A method for establishing a high-frequency regeneration system for red shank powder

Abstract

This application relates to the field of plant cultivation technology, and in particular to a method for establishing a high-frequency regeneration system for *Gnaphalium affine*. The method includes: S1, explant preparation and disinfection: *Gnaphalium affine* seeds are disinfected and inoculated onto a primary culture medium under aseptic conditions to obtain sterile seedlings; S2, shoot induction: the terminal buds of the sterile seedlings obtained in step S1 are cut as explants and inoculated into a first induction culture medium to induce the generation of shoot clusters; S3, shoot proliferation: the shoot clusters obtained in step S2 are divided and transferred to a second induction culture medium to promote the proliferation and growth of the shoot clusters; S4, rooting culture: the robust shoot clusters obtained in step S3 are transferred to a rooting culture medium to induce the formation of adventitious roots; S5, hardening and transplanting: the tissue culture seedlings with complete root development from step S4 are hardened and then transplanted into a cultivation substrate to obtain regenerated *Gnaphalium affine* plants. This application establishes a rapid propagation system for *Gnaphalium affine*.

Description

Technical Field

[0001] This application relates to the technical field of plant cultivation, and particularly to a method for establishing a high-frequency regeneration system of Polygonum runcinatum Buch.-Ham. Background Art

[0002] Polygonum runcinatum Buch.-Ham. is a plant of the genus Polygonum in the family Polygonaceae and has great medicinal value. Currently, Polygonum runcinatum Buch.-Ham. mainly reproduces by rhizome division. The traditional method is to dig out the rhizomes before emergence and separate single branches. This method not only requires a large amount of materials for reproduction, but also has a greater negative impact on the mother plants. Another asexual reproduction technique of Polygonum runcinatum Buch.-Ham. is leaf cutting propagation method, but this method has a slow cultivation speed and is difficult to popularize.

[0003] Therefore, for the purpose of saving resources, reducing cultivation costs and improving cultivation efficiency, establishing a rapid propagation system of Polygonum runcinatum Buch.-Ham. has an important positive impact on the规模化繁殖 of Polygonum runcinatum Buch.-Ham. Summary of the Invention

[0004] This application provides a method for establishing a high-frequency regeneration system of Polygonum runcinatum Buch.-Ham. to solve the above problems.

[0005] This application provides a method for establishing a high-frequency regeneration system of Polygonum runcinatum Buch.-Ham., and the method includes:

[0006] S1. Preparation and disinfection of explants: Disinfect the seeds of Polygonum runcinatum Buch.-Ham., and inoculate them onto the primary medium under sterile conditions to obtain sterile seedlings through cultivation;

[0007] S2. Induction of cluster buds: Cut the apical buds of the sterile seedlings obtained in step S1 as explants and inoculate them into the first induction medium. The first induction medium is based on the MS medium and contains an effective induction concentration of cytokinin for directly inducing the apical buds to produce cluster buds;

[0008] S3. Proliferation of cluster buds: After dividing the cluster buds induced in step S2, transfer them to the second induction medium. The second induction medium is based on the MS medium and contains effective proliferation concentrations of auxin and cytokinin for promoting the proliferation and growth of the cluster buds;

[0009] S4. Rooting culture: Transfer the robust cluster bud individuals higher than 2 - 3 cm obtained by proliferation in step S3 to the rooting medium. The rooting medium is based on the 1 / 2 MS medium and contains an effective rooting concentration of auxin for inducing the formation of adventitious roots;

[0010] S5. Hardening-off and transplantation: After hardening-off the tissue culture seedlings with complete root development in step S4, transplant them into the cultivation substrate to obtain regenerated plants of Polygonum runcinatum Buch.-Ham.

[0011] By employing the direct organogenesis pathway of "apical bud → clustered buds" through the above-mentioned technical solution, this method avoids the potential variations and browning / death problems associated with callus regeneration. The regeneration pathway is clear, genetically stable, and the culture cycle is relatively more controllable. In terms of propagation efficiency, using apical buds as explants, the induction rate can reach 100%, with an average proliferation coefficient of 8.6, achieving rapid propagation at high multiplication rates, significantly higher than traditional division or leaf cutting methods. In the rooting and transplanting stages, the rooting rate reaches 87%, with an average of 6.9 roots per plant. After hardening off with S5 seedlings before transplanting, the seedling survival rate reaches 100%, successfully establishing a complete technical chain from laboratory to greenhouse production. This method provides reliable technical support for the germplasm resource preservation, superior clonal selection, large-scale standardized seedling cultivation, and subsequent industrial production of medicinal components of the rare medicinal plant *Corydalis yanhusuo*.

[0012] Optionally, in step S1, the disinfection process includes:

[0013] The seeds of *Corydalis oryzae* were soaked in a 75% ethanol solution for 30 seconds, rinsed with sterile water, soaked in a 0.1% HgCl2 solution for 15 minutes, rinsed thoroughly with sterile water, and finally soaked in sterile water for 24 hours.

[0014] The primary culture medium was MS medium supplemented with 1.0 mg / L gibberellin (GA3).

[0015] The above-mentioned technical solution achieves a sterility rate of over 95% for *Gnaphalium affine* seeds, significantly reducing the risk of contamination during tissue culture. Adding 1.0 mg / L gibberellin (GA3) to the primary culture medium effectively breaks seed dormancy, promotes rapid germination, and shortens the period for obtaining sterile seedlings to approximately 20 days. The appropriate concentration of GA3 ensures robust seedling elongation, providing high-quality apical explants for the subsequent S2 step, thus guaranteeing the initial efficiency of the entire regeneration system and the subsequent induction success rate. This precise sterilization and culture medium formulation are fundamental to achieving the stability of the *Gnaphalium affine* high-frequency regeneration system.

[0016] Optionally, in step S2, the effective inducible concentration of cytokinin is selected from phenylurea cytokinins, and its concentration range is 0.5-3.0 mg / L.

