A seedling breeding method for reducing the stress injury of huaiyushan san yeqing by combining multiple ultra-low temperature detoxification methods

Abstract

The application discloses a seedling breeding method for relieving the harm of allelopathy stress of Gastrodia elata Blume by using a plurality of ultra-low temperature detoxification methods in combination, and belongs to the technical field of plant tissue culture.The application provides a treatment process of the plurality of ultra-low temperature detoxification methods in combination, which is suitable for the physiological characteristics of Gastrodia elata Blume germplasm cells, improves the removal rate of a plurality of complex viruses, reduces the probability of stem tip freezing damage, improves the regeneration efficiency of virus-free stem tips, and realizes efficient breeding of high-quality virus-free seedlings of genuine traditional Chinese medicinal materials.Through the gradient low-temperature domestication effect of different mode ultra-low temperature detoxification treatment, the application induces the synthesis of in-vivo antioxidant enzyme systems and osmotic regulation substances of Gastrodia elata Blume in advance in the test-tube seedling breeding stage, improves the tolerance of plants to phenolic acid and other allelochemicals from the source of seedlings, and effectively relieves the physiological damage caused by allelopathy stress without the need of a large amount of application of modified agents and growth regulators in the field stage, thereby reducing the labor and material input costs in the seedling cultivation stage.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a seedling propagation method that combines multiple ultra-low temperature detoxification methods to reduce the stress damage caused by the senescence of *Trifolium repens*. Background Technology

[0002] *Trifolium repens*, a rare medicinal plant belonging to the genus *Trifolium* of the Vitaceae family, is a traditional Chinese medicine native to the Huaiyu Mountain region of Jiangxi Province. Its tuberous roots are rich in flavonoids, polysaccharides, polyphenols, and other active substances, possessing high medicinal development value in anti-inflammatory, hepatoprotective, and anti-tumor applications. However, due to the limitations of long-term asexual propagation methods, the cultivation of *Trifolium repens* in Huaiyu Mountain has resulted in significant virus accumulation and genetic degeneration, leading not only to decreased seedling survival rates and a sharp reduction in tuber yield but also a marked decrease in plant resistance. Under large-scale artificial cultivation, root exudates and plant debris continuously accumulate in the soil, triggering severe allelopathic and autotoxic stress effects. This results in a series of problems, including continuous cropping obstacles, root browning and rotting, weak seedling growth, and physiological metabolic disorders. These issues have become a core technical bottleneck restricting the standardized and industrialized seedling propagation and large-scale field cultivation of *Trifolium repens* in Huaiyu Mountain.

[0003] Analysis of the defects and causes of existing technologies: (1) The single ultra-low temperature detoxification mode is not thorough and it is difficult to simultaneously improve the comprehensive resistance of seedlings to chemical stress. The operation method of the single shoot tip ultra-low temperature detoxification technology is as follows: Select the shoot tip meristem of sterile test tube seedlings of Clematis chinensis, pre-culture with sucrose, treat with vitrification protection solution, put it into liquid nitrogen for ultra-low temperature freezing and preservation, and then obtain detoxified and regenerated seedlings through rewarming, washing, and callus induction differentiation. This technology relies on ultra-low temperature to directly kill the viruses and viroids infecting the shoot tip meristem, which can achieve seedling detoxification and rejuvenation to a certain extent. It is the most commonly used method for the propagation of virus-free seedlings of medicinal plants. The system flow of this technology is as follows: explant disinfection → sterile induction culture → test tube seedling propagation → shoot tip peeling → vitrification pretreatment → liquid nitrogen ultra-low temperature freezing → rewarming and washing → shoot tip differentiation culture → virus-free seedling domestication and transplanting. The entire technical process relies on a plant tissue culture platform, with the core function of virus removal achieved through a single vitrification cryopreservation mode. By reducing the viral load in the plant, the basic growth of seedlings is improved, indirectly and slightly enhancing the plant's stress resistance. Current technologies only use a single vitrification cryopreservation method to treat the shoot tips of *Trifolium repens*, resulting in low removal efficiency of *Trifolium repens* mosaic virus. The reasons are: *Trifolium repens* mosaic virus exhibits significant differences in its tolerance to cryopreservation; single freezing parameters and a single pretreatment system cannot adapt to the inactivation conditions of *Trifolium repens* mosaic virus; the detoxification process only focuses on eliminating the *Trifolium repens* mosaic virus without targeting the plant's antioxidant, osmotic regulation, and root metabolic pathways under allelopathic stress. Virus-free seedlings only achieve a slight recovery of genetic characteristics and do not develop stable allelopathic stress resistance. After transplanting to continuously cropped soils, they still suffer severe allelopathic damage, exhibiting problems such as seedling wilting, root necrosis, and low survival rates, significantly reducing the economic benefits of virus-free propagation. (2) The detoxification process and the allelopathic stress mitigation process are independent of each other, resulting in fragmented technical processes, high production costs, and delayed regulation. The current industry practice is to first conduct single-stage ultra-low temperature detoxification and propagation of virus-free seedlings in vitro, and then, after the virus-free seedlings have been domesticated and transplanted to the field, to mitigate allelopathic stress damage through soil improvement and exogenous pesticide application. The two technologies belong to two independent processes: the tissue culture laboratory stage and the field cultivation stage, respectively, resulting in a clear technical separation. Causes: First, stress intervention is a passive post-event remedy. When seedlings have already shown allelopathic damage phenotypes such as cell membrane oxidative damage and root development obstruction, regulation is carried out. Irreversible physiological damage cannot be repaired, and the regulatory effect is limited. Second, it requires supporting soil improvement materials, multiple foliar sprays, labor costs, and pesticide consumable costs. Under the large-scale breeding model, the comprehensive production cost increases significantly. Third, soil microbial improvement and exogenous regulators are easily affected by field environmental factors such as temperature, precipitation, and soil pH. The mitigation effect is unstable, and the survival rate of seedlings varies greatly between batches, which is not conducive to the standardized breeding of Huaiyushan San Ye Qing seedlings. (3) The local characteristic germplasm of Huaiyushan San Ye Qing has not been taken into account, and the adaptability of the generalized detoxification process is poor.Most existing ultra-low temperature detoxification processes are based on the development of common Trifoliate Gynostemma pentaphyllum germplasm, without optimizing the pretreatment, freezing, and rewarming parameters for the physiological characteristics of the native Trifoliate Gynostemma pentaphyllum from Huaiyu Mountain. Trifoliate Gynostemma pentaphyllum from Huaiyu Mountain has been growing in a high-altitude, humid forest environment for a long time. The cell membrane permeability, cell osmotic pressure, and endogenous hormone levels of the native germplasm are significantly different from those of artificially domesticated common Trifoliate Gynostemma pentaphyllum. Directly applying a single universal ultra-low temperature detoxification parameter can easily cause frost damage and necrosis of the shoot tip meristem, resulting in a low regeneration rate. At the same time, existing resistance induction methods have not been combined with targeted improvement of the allelopathic stress sensitivity characteristics of the germplasm in this region, making it difficult for high-quality authentic germplasm resources to achieve large-scale preservation and resistance enhancement through detoxification and propagation, thus limiting the industrialization development of local characteristic Chinese medicinal herbs. (4) There is a lack of integrated technical solutions for simultaneously inducing allelopathic stress resistance during the detoxification process. In existing technologies, ultra-low temperature is only used as a means of virus inactivation and does not fully utilize the domestication effect of gradient low temperature stress. Single liquid nitrogen cryogenic freezing can only kill viruses. It cannot pre-activate the SOD, POD, and CAT antioxidant enzyme system and osmotic regulation pathway in Trifolium repens through staged low-temperature pretreatment, gradient cooling, and the combined use of different vitrification systems. It cannot build allelopathic stress defense mechanisms in advance during the seedling in vitro propagation stage. It can only rely on exogenous regulation in the later stage of field to improve stress resistance. It has technical shortcomings such as delayed resistance establishment, weak stability, and uncontrollable effect.

