters are robust and free from vitrification.

[0055] Beneficial Effects: By employing a specific ratio of NAA 0.1 mg / L and 6-BA 2.0 mg / L, this invention achieves high efficiency and stability in inducing shoot clusters in *Polygonum cuspidatum*. This formulation ensures direct shoot induction and avoids genetic variation. Experimental results show that under this formulation, the shoot cluster induction rate can reach 93.35%, with an average of at least 10 shoot clusters per explant, greatly improving propagation efficiency and providing technical support for the large-scale production of *Polygonum cuspidatum*.

[0056] In some embodiments, the culture conditions for step S2 are: temperature 25±1℃, light intensity 1000-1500lx, and light cycle 12 hours / day; after 28 days of culture, the induction rate of clustered shoots reaches 93.35%, and each explant produces at least 10 clustered shoots on average.

[0057] Technical Background and Working Principle: The efficiency of plant tissue culture depends not only on the composition of the culture medium but also on the strict control of environmental conditions. In step S2, the induction of shoot clusters, this invention optimizes the culture conditions. A temperature of 25±1℃ is the optimal temperature range for most plant tissue cultures, ensuring normal enzyme activity and cell metabolism. A light intensity of 1000-1500 lx and a photoperiod of 12 hours / day provide suitable photosynthetic conditions, ensuring carbohydrate accumulation in the shoots, promoting robust growth, and preventing yellowing. Under these optimized conditions, combined with a specific hormone ratio (such as NAA 0.1 mg / L and 6-BA 2.0 mg / L), the segmented stems of *Polygonum cuspidatum* can be efficiently induced to differentiate directly. After 28 days of culture, statistical data showed that the shoot cluster induction rate was as high as 93.35%, and each explant produced an average of at least 10 shoot clusters, indicating that the culture conditions and culture medium formulation of this invention have a very high synergistic effect and reproductive efficiency.

[0058] Technical Solution and Component Functions: This embodiment precisely sets the culture environment parameters for step S2, shoot induction 12. Temperature is controlled within a narrow range of 25±1℃ to ensure optimal physiological activity of the meristematic tissue of the *Pterocarya stenoptera* cells. Light intensity is set at 1000-1500 lx, which meets the needs of plant photosynthesis, promotes robust seedlings, and avoids scorching or inhibition caused by excessive light. The photoperiod is 12 hours of light / 12 hours of darkness, simulating a suitable diurnal environment for plant growth. Under these precisely controlled conditions, after a 28-day culture period, explants achieve efficient direct induction. The result is a shoot induction rate of 93.35%, with an average of at least 10 shoots produced per explant. These quantitative data fully demonstrate the high efficiency and stability of this invention in the shoot induction stage, providing sufficient high-quality material for subsequent proliferation 13.

[0059] Beneficial effects: Precisely controlled culture conditions (temperature 25±1℃, light intensity 1000-1500 lx, photoperiod 12 hours / day) combined with optimized culture medium formulation significantly improved the efficiency of shoot induction. An induction rate as high as 93.35% and a propagation coefficient of at least 10 shoots per explant indicate that this method has extremely high potential for large-scale production. The high induction rate and high shoot yield significantly shorten the time required to obtain a large amount of propagation material, reduce production costs, and simultaneously ensure seedling quality.

[0060] In some embodiments, the proliferation solid culture medium in step S3 is based on MS medium, with only a specific mass concentration of 6-benzylaminopurine (6-BA) added as an exogenous hormone for the continuous expansion of the clustered shoots; the specific mass concentration of 6-benzylaminopurine (6-BA) in the proliferation solid culture medium is 2.0 mg / L.

[0061] Technical Background and Working Principle: In step S3, the shoot proliferation stage, the core objective is to achieve a geometric increase in the number of shoots. According to the principles of plant hormone regulation, high concentrations of cytokinins can continuously promote cell division, maintaining the continuous germination of axillary buds, thereby achieving continuous expansion. This invention uses MS medium as a base and innovatively adds only a specific mass concentration of 6-benzylaminopurine (6-BA) as an exogenous hormone. This formulation strategy aims to maximize shoot differentiation and proliferation without adding auxins (such as NAA) to avoid auxin-induced callus differentiation or root formation, thus concentrating nutrients and energy for rapid shoot expansion. A 6-BA concentration of 2.0 mg / L is the key preferred concentration for maintaining a high proliferation coefficient (up to 8-10 times).

[0062] Technical Solution and Component Functions: This embodiment precisely defines the culture medium for the proliferation of shoot clusters in step S3. The proliferation solid culture medium 40 is based on MS medium, and its core feature is the addition of only the exogenous hormone 6-benzylaminopurine (6-BA). As a highly active cytokinin, 6-BA continuously breaks the apical dominance of the shoot, promoting the rapid germination and growth of lateral and axillary buds, thus achieving continuous expansion of the shoots. This culture medium does not contain exogenous auxin, ensuring that the shoots in the proliferation stage maintain high meristematic capacity and do not undergo root or callus differentiation. The specific mass concentration of 6-BA is set at 2.0 mg / L, which is the optimal range selected to achieve the highest proliferation rate while maintaining the robustness and green color of the shoots.