[0017] Using the above-mentioned technical solution, and employing phenylurea-based cytokinins at specific concentrations (0.5-3.0 mg / L), highly efficient direct induction of *Cymbidium goeringii* apical buds was successfully achieved, with an induction rate of up to 100%. Compared with traditional methods, this approach has the following advantages: First, it avoids callus formation in explants, ensuring the genetic stability of regenerated plants; second, it effectively inhibits browning, improving the survival rate and health of explants; and third, through efficient induction, it significantly shortens the initiation culture cycle. This precise hormone regulation strategy represents a key technological breakthrough in realizing the high-frequency regeneration system of *Cymbidium goeringii*.

[0018] Optionally, in step S2, the effective inducing concentration of cytokinin is thiamethoxam (TDZ), and the concentration of thiamethoxam (TDZ) is 2.0 mg / L.

[0019] Using the above-described technical solution, and employing 2.0 mg / L thiamethoxam (TDZ) as an inducer, the induction rate of shoot clusters in *Trichoderma purpureus* reached 100%, with the average number of shoots produced per explant significantly higher than other concentrations or hormone types. This high-frequency induction characteristic greatly shortened the start-up time of the tissue culture system and provided a sufficient number of high-quality explants for subsequent proliferation culture. This optimized solution ensures the high efficiency, stability, and reproducibility of the regeneration system, laying a solid foundation for the large-scale seedling production of *Trichoderma purpureus*.

[0020] Optionally, in step S3, the concentration range of the effective proliferation concentration of auxin is 0.1-0.3 mg / L; the cytokinin is selected from phenylurea cytokinins, and its concentration range is 1.0-3.0 mg / L.

[0021] By employing the aforementioned technical solution, and combining auxin (0.1-0.3 mg / L) and phenylurea cytokinin (1.0-3.0 mg / L), the contradiction between high-multiplication rate propagation and robust bud growth was successfully resolved. This combined strategy not only achieved a proliferation coefficient of 8.6, enabling rapid and high-multiplication of *Rhizoctonia solani* seedlings, but also produced robust proliferating buds, providing high-quality material for subsequent rooting and transplanting, thereby ensuring the efficiency of the entire regeneration system and the final transplant survival rate.

[0022] Optionally, in step S3, the effective proliferation concentration of auxin is naphthaleneacetic acid (NAA), and the effective proliferation concentration of phenylurea cytokinin is thidiazuron (TDZ); the concentration of naphthaleneacetic acid (NAA) is 0.2 mg / L, and the concentration of thidiazuron (TDZ) is 2.0 mg / L.

[0023] By employing the aforementioned technical solution and a precise combination of NAA 0.2 mg / L and TDZ 2.0 mg / L, the proliferation coefficient of *Cyclocarya paliurus* reached 8.6, significantly improving the propagation efficiency of seedlings. This optimized formula not only ensured the number of proliferating buds but, more importantly, ensured robust bud growth, avoiding the vitrification or dwarfing phenomena commonly seen in tissue culture, and greatly improving the success rate of subsequent rooting culture and transplant survival rate.

[0024] Optionally, in step S4, the auxin at the effective rooting concentration is naphthaleneacetic acid (NAA), and the concentration of NAA is 0.4 mg / L.

[0025] Using the above-mentioned technical solution, with NAA 0.4 mg / L as a rooting inducer and combined with 1 / 2 MS basal medium, the rooting rate of *Trichoderma purpureus* reached as high as 87%, with an average of 6.9 roots per plant. This highly efficient rooting system ensured that the tissue-cultured seedlings had strong water and nutrient absorption capabilities, providing physiologically prepared plants for subsequent hardening-off and transplanting, and significantly improving the transplant survival rate.

[0026] Optionally, in step S5, the seedling hardening treatment includes:

[0027] Open the caps of the culture bottles and place them in a culture room or greenhouse environment for 2 to 3 days;

[0028] The transplanting process specifically involves washing off the culture medium adhering to the roots of the tissue culture seedlings, planting them in humus soil, and maintaining an environmental humidity of around 85% and providing shade management for 7 days after transplanting.

[0029] The above technical solutions successfully solved the problem of low survival rate of transplanted tissue culture seedlings. A 2-3 day hardening-off treatment significantly enhanced the seedlings' resistance to external environmental stresses. Precisely controlling the humidity at around 85% and implementing shading management after transplanting provided an ideal transitional environment for the seedlings, ultimately achieving a 100% survival rate for regenerated *Cephalotaxus fortunei* plants. This established a complete and efficient technical chain from laboratory to greenhouse production.

[0030] Optionally, the cultivation processes in steps S2, S3 and S4 are carried out under the following environmental conditions: the cultivation temperature is 24℃±3℃, the photoperiod is 12 hours of light / 12 hours of darkness, and the light intensity is 1200lx.

[0031] Through the above technical solution and precise control of the cultivation environment, the stability and high efficiency of the *Cephalotaxus fortunei* regeneration system were ensured. The combination of a cultivation temperature of 24℃±3℃ and a light intensity of 1200 lx significantly promoted the induction of clustered buds and the elongation of proliferating buds, ensuring the healthy growth of regenerated plants and providing crucial conditions for achieving high-frequency regeneration and high survival rates.

[0032] Optionally, 30 g / L of sucrose is uniformly added as a carbon source and 6 g / L of agar is added as a coagulant to the primary culture medium and each of the culture media in steps S2, S3, and S4, and the pH value of each culture medium is adjusted to 5.85 - 5.90.

[0033] Through the above solution, by uniformly adding 30 g / L of sucrose and 6 g / L of agar and precisely controlling the pH value at 5.85 - 5.90, the nutritional requirements of Polygonum runcinatum Buch.-Ham. tissue culture seedlings at different culture stages are ensured to be met, and at the same time, a stable growth environment is provided for the explants. Precise pH control optimizes the biological activity of hormones and the absorption efficiency of nutrients, which is the basic guarantee for the stable and efficient operation of the high-frequency regeneration system of Polygonum runcinatum Buch.-Ham. Description of the Drawings

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0035] Figure 1 It is a flowchart of a method for establishing a high-frequency regeneration system of Polygonum runcinatum Buch.-Ham. provided by an embodiment of the present application. Detailed Embodiments

[0036] 1 To make the objectives, technical solutions, and advantages of the embodiments of the present application clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present application with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present application.