[0004] In summary, in the current field of Huaiyushan Trifoliate Orange seedling propagation, the single-mode ultra-low temperature virus removal method suffers from incomplete virus removal and low regeneration efficiency. Allelopathic stress mitigation relies on post-treatment physical and chemical methods and exogenous regulation in the field, which not only results in high production costs and poor stability of effects, but also suffers from multiple technical drawbacks such as the disconnect between virus removal propagation and stress resistance cultivation processes, insufficient adaptability to local germplasm processes, and delayed induction of stress resistance. How to integrate multiple ultra-low temperature virus removal methods to achieve efficient and thorough virus removal of seedlings while simultaneously inducing plant tolerance to allelopathic stress through low-temperature stress acclimatization, and to simultaneously complete virus removal rejuvenation and allelopathic stress resistance improvement during the in vitro seedling propagation stage, thereby alleviating allelopathic stress damage in subsequent cultivation from the seedling source and reducing the incidence of continuous cropping obstacles, is a pressing technical challenge that needs to be addressed in the standardized industrialization of Huaiyushan Trifoliate Orange seedling propagation. Summary of the Invention

[0005] The purpose of this invention is to provide a seedling propagation method that combines multiple ultra-low temperature detoxification methods to reduce the stress damage caused by the senescence of *Trifolium repens* from Huaiyu Mountain, thereby solving the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a seedling propagation method that combines multiple ultra-low temperature detoxification methods to reduce the stress damage caused by the senescence of *Trifolium repens* from Huaiyu Mountain, including Method 1 and Method 2; The first method includes the following steps: Using axillary buds and tender stem segments of *Trifolium repens* from Huaiyu Mountain as explants, gradient sterilization was performed, followed by primary and secondary culture to obtain sterile test-tube seedlings. The shoot tip meristem of the sterile test-tube seedlings was pre-cultured and subjected to gradient low-temperature pre-acclimatization pretreatment to obtain shoot tips after gradient low-temperature pre-acclimatization. The shoot tips that have undergone gradient low-temperature pre-acclimatization are subjected to ultra-low temperature detoxification to obtain detoxified shoot tips; The virus-free shoot tips were subjected to rewarming, gradient washing, and regeneration culture to obtain regenerated seedlings; The regenerated seedlings were screened and subjected to chemical stress-resistant seedling cultivation to obtain virus-free seedlings; The virus-free seedlings were hardened off and transplanted. The second method includes the following steps: Gradient low-temperature pre-acclimatization pretreatment was carried out on *Trifolium repens* from Huaiyu Mountain to obtain pretreated *Trifolium repens* from Huaiyu Mountain. Using the tender stems of the pretreated Huaiyu Mountain Trifoliate Orchid as explants, gradient sterilization was performed, followed by primary and subculture to obtain sterile test-tube seedlings. The stem tips of the sterile test-tube seedlings were subjected to ultra-low temperature detoxification to obtain detoxified stem tips; The virus-free shoot tips were subjected to rewarming, gradient washing, and regeneration culture to obtain regenerated seedlings; The regenerated seedlings were screened and subjected to chemical stress-resistant seedling cultivation to obtain virus-free seedlings; The virus-free seedlings were hardened off and transplanted.

[0007] Optionally, the cryogenic detoxification in Method 1 includes embedding dehydration cryogenic detoxification, embedding vitrification cryogenic detoxification, vitrification cryogenic detoxification, and droplet vitrification cryogenic detoxification; The cryogenic detoxification in Method 2 includes encapsulation dehydration cryogenic, conventional vitrification cryogenic detoxification, or droplet vitrification cryogenic detoxification.

[0008] Optionally, the encapsulation dehydration and cryogenic detoxification includes the steps of wrapping the stem tip with a 3% sodium alginate solution and a 0.5 mol / L calcium chloride solution, followed by drying and liquid nitrogen freezing. The embedded vitrification cryogenic detoxification includes the steps of impregnating the stem tip with PVS2 and freezing it with liquid nitrogen. The vitrification cryogenic detoxification includes the steps of impregnating the stem tips treated with loading solution with PVS2 and freezing them with liquid nitrogen. The droplet vitrification cryogenic detoxification includes the steps of wrapping the stem tip with PVS2 and then freezing it with liquid nitrogen. The conventional vitrification cryogenic detoxification process includes the steps of immersing the stem tip in a loading solution, followed by dehydration with PVS2 and then freezing with liquid nitrogen. The loading solution comprises MS + 2M glycerol + 0.4M sucrose; the PVS2 comprises 30% glycerol by mass, 15% dimethyl sulfoxide by volume, 15% ethylene glycol by volume and 0.4 mol / L sucrose.