[0063] Beneficial Effects: Using a solid culture medium containing only 2.0 mg / L 6-BA, this invention achieves an extremely high proliferation coefficient for *Gynostemma pentaphyllum* buds, typically reaching 8-10 times, far exceeding the propagation efficiency of existing technologies. This single-hormone formulation simplifies the culture medium preparation process and reduces costs. More importantly, because it does not contain auxin, it effectively avoids the formation of callus tissue or premature rooting during the bud proliferation process, ensuring the purity and quality of the proliferation material and providing sufficient, high-quality, robust single buds for subsequent rooting culture.

[0064] In some embodiments, the rooting solid culture medium in step S4 is based on 1 / 2 MS medium and is constructed by adding a specific mass concentration of naphthaleneacetic acid (NAA) as the sole exogenous auxin; the specific mass concentration of naphthaleneacetic acid (NAA) in the rooting solid culture medium is 0.3 mg / L.

[0065] Technical Background and Working Principle: In step S4, the rooting culture stage, the goal is to induce robust single buds to form complete and well-developed root systems. Root differentiation requires a suitable low-salt environment and a specific concentration of auxin. This invention uses 1 / 2 MS medium as the base, with its salt concentration halved, reducing osmotic pressure and promoting the formation and elongation of root primordia, while avoiding the inhibition of root development caused by high salt concentration. Naphthaleneacetic acid (NAA) is used as the sole exogenous auxin, and NAA is highly effective in inducing adventitious roots. A NAA concentration of 0.3 mg / L is an optimized concentration that efficiently induces rooting and ensures robust root systems. The absence of cytokinin is crucial, as cytokinin inhibits root development, ensuring that auxin signaling dominates the differentiation direction and achieves a high rooting rate.

[0066] Technical Solution and Component Functions: This embodiment precisely defines the culture medium for rooting culture in step S4. The rooting solid culture medium is based on 1 / 2 MS medium, with a moderate concentration of nutrients that is conducive to root differentiation. The key feature of the exogenous hormone is the addition of only naphthaleneacetic acid (NAA) as the sole auxin, and its specific mass concentration is set at 0.3 mg / L. The NAA concentration of 0.3 mg / L provides the auxin signal required to induce root primordia formation, ensuring a high rooting rate. By setting the culture medium base to 1 / 2 MS and using a single and appropriate concentration of NAA, the rooting culture is optimized, typically achieving a rooting rate of over 95% within 25 days, and forming an average of more than 10 robust roots.

[0067] Beneficial Effects: By using a combination of 1 / 2 MS basal medium and 0.3 mg / L NAA, this invention achieved a high rooting rate and robust root system in single buds of *Gynostemma pentaphyllum*. The low salt concentration (1 / 2 MS) promoted rapid root elongation and development, while the precise concentration of NAA efficiently induced the formation of adventitious roots. Experimental results showed a rooting rate of 96.67% and an average of 13.6 roots per bud, ensuring a high survival rate after transplanting. Furthermore, the use of a single exogenous auxin simplified the culture medium formulation and improved the accuracy and repeatability of the operation.

[0068] In some embodiments, the seedling hardening process in step S5 includes: opening the culture tank in a brightly lit place to allow the tissue culture seedlings to gradually adapt to the natural environment; the acclimatization substrate is a mixture of humus, garden soil and perlite in a specific volume ratio; the mixing volume ratio of the acclimatization substrate is humus: garden soil: perlite = 1:2:1.

[0069] Technical Background and Working Principle: Step S5, hardening and transplanting, is the final crucial step for successful tissue culture. Tissue culture seedlings grow in a sterile, high-humidity, low-light environment within the bottle, resulting in weak stomatal regulation and photosynthetic capacity. The hardening process aims to gradually reduce humidity, increase light, and improve ventilation, allowing the seedlings to adapt to the natural environment and enhancing their resilience. Opening the culture tank in a brightly lit area is the core operation of hardening; by gradually opening or uncovering the tank, the stomatal function of the seedlings is restored, enhancing their adaptability to the external environment. The selection of the acclimatization substrate is critical, requiring good water retention, aeration, and nutrition. This invention uses a mixture of humus, garden soil, and perlite in a 1:2:1 volume ratio. Humus provides organic matter and water retention, garden soil provides basic support and minerals, and perlite provides good aeration and drainage. This specific substrate ratio provides an ideal growth environment for the hardened tissue culture seedlings, ensuring a high survival rate after transplanting.