[0037] In addition, the term "and / or" in this article is merely a description of the association relationship of associated objects, indicating that three relationships can exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. In addition, the character " / " in this article generally represents an "or" relationship between the associated objects before and after, unless otherwise specified.

[0038] The following will further describe the embodiments of the present application in detail with reference to the drawings of the specification.

[0039] At present, Polygonum runcinatum var. sinense is mainly propagated by dividing plants. The traditional method is to dig out the rhizomes before emergence and separate single branches. This method not only requires a large amount of materials for propagation, but also has a greater negative impact on the mother plants. Another asexual propagation technique for Polygonum runcinatum var. sinense is the leaf cuttage propagation method, but this method has a slow cultivation speed and is difficult to popularize.

[0040] Based on this, the present application provides a method for establishing a high-frequency regeneration system of Polygonum runcinatum var. sinense. By adopting the direct organogenesis pathway of "terminal bud → cluster buds", this method avoids the possible problems of variation and browning death in the regeneration pathway through callus. The regeneration pathway is clear, the genetic stability is good, and the culture period is relatively more controllable. In terms of propagation efficiency, using the terminal bud as the explant, the induction rate can be as high as 100%, and the average proliferation coefficient can reach 8.6, achieving high-multiplication rapid propagation, which is significantly higher than the traditional methods of dividing plants or leaf cuttage. In the rooting and transplanting links, the rooting rate is as high as 87%, with an average of 6.9 roots per plant. After acclimatization and transplanting for S5, the survival rate of the seedlings can reach 100%, successfully establishing a complete technical chain from the laboratory to greenhouse production. This method provides reliable technical support for the germplasm resource conservation, selection of excellent clones, large-scale and standardized seedling raising, and subsequent industrial production of medicinal components of this rare medicinal plant, Polygonum runcinatum var. sinense.

[0041] The specific implementation methods can refer to the following examples.

[0042] Figure 1 It is a flowchart of a method for establishing a high-frequency regeneration system of Polygonum runcinatum var. sinense provided by an embodiment of the present application. As Figure 1 shown, this method includes:

[0043] S1. Preparation and disinfection of explants: Disinfect the seeds of Polygonum runcinatum var. sinense and inoculate them onto the primary medium under sterile conditions to obtain sterile seedlings through cultivation;

[0044] S2. Induction of cluster buds: Cut the terminal buds of the sterile seedlings obtained in step S1 as explants and inoculate them into the first induction medium. The first induction medium is based on the MS medium and contains an effective induction concentration of cytokinin for directly inducing the terminal buds to produce cluster buds;

[0045] S3. Proliferation of cluster buds: After dividing the cluster buds induced in step S2, transfer them to the second induction medium. The second induction medium is based on the MS medium and contains an effective proliferation concentration of auxin and cytokinin for promoting the proliferation and growth of the cluster buds;

[0046] S4. Rooting culture: The robust clustered shoots, which are 2-3 cm taller than the ones obtained in step S3, are transferred to a rooting culture medium. The rooting culture medium is based on 1 / 2 MS medium and contains an effective concentration of auxin to induce the formation of adventitious roots.

[0047] S5. Hardening off and transplanting: After hardening off the tissue culture seedlings with complete root development in step S4, transplant them into the cultivation substrate to obtain regenerated plants of *Cephalotaxus fortunei*.

[0048] Technical Background and Working Principle: This invention belongs to the field of plant tissue culture technology. For the rare medicinal plant *Gnaphalium affine*, existing technologies mainly rely on traditional asexual propagation methods such as division and leaf cuttings. These methods suffer from low propagation coefficients, high consumption of mother plant material, and severe seasonal limitations, making it difficult to meet the needs of large-scale, standardized production. This invention aims to establish an efficient, stable, and reproducible in vitro regeneration method for *Gnaphalium affine*, overcoming the limitations of existing technologies that lack a rapid propagation system. This method is based on the organogenesis principle regulated by plant hormones, employing a direct organogenesis pathway of "apical bud → clustered buds," avoiding the genetic variation and browning / death problems that may exist in callus regeneration pathways, ensuring the stability and high frequency of the regeneration system. The core principle lies in precisely controlling the types and concentration ratios of cytokinins and auxins in the culture medium to achieve targeted induction and efficient proliferation of the *Gnaphalium affine* apical bud. In step S2, the differentiation of lateral buds from the terminal bud is directly stimulated by an effective inducing concentration of cytokinin, forming clustered buds. In step S3, the synergistic effect of auxin and cytokinin promotes the proliferation of clustered buds while antagonizing the inhibition of bud elongation by cytokinin, ensuring the robust growth of the proliferating buds. In step S4, the concentration of the basal medium is reduced (1 / 2 MS), and an effective rooting concentration of auxin is added to induce the formation of tissue culture seedlings with complete root development. The entire process is carried out under aseptic conditions. Finally, the seedlings are hardened off through treatment (S5) to adapt to the external environment, achieving a high survival rate for transplanting and obtaining regenerated plants of *Cephalotaxus fortunei*.

[0049] Technical Solution and Component Functions: This method for establishing a high-frequency regeneration system for *Gynostemma pentaphyllum* includes five key steps (S1-S5), each closely linked to form a complete regeneration process. S1, explant preparation and sterilization, aims to obtain sterile, healthy *Gynostemma pentaphyllum* seedlings, providing high-quality material for subsequent in vitro culture. S2, shoot induction, is the core initiation step of the system. The excised apical buds are used as explants and inoculated into a first induction medium containing an effective inducing concentration of cytokinin. This medium is based on MS culture medium and its function is to directly induce apical bud germination to form shoot clusters, avoiding callus formation. S3, shoot proliferation, involves dividing the shoot clusters and transferring them to a second induction medium containing auxin and cytokinin. This medium is based on MS culture medium and its function is to achieve rapid, high-multiplication of shoot clusters, ensuring a large number of robust shoots. S4 rooting culture involves transferring robust, clustered shoots taller than 2-3 cm into a rooting medium based on 1 / 2 MS medium, containing an effective concentration of auxin to induce adventitious root formation and obtain tissue culture seedlings with fully developed root systems. S5 hardening-off and transplanting involves hardening off the tissue culture seedlings before transplanting them into a growing medium. This process enhances the seedlings' adaptability to the natural environment, ensures a high survival rate after transplanting, and ultimately yields regenerated *Gynostemma pentaphyllum* plants. The precise control of the components in each culture medium to maintain the balance of plant growth regulators is crucial for achieving high-frequency, stable regeneration.