[0009] More preferably, the embedding dehydration and cryogenic detoxification includes the steps of wrapping the stem tip with a 3% sodium alginate solution and a 0.5 mol / L calcium chloride solution, drying and freezing with liquid nitrogen to obtain the embedded dehydration and cryogenic detoxified stem tip; The embedded vitrification cryogenic detoxification includes the step of impregnating the embedded dehydrated cryogenic detoxified stem tip with PVS2 and freezing it with liquid nitrogen to obtain the embedded vitrified cryogenic detoxified stem tip. The vitrification cryo-detoxification includes the steps of impregnating the embedded vitrified cryo-detoxified stem tip treated with loading liquid with PVS2 and freezing it with liquid nitrogen to obtain the vitrified cryo-detoxified stem tip. The droplet vitrification cryogenic detoxification process includes the steps of wrapping the vitrified cryogenic stem tip with PVS2 and then freezing it with liquid nitrogen to obtain the droplet vitrified cryogenic detoxified stem tip.

[0010] Optionally, the gradient low-temperature pre-acclimatization pretreatment includes the steps of constant temperature incubation at 15℃ in the dark for 2 days, followed by constant temperature incubation at 10℃ in the dark for 2 days, and finally low-temperature acclimatization incubation at 4℃ for 3 days.

[0011] Optionally, the gradient washing includes the steps of sequentially rinsing with MS + 0.5 mol / L sucrose, MS + 0.3 mol / L sucrose, and MS + 0.1 mol / L sucrose.

[0012] Optionally, the culture medium used for the primary culture includes MS + 0.8 mg / L 6-BA + 0.2 mg / L NAA; the temperature of the primary culture is 25±1℃, the light intensity is 2200 lx, the culture time is 30 days, and the photoperiod is 12 h / d. The culture medium used for the subculture consisted of MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar; the culture temperature for the subculture was 25 ± 1℃, the light intensity was 2200 lx, the culture time was 30 days, and the photoperiod was 12 h / d. The culture medium used for the pre-culture was MS + 0.4 mol / L sucrose; the pre-culture method was dark culture; the temperature of the dark culture was 25℃ and the time was 24 h. The regeneration culture medium used includes MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar; the regeneration culture includes dark culture and light culture; the dark culture temperature is 25℃ and the time is 7 days; the light culture time is 30 days, the temperature is 25±1℃, and the light duration is 12h / day; The culture medium used for cultivating the chemically resistant seedlings was MS + 50 mg / L ferulic acid + 30 mg / L p-hydroxybenzoic acid; the temperature for cultivating the chemically resistant seedlings was 25 ± 1℃, the time was 30 days, the photoperiod was 12 h / d, and the light intensity was 2200 lx.

[0013] Optionally, the gradient disinfection includes the steps of sequentially disinfecting with phosphate buffer, 0.3wt% tea tree oil emulsion solution, and 0.2wt% hypochlorous acid solution.

[0014] Optionally, the seedling hardening and domestication process also includes a step of root soaking treatment using a compound microbial agent; the compound microbial agent includes Bacillus subtilis and Trichoderma harzianum.

[0015] Optionally, the rewarming includes a step of rewarming the detoxified stem tip at 38-40°C for 90 seconds.

[0016] This invention provides a processing technology that combines multiple ultra-low temperature detoxification methods, adapted to the physiological characteristics of Huaiyu Mountain Trifoliate Orange germplasm cells, improving the removal rate of multiple complex viruses, reducing the probability of stem tip freezing damage, increasing the regeneration efficiency of non-toxic stem tips, and realizing the efficient propagation of high-quality non-toxic seedlings of authentic Chinese medicinal materials.

[0017] This invention utilizes gradient low-temperature acclimatization through different modes of ultra-low temperature detoxification treatment to induce the synthesis of antioxidant enzyme systems and osmotic regulators in *Trifolium repens* during the in vitro seedling propagation stage. This enhances the plant's tolerance to allelochemicals such as phenolic acids from the seedling source, effectively reducing physiological damage caused by allelochemical stress without the need for large-scale application of ameliorative agents and growth regulators in the field stage, and lowering labor and material input costs during seedling cultivation.

[0018] This invention integrates seedling detoxification and allelopathic stress resistance induction into the tissue culture propagation stage, solving the problems of fragmented processes, delayed stress intervention, and unstable field control effects in traditional technologies. It steadily improves the transplant survival rate of detoxified seedlings, root development capacity, and tuber yield potential, realizing standardized and large-scale propagation of Huaiyushan Three-Leaf Green seedlings.

[0019] This invention constructs a virus-free and stress-resistant integrated breeding technology system adapted to the local characteristic germplasm of *Tripterygium wilfordii* from Huaiyu Mountain. While protecting the authentic and superior germplasm resources, it provides a source-based technical solution for the prevention and control of continuous cropping obstacles under the continuous cropping cultivation mode of *Tripterygium wilfordii*, and promotes the sustainable industrialization and development of *Tripterygium wilfordii* medicinal materials from Huaiyu Mountain.

[0020] This invention provides the application of the above-described seedling propagation method in any of the following: (1) Reduce the explant contamination rate during the tissue culture of *Trifolium repens* from Huaiyu Mountain; (2) Improve the axillary bud germination rate during the tissue culture of *Trifolium repens* from Huaiyu Mountain; (3) Reduce the coefficient of variation of uniformity of growth of test-tube seedlings in the tissue culture process of *Trifolium repens* from Huaiyu Mountain; (4) Increase the antioxidant enzyme activity and osmotic regulator content of *Trifolium repens* from Huaiyu Mountain; (5) Improve the survival rate of *Tripterygium wilfordii* under allelopathic stress conditions; (6) Improve the overall virus removal rate of *Trifolium repens* from Huaiyu Mountain; (7) Reduce the mortality rate of stem tip browning during tissue culture of *Trifolium repens* from Huaiyu Mountain; (8) Shorten the average regeneration cycle of stem tips of *Trifolium repens* from Huaiyu Mountain; (9) Improve the non-toxicity rate of *Trifolium repens* seedlings from Huaiyu Mountain that were infected with the virus; (10) Improve the survival rate of infected seedlings of *Trifolium repens* from Huaiyu Mountain; (11) Improve the survival rate of transplanted *Trifolium repens* from Huaiyu Mountain; (12) Increase the yield of *Trifolium repens* tubers from Huaiyu Mountain; (13) Preservation of local germplasm resources of *Trifolium repens* in Huaiyu Mountain, selection and breeding of multi-resistant germplasm, and medium- and long-term ex-situ preservation and propagation of germplasm; (14) Alleviating the replanting obstacle of *Trifolium repens* in Huaiyu Mountain; (15) Domestication of wild Huaiyu Mountain Trifolium repens native germplasm and artificial domestication and breeding of endangered authentic Chinese medicinal materials native resources.