[0070] Technical Solution and Component Functions: This implementation precisely defines the operational details of step S5, hardening and transplanting, and the formulation of the acclimatization substrate. The specific operation of the hardening process is as follows: After rooting culture is completed, the culture tank is moved to a brightly lit area, and the lid is gradually opened or uncovered. For example, the lid is opened for 1 hour on the first day, 2 hours on the second day, and the opening time is gradually extended for 3-5 days, allowing the tissue culture seedlings to gradually adapt to lower humidity and stronger light. The acclimatization substrate is a mixture of three components: humus, garden soil, and perlite. Humus provides a loose structure and abundant organic matter; garden soil provides stable support and basic nutrients; perlite is a porous material that ensures good aeration and drainage of the substrate, preventing root rot. The volume ratio of these three components is 1:2:1. This precise ratio is the optimal solution selected through screening, balancing water retention and aeration to ensure normal respiration and growth of the roots after transplanting.

[0071] Beneficial effects: Through gradual hardening-off, the stomatal regulation function of tissue-cultured seedlings is restored, drought resistance is enhanced, and mortality caused by sudden environmental changes during transplanting is effectively avoided. The acclimatization substrate ratio of humus:garden soil:perlite = 1:2:1 has optimal water retention and aeration properties, providing a suitable growth environment for the roots of tissue-cultured seedlings, thereby increasing the transplant survival rate to over 90%, significantly better than the survival rate of traditional single-substrate methods. This optimized hardening-off and transplanting program is the key guarantee for the entire rapid propagation system to achieve a high survival rate of regenerated plants.

Claims

1. A method for rapid propagation of Gynostemma pentaphyllum in vitro, characterized by, The method comprises the following steps: S1, obtaining sterile materials: taking the stem tip type explants of the plant, after disinfection, inoculating into the primary solid culture medium containing a first specific concentration of auxin and cytokinin for culture to obtain sterile shoots; S2, inducing shoots: cutting the stem segments of the sterile shoots obtained in step S1, inoculating into the shoot induction solid culture medium containing a second specific concentration of auxin and cytokinin to directly induce shoots; S3, shoot proliferation: cutting the shoots induced in step S2, and then transferring to the proliferation solid culture medium containing a specific concentration of cytokinin for subculture to realize the mass proliferation of shoots; S4, rooting culture: selecting the healthy single shoots obtained in step S3, inoculating into the rooting solid culture medium containing a suitable concentration of auxin to induce rooting and obtain complete tissue culture seedlings; S5, hardening and transplanting: hardening the rooted tissue culture seedlings obtained in step S4, and then transplanting into the acclimatization substrate to obtain regenerated plants.

2. The method of claim 1, wherein, In step S1, the disinfection treatment is performed on the stem tip type explants by using a gradient disinfection method with increasing concentration. The primary solid culture medium is constructed by adding a specific mass concentration of naphthalene acetic acid (NAA) and 6-benzylaminopurine (6-BA) as exogenous hormones based on MS medium.

3. The method of claim 1, wherein, In the primary solid culture medium, the specific mass concentration of naphthalene acetic acid (NAA) is 0.2 mg / L, and the specific mass concentration of 6-benzylaminopurine (6-BA) is 1.0 mg / L.

4. The method of claim 2, wherein, The gradient disinfection method comprises: firstly, soaking in 75% ethanol solution for 1 minute, then soaking in 0.1% mercury chloride (HgCl2) solution for 5-6 minutes, and rinsing with sterile water for 3-4 times after each disinfection to remove residual disinfectant.

5. The method of claim 1, wherein, The shoot induction solid culture medium in step S2 is constructed by adding a specific mass concentration of naphthalene acetic acid (NAA) and a higher concentration of 6-benzylaminopurine (6-BA) based on MS medium. The direct induction refers to the process of directly germinating and proliferating axillary buds from the stem segments to form clusters of shoots without passing through the stage of callus differentiation.

6. The method of claim 4, wherein, In the shoot induction solid culture medium, the specific mass concentration of naphthalene acetic acid (NAA) is 0.1 mg / L, and the specific mass concentration of 6-benzylaminopurine (6-BA) is 2.0 mg / L.

7. The method of claim 5, wherein, The culture conditions in step S2 are as follows: temperature 25±1℃, light intensity 1000-1500lx, light cycle 12 hours / day; after 28 days of culture, the shoot induction rate reaches 93.35%, and at least 10 shoots are produced from each explant on average.

8. The method of claim 5, wherein, The proliferation solid culture medium in step S3 is constructed by adding a specific mass concentration of 6-benzylaminopurine (6-BA) as exogenous hormone based on MS medium, which is used for the continuous expansion of the shoots. In the proliferation solid culture medium, the specific mass concentration of 6-benzylaminopurine (6-BA) is 2.0 mg / L.

9. The method of claim 7, wherein, The rooting solid culture medium in step S4 is constructed based on 1 / 2MS medium by adding a specific mass concentration of naphthalene acetic acid (NAA) as the only exogenous auxin; The specific mass concentration of the naphthalene acetic acid (NAA) in the rooting solid culture medium is 0.3 mg / L.

10. The method of claim 8, wherein, The seedling process in step S5 comprises: opening the culture tank in a bright light place to make the tissue culture seedlings gradually adapt to the natural environment; and the acclimatization substrate is mixed by humus soil, garden soil and perlite according to a specific volume ratio. The mixed volume ratio of the acclimatization substrate is humus soil: garden soil: perlite = 1:2:1.

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