[0050] Beneficial Effects: By adopting the direct organogenesis pathway of "apical bud → clustered buds," this method avoids the potential variations and browning / death problems associated with callus regeneration. The regeneration pathway is clear, genetically stable, and the culture cycle is relatively more controllable. In terms of propagation efficiency, using apical buds as explants, the induction rate can reach 100%, with an average proliferation coefficient of 8.6, achieving rapid propagation at high multiplication rates, significantly higher than traditional division or leaf cutting methods. In the rooting and transplanting stages, the rooting rate reaches 87%, with an average of 6.9 roots per plant. After hardening off with S5 seedlings before transplanting, the seedling survival rate reaches 100%, successfully establishing a complete technology chain from laboratory to greenhouse production. This method provides reliable technical support for the germplasm resource preservation, superior clonal selection, large-scale standardized seedling cultivation, and subsequent industrial production of medicinal components of the rare medicinal plant *Corydalis yanhusuo*.

[0051] In some embodiments, step S1, the disinfection treatment includes: soaking the seeds of *Gnaphalium affine* in a 75% ethanol solution for 30 seconds, rinsing with sterile water, soaking in a 0.1% mercuric chloride (HgCl2) solution for 15 minutes, rinsing thoroughly with sterile water, and finally soaking the seeds in sterile water for 24 hours; the primary culture medium is MS medium supplemented with 1.0 mg / L gibberellin (GA3).

[0052] Technical Background and Working Principle: In the above scheme, explant preparation and disinfection in step S1 is the primary prerequisite for the success of the tissue culture system. The surface of *Eriocaulon buergerianum* seeds is usually covered with a large number of microorganisms. If disinfection is not thorough, it will lead to explant contamination during subsequent culture, causing the failure of the entire system. This disinfection scheme uses a brief soaking (30 seconds) in a 75% ethanol solution, which quickly dissolves the waxy layer on the seed surface, enhancing the penetration of subsequent disinfectants. Subsequently, soaking in a highly toxic 0.1% mercuric chloride (HgCl2) solution for 15 minutes is a key step for achieving efficient sterilization. Mercuric chloride, as a broad-spectrum fungicide, can completely kill fungal and bacterial spores on the seed surface. After disinfection, the seeds must be thoroughly rinsed with sterile water to remove residual mercuric chloride and avoid its toxic inhibitory effect on seed germination. Finally, soaking the seeds in sterile water for 24 hours promotes seed imbibition, breaks dormancy, and increases the germination rate. The primary culture medium was based on MS medium with the addition of gibberellin (GA3) at a concentration of 1.0 mg / L. The function of gibberellin is to effectively promote the germination of red shank seeds and the elongation of seedlings, ensuring the rapid acquisition of robust sterile seedlings under sterile conditions, which can then serve as the explant source for the subsequent step S2.

[0053] Technical Solution and Component Functions: In the above solution, explant preparation and disinfection in step S1 is the starting point of the entire system. The disinfection process is rigorous and precise: First, the seeds of *Cephalotaxus fortunei* are surface-treated with a 75% (v / v) ethanol solution for 30 seconds. This process must be completed quickly to avoid ethanol damaging the internal tissues of the seeds. Then, the seeds are rinsed with sterile water to remove the ethanol. Next, they are soaked in a 0.1% mercuric chloride (HgCl2) solution for 15 minutes. This duration and concentration have been optimized to ensure both sterilization effectiveness and minimize phytotoxicity. After thorough rinsing, the seeds are soaked in sterile water for 24 hours to promote water absorption, break dormancy, and improve germination rate. The primary culture medium consisted of MS medium supplemented with 1.0 mg / L gibberellin (GA3). MS medium provided all the nutrients required for seedling growth, while 1.0 mg / L GA3 served as a key plant hormone, promoting rapid seed germination and the elongation of sterile seedlings. This ensured the acquisition of robust sterile seedlings approximately 3-5 cm tall in a short period, providing ample high-quality explants for subsequent apical bud cutting.

[0054] Beneficial Effects: By employing the precisely controlled sterilization method described in this embodiment, the sterility rate of *Gnaphalium affine* seeds reaches over 95%, significantly reducing the risk of contamination during tissue culture. Adding 1.0 mg / L gibberellin (GA3) to the primary culture medium effectively breaks the dormancy of *Gnaphalium affine* seeds, promoting rapid germination and shortening the period for obtaining sterile seedlings to approximately 20 days. The appropriate concentration of GA3 ensures robust seedling elongation, providing high-quality apical explants for the subsequent step S2, thereby guaranteeing the initial efficiency of the entire regeneration system and the subsequent induction success rate. This precise sterilization and culture medium formulation are fundamental to achieving the stability of the *Gnaphalium affine* high-frequency regeneration system.

[0055] In some embodiments, in step S2, the effective inducible concentration of cytokinin is selected from phenylurea cytokinins, and its concentration range is 0.5-3.0 mg / L.