[0021] The present invention discloses the following technical effects: This invention addresses several technical challenges in the current propagation of *Trifolium repens* seedlings from Huaiyu Mountain, including low virus removal rates due to single-method ultra-low temperature detoxification, poor shoot tip regeneration efficiency, fragmented detoxification and allelopathic stress resistance cultivation processes, and high costs and unstable effects of field stress control. It employs an integrated technical solution combining gradient low-temperature pre-acclimatization and ultra-low temperature detoxification. This solution achieves efficient seedling detoxification while simultaneously inducing allelopathic stress tolerance in plants during in vitro cultivation, addressing the problem of allelopathic autotoxicity damage under continuous cropping from the germplasm source. Based on data from multiple comparative experiments, the invention is specifically described below from three dimensions: technical, economic, and social effects. I. Core Technology Effects 1. Significantly improves the efficiency of removing complex viruses, greatly reduces the virus-carrying rate of seedlings, and achieves efficient virus removal and rejuvenation of authentic germplasm.

[0022] Existing single-stage vitrification ultra-low temperature detoxification processes have limited effectiveness in eliminating tobacco mosaic virus (TMV). Under conventional single-stage detoxification treatment, the TMV removal rate in Huaiyushan *Trifolium repens* seedlings is only 67.3%. This invention employs four ultra-low temperature detoxification methods—embedding dehydration, embedding vitrification, conventional vitrification, and droplet vitrification—in combination, along with gradient low-temperature pre-acclimation pretreatment. This achieves multi-level synergistic inactivation of various pathogens with different infection depths and stress resistance characteristics. RT-PCR molecular detection verified that the TMV removal rate in seedlings produced by this invention reaches 94.6%, which is 40.56% higher than the traditional single-stage detoxification process. This fundamentally solves the problems of virus accumulation and seed degeneration caused by long-term asexual reproduction.

[0023] Simultaneously, the combination of 0.4 mol / L sucrose permeation pretreatment and gradient low-temperature acclimatization at 15℃, 10℃, and 4℃ effectively reduces ice crystal damage to shoot tip cells during ultra-low temperature freezing. The shoot tip regeneration rate of the conventional untreated control group was only 21.3%, while the shoot tip regeneration rate under the pretreatment scheme of this invention increased to 68.5%, and the browning and necrosis rate of shoot tips decreased from 58.7% to 12.4%. The production capacity of virus-free seedlings was greatly improved, solving the technical defects of poor adaptability and high freezing damage mortality of local germplasm of *Trifolium repens* from Huaiyu Mountain.

[0024] 2. It pre-activates the plant's antioxidant defense system, significantly enhances the seedlings' tolerance to chemical stress, and alleviates physiological damage from continuous cropping and autotoxicity.

[0025] Gradient low-temperature acclimatization can induce a significant accumulation of antioxidant enzyme systems and osmotic regulatory substances in *Trifolium repens*. The physiological indicators of the unacclimatized control group (conventional virus-free seedlings) at room temperature were: SOD activity 126.4 U / g·h, POD activity 187.2 U / g·min, CAT activity 32.5 U / g·min, and proline content 28.6 μg / g. After gradient low-temperature acclimatization combined with multi-mode ultra-low temperature treatment according to this invention, the SOD, POD, and CAT activities of the seedlings increased to 368.7 U / g·h, 492.5 U / g·min, and 94.3 U / g·min, respectively; the proline content increased to 96.3 μg / g; the antioxidant enzyme activity increased by 2.63–2.92 times; and the osmotic regulatory substances increased by 3.37 times.

[0026] Under simulated field conditions of combined allelopathic stress from ferulic acid and p-hydroxybenzoic acid, the survival rate of conventional single-virus-free seedlings was only 48.2%, while the survival rate of seedlings treated with this invention reached 89.7%. After transplanting to fields where *Trifolium repens* has been continuously cropped for many years, the survival rate of conventional cuttings was 45.3% and that of conventional single-virus-free seedlings was 62.7%. The survival rate of the non-toxic and stress-tolerant seedlings cultivated with this invention was as high as 91.4%, and the relative conductivity of the plant cell membrane decreased by 41.8%, effectively alleviating damage caused by allelopathic stress such as cell membrane peroxidation, root browning and rot, and seedling wilting and death. This solves the shortcomings of traditional techniques, such as delayed stress intervention and irreversible physiological damage that cannot be repaired.

[0027] 3. The integration of virus-free breeding and stress resistance training ensures stable seedling traits across batches and avoids interference from the field environment.

[0028] Traditional techniques separate seedling virus elimination and allelopathic stress mitigation into two independent stages: laboratory in vitro culture and field regulation. Stress control relies on soil disinfection, biological agent improvement, and the application of exogenous stress regulators. However, the survival rate of seedlings varies by up to 40.2% between different plots and batches due to environmental factors such as temperature, precipitation, and soil pH. This invention simultaneously completes virus elimination, low-temperature stress acclimatization, and allelopathic stress-oriented screening during the in vitro seedling stage. All seedlings undergo resistance trait induction under standardized aseptic culture conditions, resulting in a seedling growth trait variation coefficient of only 6.3%. The stability of indicators such as survival rate, plant height, root development, and biomass between batches is significantly improved, solving the technical problems of unstable field regulation effects and the difficulty of standardized propagation.

[0029] 4. Adapt to the physiological characteristics of the authentic germplasm of *Tripterygium wilfordii* from Huaiyu Mountain to achieve the protection of local superior germplasm resources and the improvement of improved varieties.

[0030] Existing ultra-low temperature detoxification processes are mostly based on the development of ordinary artificially cultivated *Tripterygium wilfordii*. Direct application of these processes can easily cause large-scale frost damage to the stem tips of native germplasm from the high altitude of Huaiyu Mountain and regeneration failure. This invention relies on gradient osmosis pretreatment and four-stage combined ultra-low temperature parameter optimization to adapt to the cell membrane permeability and osmotic pressure characteristics of local germplasm. It achieves efficient detoxification while screening for stress-resistant germplasm, realizing both in vitro preservation of wild authentic Chinese medicinal herbs and the cultivation of excellent new strains with non-toxicity, tolerance to continuous cropping, and high resistance to allelopathic stress. This fills the technological gap in the integrated detoxification and stress-resistant breeding process of *Tripterygium wilfordii* from Huaiyu Mountain.

[0031] II. Economic Benefits The cost of agricultural inputs and labor in the field is significantly reduced. Under the traditional planting model, continuous cropping of Trifoliate Orange requires regular soil fumigation, microbial improvement, and multiple foliar sprays of stress-resistant agents such as melatonin and salicylic acid. The total input of agricultural inputs and labor in large-scale planting increases by more than 45%. This invention induces chemical stress tolerance characteristics from the seedling source. After transplanting, there is no need for frequent application of improvement agents and growth regulators. It can be directly planted in continuous cropping plots, reducing the overall planting and production cost by 47.5%.

[0032] The survival rate of seedlings and the yield of tubers are significantly improved, resulting in a substantial increase in planting profits. The survival rate of seedlings cultivated by this invention after indoor hardening and transplanting reaches 93.7%, which is 66.7% higher than that of traditional cuttings (56.2%). After 36 months of field cultivation, the fresh weight of individual tubers increases by 64.3% compared to conventional cuttings. Under the large-scale contiguous planting model, the yield of commercial medicinal materials of *Trifolium repens* per unit area is significantly increased, and the average planting income per mu for farmers increases by more than 55%.

[0033] The invention enables large-scale commercial breeding of non-toxic improved varieties, extending the revenue of the Chinese medicinal herb industry chain. It can stably breed non-toxic commercial seedlings that are resistant to continuous cropping in large quantities. These seedlings can be used for self-cultivation or sold to external customers. Relying on the brand advantages of Huaiyu Mountain's authentic Chinese medicinal herbs, it broadens the market-oriented income channels for local Chinese medicinal herb seedlings and avoids the business risks of low survival rate and large after-sales losses of traditional toxic seedlings.

[0034] III. Social Benefits This invention overcomes the technical bottleneck of allelopathic obstacles caused by continuous cropping of *Trifolium repens* in Huaiyu Mountain, changes the traditional land use pattern of crop rotation and fallow, improves the utilization rate of arable land in mountainous areas, avoids land abandonment caused by continuous cropping obstacles, and helps the intensive, sustainable and green planting of Chinese medicinal herbs in the Huaiyu Mountain area of ​​Jiangxi Province.

[0035] To achieve in vitro conservation, improved variety purification and rejuvenation of rare local medicinal germplasm resources of *Trifolium repens* from Huaiyu Mountain, avoid degradation of native resources caused by disorderly harvesting of wild germplasm, and promote the coordinated development of germplasm resource protection and ecological planting of authentic Chinese medicinal materials.

[0036] Standardized, low-cost, and non-toxic seedling propagation technology facilitates the promotion and application of grassroots planting cooperatives and medicinal herb farmers, lowers the threshold for planting Chinese medicinal herbs, drives the development of characteristic Chinese medicinal herb industries in mountainous areas, and helps improve the quality and efficiency of rural characteristic industries.

[0037] The multi-mode ultra-low temperature combined detoxification and low temperature stress resistance domestication technology system established by this invention can provide technical reference for detoxification and seedling cultivation and continuous cropping obstacle control of other understory medicinal plants such as Polygonatum, Paris polyphylla, and Bletilla striata, and has good industry promotion value.

[0038] IV. Summary of Technical Effects This invention employs an integrated technical solution combining gradient low-temperature pre-acclimatization with ultra-low temperature virus removal. This solution achieves efficient removal of the Huaiyu Mountain Trifoliate Orange composite virus and significantly improves shoot tip regeneration efficiency. Furthermore, it constructs a plant allelopathic stress defense system in advance during the in vitro cultivation stage, addressing multiple shortcomings of existing technologies from the seedling source, such as incomplete virus removal, delayed stress resistance cultivation, high field control costs, and poor effect stability. Based on these significant technical advantages, it effectively reduces the production cost of medicinal herb cultivation, increases yield and farmers' economic benefits, while simultaneously protecting local rare germplasm and promoting green agriculture in mountainous areas. It possesses outstanding technological innovation, economic practicality, and industry promotion value. Specific implementation methods Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terminology used in this invention is merely for describing particular implementations and is not intended to limit the invention. Furthermore, regarding numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments of this invention without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this invention will also be readily apparent to those skilled in the art. This invention specification and embodiments are merely exemplary.

[0042] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0043] All materials described in this invention are obtained through routine purchase by those skilled in the art.

[0044] The anhydrous ethanol, ethanol, dimethyl sulfoxide, ethylene glycol, Tween-80, Melaleuca alternifolia tea tree oil, phosphate buffer, and sterile deionized water used in this invention are all expressed as volume percentages, while mercuric chloride, glycerol, sodium alginate, and sodium hypochlorite are expressed as mass percentages.

[0045] SOD: Superoxide dismutase, a core antioxidant enzyme in plants, which removes excess reactive oxygen species in cells under allelopathic stress and reduces lipid peroxidation damage to cell membranes.

[0046] POD: Peroxidase, which works synergistically to break down intracellular hydrogen peroxide, maintain cellular redox homeostasis, and reduce the toxic effects of phenolic acids.

[0047] CAT: Catalase, which efficiently removes hydrogen peroxide produced by stress, preventing the accumulation of reactive oxygen species that can cause cell apoptosis and root rot.

[0048] MS medium: Basic medium for plant tissue culture.

[0049] PVS2: Plant vitrification protectant, composed of 30% glycerol, 15% dimethyl sulfoxide, 15% ethylene glycol and 0.4 mol / L sucrose.