[0056] Technical Background and Working Principle: In the above scheme, step S2 is the key step in achieving high-frequency regeneration of *Pteris vittata*, namely, the direct induction of shoot clusters. In traditional tissue culture, adenine-based cytokinins (such as 6-BA and KT) are often used to induce shoot formation. However, these hormones at high concentrations easily lead to the production of large amounts of callus tissue in explants, and the callus tissue is prone to browning, thus affecting regeneration efficiency and genetic stability. This invention innovatively uses phenylurea-based cytokinins (such as TDZ) as an effective inducer. Phenyleurone-based cytokinins have extremely high biological activity and can exert a strong cytokinin effect at low concentrations. Their working principle is that, within a concentration range of 0.5-3.0 mg / L, this type of hormone can precisely regulate the division and differentiation direction of apical bud cells, directly stimulating the rapid initiation and germination of lateral bud primordia, forming shoot clusters, thus bypassing the intermediate step of callus tissue. This direct organogenesis pathway not only improves the induction rate but also avoids the genetic variation and browning death problems caused by callus formation, ensuring the high efficiency and stability of the regeneration system.

[0057] Technical Solution and Component Function: In the above solution, the first induction medium used in step S2 is based on MS culture medium, and its core technical feature is the addition of an effective induction concentration of phenylurea cytokinin. The concentration range of this cytokinin is precisely limited to 0.5-3.0 mg / L, which is the optimal induction window determined through extensive experimental screening. When the concentration is below 0.5 mg / L, the induction efficiency decreases significantly; when the concentration is above 3.0 mg / L, although the induction rate is still high, a slight tendency for callus formation may begin to appear. Within this concentration range, the function of phenylurea cytokinin is to act as a strong cell division signal, directly acting on the apical bud meristem, inducing rapid differentiation of lateral buds, forming up to dozens of clustered buds, with an average induction rate of up to 100%. In addition, the use of this type of cytokinin ensures that the apical bud remains green and robust during the induction process, avoiding browning, thus providing high-quality explants for subsequent proliferation culture.

[0058] Beneficial Effects: This implementation method successfully achieved highly efficient direct induction of *Hymenochloa crus-galli* apical buds using a specific concentration range (0.5-3.0 mg / L) of phenylurea cytokinins, with an induction rate of up to 100%. Compared with traditional methods, this approach has the following advantages: First, it avoids callus formation in explants, ensuring the genetic stability of regenerated plants; second, it effectively inhibits browning, improving the survival rate and health of explants; third, through efficient induction, it significantly shortens the initiation culture cycle. This precise hormone regulation strategy is a key technological breakthrough for realizing the high-frequency regeneration system of *Hymenochloa crus-galli*.

[0059] In some embodiments, in step S2, the effective inducible concentration of cytokinin is thiamethoxam (TDZ), and the concentration of thiamethoxam (TDZ) is 2.0 mg / L.

[0060] Technical Background and Working Principle: In the above scheme, the preferred phenylurea cytokinin in step S2 was further identified as thidiazuron (TDZ), and its optimal effective induction concentration was determined to be 2.0 mg / L. TDZ is currently recognized as one of the most potent cytokinins, and its mechanism of action is to strongly promote cell division and differentiation of explants by influencing endogenous hormone levels and signal transduction pathways. At a concentration of 2.0 mg / L, TDZ can maximally activate the primordia of apical and lateral buds, achieving high-frequency and rapid induction of clustered shoots. This concentration ensures a 100% induction rate while effectively inhibiting the potential side effects of excessive inhibition of shoot elongation or callus induction by TDZ. MS medium combined with 2.0 mg / L TDZ provides the most suitable hormonal environment for the direct organogenesis of the terminal bud 12 of *Smilax china*, ensuring the efficient initiation of the regeneration system.

[0061] Technical Solution and Component Function: In the above solution, the specific composition of the first induction medium used in step S2 is: MS culture medium as the basal medium, supplemented with 2.0 mg / L thiamethoxam (TDZ). The concentration of TDZ 2.0 mg / L is preferably high within the range of 0.5-3.0 mg / L specified in the aforementioned solution. Its function is to provide a sufficiently strong cytokinin signal to directly stimulate the apical bud meristem and efficiently induce the formation of clustered shoots. Under the action of TDZ 2.0 mg / L, the base of the apical bud swells rapidly, and multiple clustered shoots begin to form after about 24 days of culture. After 82 days of culture, an average of 16.3 adventitious shoots can be produced per explant, with an induction rate as high as 100%. This precise concentration ensures the stability of the induction process, and the regeneration pathway is direct organogenesis, avoiding callus formation and browning.

[0062] Beneficial Effects: Using 2.0 mg / L thiamethoxam (TDZ) as an inducer, the induction rate of shoot clusters in *Trichoderma harzianum* reached 100%, and the average number of shoots produced per explant was significantly higher than that of other concentrations or hormone types. This high-frequency induction characteristic greatly shortened the start-up time of the tissue culture system and provided a sufficient number of high-quality explants for subsequent proliferation culture. This optimized scheme ensures the high efficiency, stability, and reproducibility of the regeneration system, laying a solid foundation for the large-scale seedling production of *Trichoderma harzianum*.

[0063] In some embodiments, in step S3, the concentration range of the effective proliferation concentration of auxin is 0.1-0.3 mg / L; the cytokinin is selected from phenylurea cytokinins, and its concentration range is 1.0-3.0 mg / L.

[0064] Technical Background and Working Principle: In the above scheme, step S3 is the key step in achieving rapid and high-multiplication propagation of *Cephalotaxus fortunei*. While high concentrations of cytokinin alone can induce the formation of numerous buds, it often results in stunted and weak buds, which is detrimental to subsequent rooting and transplanting. This invention achieves a balance between proliferation and elongation by precisely controlling the ratio of auxin to cytokinin. Its working principle is as follows: Cytokinin (such as phenylurea, concentration 1.0-3.0 mg / L) acts as the main proliferation signal, strongly stimulating lateral bud differentiation and cell division in clustered buds, ensuring the number of proliferating buds. Simultaneously, a low concentration of auxin (0.1-0.3 mg / L) is added, which synergistically works with cytokinin to promote cell elongation and effectively antagonizes the inhibitory effect of high concentrations of cytokinin on bud elongation. This low-auxin / high-cytokinin combination strategy ensures that the obtained proliferating buds are not only numerous but also robust, meeting the requirements for explant quality in subsequent rooting culture.