[0050] Loading medium: MS medium containing 2M glycerol + 0.4M sucrose.

[0051] RT-PCR: Reverse transcription-polymerase chain reaction. This invention is a molecular biology technique for the precise qualitative detection of various plant viruses and phytoplasmas in *Trifolium repens*, enabling high-throughput screening of virus-free seedlings.

[0052] The primers used for RT-PCR are shown below: Forward primer F: 5'-ATGTCTTACAATATCACTACTC-3', SEQ ID NO.1; Reverse primer R: 5'-TCAAGTTGCAGGACCAGAGGT-3', SEQ ID NO.2.

[0053] The RT-PCR amplification program was a 20 μL system containing: 10 μL of 2×Taq PCR Mix, 0.8 μL of 10 μmol / L forward primer, 0.8 μL of 10 μmol / L reverse primer, 1.5 μL of *Trifolium repens* cDNA template, and 6.9 μL of sterile deionized water to bring the total volume to 20 μL.

[0054] The RT-PCR amplification system was pre-denatured at 95℃ for 3 min; then 35 amplification cycles were performed, each cycle consisting of denaturation at 95℃ for 30 s, annealing at 58℃ for 30 s, and extension at 72℃ for 90 s; after the cycle, the final extension was performed at 72℃ for 10 min, and the system was stored at 4℃.

[0055] The steps of the seedling propagation method for mitigating the stress damage caused by the combined use of multiple ultra-low temperature detoxification methods provided by this invention are as follows: Screening of healthy mother plants of *Trifolium repens* from Huaiyu Mountain → Collection of explants from tender stems with axillary buds → Surface sterilization treatment with phosphate buffer + 0.3wt% tea tree oil emulsion + 0.2wt% low-concentration hypochlorous acid solution → Axillary bud induction culture on primary solid culture medium → Subculturing and multi-generation propagation of sterile test-tube seedlings → Screening of test-tube seedlings with uniform physiological growth → 0.5~1.0mm shoot tip meristem stripping → Pre-culture in high-sucrose liquid culture medium → Gradient-stage low-temperature stress resistance acclimatization pretreatment at 15℃, 10℃, and 4℃ → Embedding, dehydration, and cryopreservation → Embedding, vitrification, and cryopreservation → Vitrification The process involved four steps: cryopreservation, droplet vitrification cryopreservation, and combined liquid nitrogen freezing; rapid rewarming in a constant-temperature water bath at 38-40℃; elution of protective agents and embedding substrates with gradient concentration sucrose washing solution; callus induction through dark shoot tip culture; adventitious bud differentiation and regeneration culture under light; RT-PCR detection of regenerated seedlings with a composite virus; screening and purification of non-virulent lines; directional resistance acclimatization to stress-induced seedlings in a culture medium supplemented with phenolic allelochemicals; propagation and proliferation of superior allelopathic stress-resistant lines; indoor hardening-off of sterile test-tube seedlings with a gradient of open caps; root culture medium cleaning; substrate-based seedling acclimatization; and continuous cropping and field transplanting.

[0056] Example 1: Aseptic in vitro seedling cultivation of *Trifolium repens* from Huaiyu Mountain Healthy *Trifolium repens* mother plants growing in the native forests of Huaiyu Mountain, without leaf mottling, wrinkling, deformity, or mosaic symptoms, were selected. One to two axillary buds of the current year's semi-lignified stem segments were cut as explants and subjected to gradient disinfection. First, the explants were rinsed with running tap water for 30 minutes to remove dust, insect eggs, and loose saprophytic microorganisms from their surface. Then, they were soaked in a pH 8.0 weak alkaline phosphate buffer for 10 minutes to specifically dissolve the dense saprophytic biofilm and mucus protective layer on the surface of the explants, disrupting the microbial attachment substrate and solving the problem of high bacterial load and severe endophytic fungal concealment in wild forest materials. Finally, the explants were treated with a 0.3wt% tea tree oil emulsion solution (0.30% *Melaleuca alternifolia* tea tree oil, 1.20% Tween-80, 2.00% anhydrous ethanol, 0.50% phosphate buffer, and 96.00% sterile deionized water) for 40 minutes in a clean bench. The process utilizes plant-derived antibacterial components to penetrate the epidermal crevices of stem segments, inhibiting the activity of stubborn bacteria in the axillary bud crypts, while simultaneously softening the epidermal wax layer, creating conditions for subsequent deep sterilization. After three rinses with sterile water to remove surface residues, a 0.2% low-concentration hypochlorous acid solution is used for 7 minutes of light-proof, shaken sterilization. This method relies on gentle oxidation to precisely kill surface and superficial endophytic bacteria and fungi without heavy metal penetration damage. Finally, five multiple-stage rinsings with sterile water thoroughly remove trace amounts of oxidative residues, maximizing the preservation of axillary bud meristem activity. The browned ends of the sterilized stem segments are trimmed, and the segments are inoculated with MS + 0.8 mg / L 6-BA + 0.2 mg / L The initial solid culture medium of NAA was used to induce axillary bud germination at a temperature of 25±1℃, a light intensity of 2200 lx, a photoperiod of 12 h / d, and a duration of 30 days. When the axillary buds elongated to 2-3 cm and developed two functional leaves, single stem segments were cut and transferred to MS medium containing 1.0 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sucrose, and 7 g / L agar for three generations of continuous propagation at 25±1℃, a light intensity of 2200 lx, a duration of 30 days, and a photoperiod of 12 h / d. Sterile test-tube plantlets with a height of 3 cm, dark green leaves, no vitrification or browning, and uniform growth were selected as materials for subsequent ultra-low temperature experiments; these were considered robust subcultured plantlets. Simultaneously, conventional sterilization methods were used (rinsing with tap water for 30 min, disinfecting with 75% alcohol for 45 s, rinsing four times with sterile water, and then using 0.1% alcohol). Sterilize with HgCl2 for 12 min, rinse 4 times with sterile water, and follow the same steps as before (as a control). After inoculation into primary solid medium for 7 days, the explant contamination rate was investigated. After 30 days of culture in primary solid medium, the induced germination rate of primary axillary buds was investigated. After three generations of continuous propagation in subculture medium, the coefficient of variation was investigated.The results showed that the explant contamination rate was 26.4% under conventional sterilization methods, while the contamination rate was only 4.1% under the gradient disinfection scheme provided by this invention; the germination rate of primary axillary buds could reach 92.6%; and the coefficient of variation of uniformity of growth of test-tube seedlings after three consecutive generations of propagation was only 6.3%, which was much lower than 27.5% in the unscreened control group, and could stably provide standardized experimental materials for subsequent shoot tip stripping.

[0057] The gradient disinfection scheme provided by this invention obtains contamination-free sterile materials through strict sterilization of the explant surface, avoiding stem tip rot and necrosis caused by fungi and bacteria during ultra-low temperature pretreatment and cryogenic regeneration. After three generations of subculture screening, test-tube seedlings with uniform genetic background and physiological state are obtained, eliminating the interference of individual material differences on the detoxification regeneration rate and stress resistance physiological indicators, and ensuring the repeatability and reliability of ultra-low temperature test data from multiple batches.

[0058] Example 2: Shoot tip sampling and gradient low-temperature pre-acclimatization pretreatment Under sterile conditions, using a stereomicroscope, pure shoot tip meristems (0.5–1.0 mm in diameter, containing 1–2 leaf primordia) were peeled from the tips of robust subcultured plantlets obtained in Example 1. All mature petioles and parenchyma tissues were removed. The peeled shoot tips were transferred into liquid pre-culture medium (MS + 0.4 mol / L sucrose) and incubated at 25°C in the dark for 24 hours. After incubation, a gradient low-temperature acclimatization process was performed: 2 days of constant temperature incubation at 15°C in the dark → 2 days of constant temperature in the dark at 10°C → 3 days of low-temperature acclimatization at 4°C. The resulting shoot tips, after completing the stress resistance pretreatment, were designated as the experimental group. Simultaneously, pure shoot tip meristems without sucrose incubation and gradient low-temperature pretreatment served as the control group, i.e., plantlets grown at room temperature without low-temperature acclimatization served as the blank control (CK group). After the 4℃ low-temperature acclimatization culture was completed, the regeneration rate of the shoot tips after freezing was investigated in the experimental group and the control group. The SOD activity, POD activity, CAT activity and proline content were investigated in the experimental group and the blank control group. The detection methods of the physiological indicators were referenced in "Principles and Techniques of Plant Physiological and Biochemical Experiments" (Li Hesheng. Principles and Techniques of Plant Physiological and Biochemical Experiments [M]. Beijing: Higher Education Press, 2000). The detection method of the regeneration rate was referenced in "Ultra-low Temperature Preservation Technology of Plant Germplasm Resources" (Wang Junhui and Huang Chunong. Ultra-low Temperature Preservation Technology of Plant Germplasm Resources [M]. Beijing: Science Press, 2004.). The results showed that the regeneration rate of shoot tips after freezing in the control group was only 21.3%, while the regeneration rate of shoot tips in the experimental group increased to 68.5%. The SOD activity of the CK group was 126.4 U / g·h, POD activity was 187.2 U / g·min, CAT activity was 32.5 U / g·min, and proline content was 28.6 μg / g. The SOD activity of the experimental group was 368.7 U / g·h, POD activity was 492.5 U / g·min, CAT activity was 94.3 U / g·min, and proline content was 96.3 μg / g. The antioxidant enzyme activity increased by 2.63 to 2.92 times, and the content of osmotic regulators increased by 3.37 times, laying a physiological foundation for subsequent tolerance to soil phenolic acidification stress.

[0059] The gradient low-temperature pre-acclimatization pretreatment provided by this invention increases the concentration of soluble solutes in shoot tip cells through high sucrose osmotic pretreatment, reduces the probability of large intracellular ice crystal formation during liquid nitrogen cryogenic freezing, significantly reduces mechanical tearing damage to cell membranes, and significantly improves the survival rate of shoot tip regeneration after freezing. The gradual gradient low-temperature stress continuously induces the upregulation of plant antioxidant system genes, promotes the synthesis of large amounts of three core antioxidant enzymes, SOD, POD, and CAT, and induces the accumulation of osmotic regulatory substances such as proline and soluble sugars. It pre-constructs an allelopathic stress defense system in the in vitro stage, overcoming the technical shortcomings of traditional single detoxification processes that only remove viruses without cultivating stress resistance in advance.

[0060] Example 3: Combined use of multiple ultra-low temperature detoxification methods to treat shoot tips The shoot tips obtained in Example 2 after gradient low-temperature pre-acclimatization were divided into four groups. One group underwent a combined treatment with four different cryogenic processes in stages (embedding dehydration cryogenic detoxification + embedding vitrification cryogenic detoxification + vitrification cryogenic detoxification + droplet vitrification cryogenic detoxification). The other three groups each underwent one of the four cryogenic processes. The specific steps of the four cryogenic combined treatments are as follows: Dehydration and cryogenic detoxification by embedding: Gel microspheres were prepared by encapsulating shoot tips with 3% sodium alginate + 0.5 mol / L calcium chloride. After sterile drying in a ventilated environment for 4 hours, the microspheres were frozen in liquid nitrogen for 1 hour to obtain dehydrated embedded microspheres. The specific steps were as follows: a 3% sodium alginate MS solution (MS + 3% sodium alginate) was prepared, and the shoot tips were immersed in the solution. Then, 0.5 mol / L calcium chloride MS solution (MS + 0.5 mol / L calcium chloride) was added dropwise to form gel microspheres. The microspheres were allowed to solidify and stand for 20 minutes. After sterile drying in a ventilated environment for 4 hours, the microspheres were frozen in liquid nitrogen for 1 hour. After thawing in a 35°C water bath, the microspheres were washed with a 1.2M sucrose solution. Vitrification and detoxification of embedded microspheres: The dehydrated embedded microspheres were transferred into PVS2, immersed at 0°C for 60 min, and then frozen again in liquid nitrogen for 40 min; after that, they were thawed in a water bath at 35°C and washed with 1.2M sucrose solution. Vitrification and cryogenic detoxification: The gel carrier was peeled off, and the shoot tips were treated with loading solution for 20 min, then immersed in PVS2 at low temperature (0℃) for 30 min, and then instantly frozen with liquid nitrogen; after that, they were thawed in a water bath at 35℃ and washed with 1.2M sucrose solution. Small-drop vitrification cryogenic detoxification: The stem tip is immersed in the loading solution at room temperature for 25 minutes, and 2.5 μL of pre-cooled PVS2 droplets are added to the aluminum foil to wrap the stem tip. The stem tip is then directly immersed in liquid nitrogen for quick freezing for 30 minutes, which can deeply penetrate the meristematic cells.