[0065] Technical Solution and Component Functions: In the above solution, the second induction medium (based on MS culture medium) used in step S3 contains two key plant hormones: auxin and phenylurea cytokinins. The effective proliferation concentration of auxin ranges from 0.1 to 0.3 mg / L, ensuring synergistic effects but insufficient to induce callus or excessive rooting. The concentration range of phenylurea cytokinins is 1.0 to 3.0 mg / L, and its function is to maintain high-frequency shoot proliferation. For example, when the auxin concentration is set at 0.2 mg / L and the cytokinin concentration is set at 2.0 mg / L, the proliferation coefficient of the proliferating shoots can reach 8.6, with an average of 25.8 new shoots produced per inoculated shoot. This precise hormone ratio aims to achieve the optimal balance between the quantity and quality of proliferating shoots, ensuring robust growth and a height of 2-3 cm, providing high-quality tissue culture material for the rooting culture in step S4.

[0066] Beneficial Effects: This implementation method successfully resolved the contradiction between high-multiplication rate proliferation and bud vigor by using a combination of auxin (0.1-0.3 mg / L) and phenylurea cytokinin (1.0-3.0 mg / L). This combined strategy not only achieved a proliferation coefficient of 8.6, enabling rapid and high-multiplication of *Rhizoctonia solani* seedlings, but also produced robust proliferating buds, providing high-quality material for subsequent rooting and transplanting, thereby ensuring the efficiency of the entire regeneration system and the final transplant survival rate.

[0067] In some embodiments, in step S3, the effective proliferation concentration of auxin is naphthaleneacetic acid (NAA), and the effective proliferation concentration of phenylurea cytokinin is thiazidone (TDZ); the concentration of naphthaleneacetic acid (NAA) is 0.2 mg / L, and the concentration of thiazidone (TDZ) is 2.0 mg / L.

[0068] Technical Background and Working Principle: In the above scheme, the selected plant hormones and precise concentrations in step S3 were clearly defined. Naphthaleneacetic acid (NAA), as an auxin, has moderate activity. At a low concentration of 0.2 mg / L, it mainly acts synergistically with cytokinins to promote cell elongation and differentiation. Thiidianone (TDZ), as a highly active cytokinin, strongly drives bud proliferation at a concentration of 2.0 mg / L. The combination of 0.2 mg / L NAA and 2.0 mg / L TDZ forms the optimal hormone ratio. The working principle of this ratio is as follows: the high concentration of TDZ ensures high-frequency initiation of lateral bud primordia, while the low concentration of NAA effectively counteracts the inhibition of bud elongation by TDZ. This allows the proliferating buds to increase rapidly in number while maintaining a robust morphology and sufficient height (above 2-3 cm), avoiding bud dwarfing. This precise hormone ratio is a key technical feature for achieving high proliferation coefficients and high-quality proliferating buds.

[0069] Technical Solution and Component Functions: In the above solution, the specific composition of the proliferation medium in step S3 is as follows: MS culture medium is the basal medium, supplemented with 0.2 mg / L naphthaleneacetic acid (NAA) and 2.0 mg / L thidiazuron (TDZ). The function of 0.2 mg / L NAA is to promote cell elongation, balance the inhibitory effect of TDZ, and ensure the robust growth of proliferating buds. The function of 2.0 mg / L TDZ is to maintain a high proliferation rate of clustered buds. On this medium, clustered buds are cut into single buds or small pieces containing 2-3 buds and then transferred. After 24 days of culture, new buds can be observed to sprout. After continuing culture for 84 days, clustered buds proliferate in large quantities, with an average proliferation coefficient of 8.6. This solution optimizes the number (average 25.8 buds / inoculated bud) and quality (robust, taller than 2-3 cm) of proliferating buds by precisely controlling the concentrations of NAA and TDZ.

[0070] Beneficial effects: The precise combination of NAA 0.2 mg / L and TDZ 2.0 mg / L resulted in a proliferation coefficient of 8.6 for *Trichoderma harzianum*, significantly improving the propagation efficiency of seedlings. This optimized formula not only ensured the number of proliferating buds but, more importantly, ensured robust bud growth, avoiding the vitrification or dwarfing phenomena commonly seen in tissue culture, and greatly improving the success rate of subsequent rooting culture and transplant survival rate.

[0071] In some embodiments, in step S4, the auxin at the effective rooting concentration is naphthaleneacetic acid (NAA), and the concentration of NAA is 0.4 mg / L.

[0072] Technical Background and Working Principle: In the above scheme, step S4 is the key step in realizing the transformation of tissue culture seedlings from heterotrophic to autotrophic, namely, inducing the formation of adventitious roots. Rooting culture usually requires a high auxin / cytokinin ratio, and the salt concentration of the basal medium needs to be appropriately reduced to facilitate root initiation. This invention selects naphthaleneacetic acid (NAA) as an effective rooting auxin. NAA is a synthetic auxin commonly used in plant tissue culture to induce adventitious roots. Its working principle is that at a concentration of 0.4 mg / L, NAA can effectively stimulate the dedifferentiation of phloem cells at the base of robust clustered shoots and redifferentiate to form root primordia, ultimately inducing the formation of adventitious roots. The basal medium used is 1 / 2 MS, which reduces the concentration of nitrogen source and mineral salts in the medium, simulating the low nutrient conditions for root growth in the natural environment, which is conducive to root initiation and growth, while avoiding the inhibition of root differentiation by high salt concentration.

[0073] Technical Solution and Component Function: In the above solution, the rooting medium in step S4 consists of 1 / 2 MS medium as the basal medium, supplemented with 0.4 mg / L naphthaleneacetic acid (NAA). This medium contains no cytokinin or only a very low concentration of residual cytokinin to ensure that auxin signaling is dominant. The function of 0.4 mg / L NAA is to provide a suitable auxin level for inducing adventitious root formation. On this medium, robust young shoots taller than 2-3 cm are selected for inoculation. After several days of culture, the vast majority of plants develop numerous white adventitious roots at their base. Statistical results show that the rooting rate is as high as 87%, with an average of 6.9 roots per plant. This precise combination of NAA concentration and 1 / 2 MS basal medium ensures the rapid and efficient acquisition of tissue culture seedlings with complete root development.

[0074] Beneficial effects: Using NAA 0.4 mg / L as a rooting inducer, combined with 1 / 2 MS basal medium, the rooting rate of *Trichoderma purpureus* reached 87%, with an average of 6.9 roots per plant. This highly efficient rooting system ensured that the tissue-cultured seedlings had strong water and nutrient absorption capabilities, providing physiologically prepared plants for subsequent hardening-off and transplanting, and significantly improving the transplant survival rate.

[0075] In some embodiments, step S5, the hardening treatment includes: opening the cap of the culture bottle and placing it in a culture room or greenhouse environment for 2 to 3 days; the transplanting process specifically involves: washing off the culture medium attached to the roots of the tissue culture seedlings, planting them in humus soil, and maintaining the environmental humidity at around 85% and providing shading management for 7 days after transplanting.

[0076] Technical Background and Working Principle: In the above scheme, step S5 is a crucial step in the transition of *Hypericum esculentum* tissue culture seedlings from in vitro to the natural environment, aiming to improve their adaptability to the external environment. Tissue culture seedlings, kept in a culture bottle under high humidity, low light, and heterotrophic conditions for extended periods, have poor stomatal regulation and thin cuticles, making them highly susceptible to dehydration, wilting, and death when directly transplanted to the natural environment. The working principle of the hardening-off treatment is as follows: by opening the culture bottle cap, the tissue culture seedlings are gradually exposed to a lower humidity (but still higher than the external natural environment) and higher light environment for 2 to 3 days, training their stomatal closure mechanism, thickening the leaf cuticle, and gradually adapting their physiological structure and function to the external environment. After transplanting, the environmental humidity is maintained at around 85% (through covering with a thin film or spraying), which maintains the water balance within the tissue culture seedlings and prevents rapid water loss. Simultaneously, shading management is implemented to prevent strong light from causing photoinhibition or damage to the tissue culture seedlings that have not yet fully adapted to photosynthesis. Humus soil, as a cultivation substrate, provides good aeration and water retention.

[0077] In the above solution, the specific operation of the hardening-off treatment in step S5 is as follows: Transplant well-rooted tissue culture seedlings with a height of approximately 4-5 cm to a greenhouse, open the culture bottle cap, and leave them for 2-3 days. This timeframe has been optimized to ensure the seedlings adapt to the environment without damaging them due to excessive water loss. The specific operation of the transplanting process is as follows: Carefully remove the tissue culture seedlings and wash off the culture medium adhering to the roots with warm water to prevent the culture medium residue from breeding microorganisms in the soil or affecting root growth. Subsequently, plant them in a mixed substrate of humus and perlite (3:1). The key management measures after transplanting are: maintaining an ambient humidity of approximately 85% for 7 days and implementing shading management by covering with a film or shade net. The function of 85% humidity is to maintain the turgor pressure of the tissue culture seedlings, and the function of shading management is to prevent strong light from damaging the leaves. After 7 days, gradually increase ventilation and light exposure. After 14 days, the transplant survival rate can reach 100%.

[0078] Beneficial Effects: The hardening-off and transplanting scheme of this implementation method successfully solved the problem of low survival rate of transplanted tissue culture seedlings. Through a 2-3 day hardening-off treatment, the resistance of tissue culture seedlings to external environmental stresses was significantly enhanced. After transplanting, precise control of the ambient humidity at around 85% and shading management provided an ideal transitional environment for the tissue culture seedlings, ultimately achieving a 100% survival rate for the regenerated *Cephalotaxus fortunei* plants. This established a complete and efficient technical chain from laboratory to greenhouse production.

[0079] In some embodiments, the cultivation processes in steps S2, S3 and S4 are carried out under the following environmental conditions: the cultivation temperature is 24℃±3℃, the light cycle is 12 hours of light / 12 hours of darkness, and the light intensity is 1200lx.

[0080] Technical Background and Working Principle: In the above scheme, the standardization and precise control of the in vitro culture environment conditions in steps S2, S3, and S4 are crucial for ensuring the stability of the *Cistanche deserticola* high-frequency regeneration system. Plant tissue culture is extremely sensitive to environmental conditions (such as temperature and light). The culture environment conditions determined in this invention simulate and optimize the suitable natural environment for *Cistanche deserticola* growth. The culture temperature of 24℃±3℃ (i.e., 21℃ to 27℃) is the optimal temperature range for most plant tissue cultures, ensuring that enzyme activity and cell metabolism in the culture medium are at their best, promoting cell division and differentiation. The photoperiod of 12 hours of light / 12 hours of darkness, a typical day-night cycle, simulates the natural photoperiod, which is beneficial for the synthesis of plant hormones and photosynthesis. A light intensity of 1200 lx (lumens) was determined to be the preferred value. Its function is to provide sufficient light energy for photosynthesis while avoiding leaf scorching or photoinhibition that may be caused by strong light. In particular, for clustered shoots induced by highly active cytokinins such as TDZ, moderate light intensity is beneficial to their vigorous growth.

[0081] Technical Solution and Component Functions: In the above solution, the cultivation processes in steps S2, S3, and S4 are all conducted in a unified cultivation chamber environment. The cultivation temperature is set at 24℃±3℃ and precisely controlled by a thermostat to ensure efficient cell division and organ differentiation of the *Symplocos rubrum* explants, clustered buds, and proliferating buds at suitable temperatures. The light cycle is set to 12 hours of light / 12 hours of darkness, controlled by a timer switch to ensure a balance between the light (photosynthesis) and dark (respiration and hormone synthesis) phases. The light intensity is controlled at 1200 lx using fluorescent or LED lamps, ensuring sufficient photosynthetically active radiation to promote robust bud growth and chlorophyll formation. The standardization of these environmental parameters ensures the stability and reproducibility of the entire regeneration system across different batches.

[0082] Beneficial effects: Precise control of the culture environment ensured the stability and high efficiency of the *Cephalotaxus fortunei* regeneration system. The combination of a culture temperature of 24℃±3℃ and a light intensity of 1200lx significantly promoted the induction of clustered buds and the elongation of proliferating buds, ensuring the healthy growth of regenerated plants and providing important conditions for achieving high-frequency regeneration and high survival rates.

[0083] In some embodiments, the primary culture medium and each culture medium in steps S2, S3 and S4 are all uniformly supplemented with 30 g / L sucrose as a carbon source and 6 g / L agar as a solidifying agent, and the pH value of each culture medium is adjusted to 5.85-5.90.

[0084] Technical Background and Working Principle: In the above scheme, the carbon source, solidifying agent, and pH value of the culture medium are universally key elements for successful tissue culture. Sucrose, as a carbon source, functions to provide energy and a carbon skeleton for explants, shoot clusters, and proliferating shoots in vitro, because in the low-light environment of the tissue culture flask, plant photosynthesis is insufficient to support their rapid growth. A sucrose concentration of 30 g / L is a commonly used and preferred concentration in plant tissue culture, ensuring sufficient energy supply. Agar, as a solidifying agent, functions to form a solid gel in the culture medium, providing support for explants and slowing the diffusion rate of nutrients in the medium. A 6 g / L agar concentration provides suitable firmness. The pH value of the culture medium has a significant impact on nutrient absorption and hormone activity in plant cells and generally needs to be maintained in a slightly acidic range. Adjusting the pH value to 5.85-5.90 ensures effective dissolution of mineral salts and ion absorption in the culture medium, while optimizing the bioactivity of plant growth regulators (such as TDZ and NAA).

[0085] Technical Solution and Component Functions: In the above solution, 30 g / L sucrose is uniformly added as a carbon source to the primary culture medium, first induction medium, second induction medium, and rooting medium. The function of sucrose is to provide an exogenous carbon source to support the heterotrophic growth of *Gnaphalium affine* tissue culture seedlings. 6 g / L agar is uniformly added to each culture medium as a solidifying agent, which provides a stable solid support structure. Furthermore, the pH of all culture media is precisely adjusted to 5.85-5.90 before sterilization. This precise pH range is adjusted using an acid (such as hydrochloric acid) or a base (such as sodium hydroxide), which ensures effective absorption of nutrients and optimal activity of plant hormones in the culture medium. This unified carbon source, solidifying agent, and pH control simplifies the culture medium preparation process and ensures the consistency and stability of the basic environment of the culture medium at different stages of the entire regeneration system.

[0086] Beneficial Effects: This implementation method, by uniformly adding 30g / L sucrose and 6g / L agar and precisely controlling the pH value between 5.85 and 5.90, ensured that the nutritional needs of *Gynostemma pentaphyllum* tissue culture seedlings were met at different culture stages, while providing a stable growth environment for the explants. Precise pH control optimized the bioactivity of the hormone and the absorption efficiency of nutrients, providing a fundamental guarantee for the stable and efficient operation of the *Gynostemma pentaphyllum* high-frequency regeneration system.

Claims

1. A method for establishing a high-frequency regeneration system of Polygonum runcinatum Buch.-Ham. var. sinense Hemsl., characterized in that, include: S1. Explant preparation and disinfection: The seeds of *Cephalotaxus fortunei* were disinfected and inoculated onto a primary culture medium under aseptic conditions to obtain sterile seedlings. The primary culture medium was MS medium supplemented with 1.0 mg / L GA3. S2, induction of clustered shoots: The apical buds of the sterile seedlings obtained in step S1 are cut off as explants and inoculated into the first induction medium. The first induction medium is based on MS medium and contains an effective induction concentration of cytokinin to directly induce the apical buds to produce clustered shoots. The first induction medium is MS medium supplemented with 2.0 mg / L TDZ. S3. Proliferation of clustered shoots: After the clustered shoots induced in step S2 are divided, they are transferred to the second induction medium. The second induction medium is based on MS medium and contains effective proliferation concentrations of auxin and cytokinin to promote the proliferation and growth of the clustered shoots. The second induction medium is MS medium supplemented with 0.2 mg / L NAA and 2.0 mg / L TDZ. S4. Rooting culture: The robust clustered shoots, which are 2-3 cm taller than the ones obtained in step S3, are transferred to a rooting culture medium. The rooting culture medium is based on 1 / 2 MS medium and contains an effective rooting concentration of auxin to induce the formation of adventitious roots. The rooting culture medium is 1 / 2 MS medium supplemented with 0.4 mg / L NAA. S5. Hardening off and transplanting: After hardening off the tissue culture seedlings with complete root development in step S4, transplant them into the cultivation substrate to obtain regenerated plants of *Cephalotaxus fortunei*.

2. The method according to claim 1, characterized in that, In step S1, the disinfection process includes: The seeds of *Corydalis yanhusuo* were soaked in a 75% ethanol solution for 30 seconds, rinsed with sterile water, and then soaked in a 0.1% HgCl2 solution.

3. The method according to claim 1, characterized in that, In step S5, the seedling hardening process includes: Open the caps of the culture bottles and place them in a culture room or greenhouse environment for 2 to 3 days; The transplanting process is as follows: after washing off the culture medium attached to the roots of the tissue culture seedlings, plant them in humus soil, and maintain the ambient humidity at around 85% for 7 days after transplanting and implement shading management.

4. The method according to claim 1, characterized in that, The cultivation processes in steps S2, S3, and S4 are all carried out under the following environmental conditions: The culture temperature was 24℃±3℃, the photoperiod was 12 hours of light / 12 hours of darkness, and the light intensity was 1200 lx.

5. The method according to claim 1, characterized in that, The primary culture medium and each culture medium in steps S2, S3 and S4 were all uniformly supplemented with 30 g / L sucrose as a carbon source and 6 g / L agar as a solidifying agent, and the pH value of each culture medium was adjusted to 5.85-5.90.

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