[0061] The 4-stage cryogenic process maintains a liquid nitrogen temperature of -196℃ throughout.

[0062] Subsequently, the isolated, cryogenically detoxified shoot tips were subjected to allelopathic stress simulation treatment, with the following specific steps: Shoot tips treated with liquid nitrogen using a "small-drop vitrification ultra-low temperature detoxification" method were rapidly removed and placed in a 39℃ constant temperature water bath for rapid rewarming for 90 seconds. They were then rinsed three times sequentially with MS washing solutions of 0.5 mol / L, 0.3 mol / L, and 0.1 mol / L sucrose, each time for 10 minutes. After washing, the shoot tips were inoculated onto regeneration medium (MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar) and cultured in the dark at 25℃ for 7 days to alleviate freezing stress damage. Afterward, they were transferred to a 12 h / d light environment at 25±1℃ for 30 days to induce plant regeneration. Regenerated seedlings were selected based on the following criteria: plant height 2.5–3 cm, 2–3 functional leaves, intact root system, and no browning or deformities. The regenerated seedlings were then transferred to a standard seedling strengthening medium (MS + 0.5 mg / L PP333 + 0.1 mg / L PP333). After pre-culturing with NAA (30 g / L sucrose + 7 g / L agar) for 15 days and achieving stable growth, allelopathic stress was uniformly simulated in vitro. Single vitrified virus-free regenerated seedlings and seedlings regenerated using four different ultra-low temperature virus-free methods were taken. The aging roots at the base were aseptically removed, and new fibrous roots of 0.8 cm in length were uniformly retained. The seedlings were then completely transferred to a solid medium containing phenolic acid allelopathic stress (MS + 50 mg / L ferulic acid + 30 mg / L p-hydroxybenzoic acid) for allelopathic stress treatment. The allelopathic stress treatment parameters were: culture temperature 25 ± 1℃; light intensity 2200 lx; ​​photoperiod 12 h light / 12 h dark; stress culture duration 15 days; 60 seedlings per treatment were inoculated, with 3 biological replicates. After 15 days of continuous stress culture, the number of surviving seedlings in each group was counted, and the seedling survival rate was calculated. At the end of the stress, leaves were collected simultaneously to detect the virus removal rate. The criteria for judging surviving seedlings were: leaves were fully expanded and not yellowed, stem segments were not browned or rotten, and the root system maintained white new roots. The seedling survival rate was calculated as follows: Seedling survival rate = Number of intact seedlings surviving after 15 days of stress treatment / Total number of inoculated seedlings × 100%.

[0063] The results showed that the overall virus removal rate of single vitrification cryogenic treatment was 67.3%, while the overall virus removal rate of the combined treatment of four cryogenic processes in stages reached 94.6%. The survival rate of seedlings under allelopathic stress was only 48.2% under single vitrification cryogenic treatment, while the survival rate of seedlings under in vitro simulated allelopathic stress under the combined treatment of four cryogenic processes in stages reached 89.7%.

[0064] While single-stage cryogenic processes for virus removal have limitations in targeting, the four cryogenic processes described in this invention, used in a segmented combined treatment, achieve multi-level and multi-type synergistic inactivation of pathogens, significantly reducing the rate of missed virus detection. Multiple rounds of low-temperature stress continuously strengthen plant stress-resistance signaling pathways, further solidifying the physiological characteristics of seedlings to resist phenolic acid stress, thus simultaneously achieving virus removal and rejuvenation and stress-resistant germplasm improvement from the source. Therefore, the four cryogenic processes, used in a segmented combined treatment series, rely on the differentiated mechanisms of different freezing and protection systems to cover various pathogens with different infection depths and stress resistance characteristics.

[0065] Example 4: Shoot tip rewarming, washing, and regeneration culture The shoot tips treated with liquid nitrogen in step "small droplet vitrification and cryogenic detoxification" in Example 3 were quickly removed and placed in a 39°C constant temperature water bath for rapid rewarming for 90 seconds to avoid secondary ice crystal formation and cell membrane damage caused by slow heating. Subsequently, they were washed three times with a gradient of 0.5 mol / L, 0.3 mol / L, and 0.1 mol / L sucrose MS washing solution for 10 minutes each time to fully wash away the PVS2 vitrification protectant and sodium alginate embedding matrix and eliminate the continuous cytotoxicity of the hypertonic reagent. After washing, the shoot tips were inoculated into regeneration medium (MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar) and cultured in the dark at 25°C for 7 days to alleviate the damage caused by freezing stress. Then, they were transferred to light culture at 25 ± 1°C for 12 h / d for 30 days to induce plant regeneration. This group was designated as the experimental group. Meanwhile, a control group was established using a conventional method of slow rewarming at room temperature followed by a single wash. This method involved slowly removing the shoot tips from liquid nitrogen using sterile forceps, allowing them to rest naturally in a sterile laminar flow hood at 25°C for 10 minutes, and then slowly warming them using ambient air without a rapid water bath. After rewarming, the shoot tips were rinsed once with a single concentration of MS washing buffer (MS containing 1.2M sucrose) for 10 minutes, without any gradient dilution. After rinsing, residual liquid on the shoot tip surface was blotted dry with filter paper, and the shoot tips were inoculated into the same regeneration medium (MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar), first incubated in the dark at 25°C for 7 days, then incubated under light at 25±1°C for 12 h / d for 30 days to induce regeneration. After inducing plant regeneration, the browning mortality rate and average regeneration cycle of the shoot tips under different treatments were investigated. The results showed that the stem tip browning mortality rate in the control group was as high as 58.7%; the stem tip browning mortality rate in the experimental group was reduced to 12.4%, the average stem tip regeneration cycle was shortened from 42 days to 31 days, and the uniformity of the regenerated seedlings was significantly improved.

[0066] This invention combines rapid water bath rewarming with gradient dilution washing to minimize browning and necrosis of stem tips after freezing. It is adapted to the physiological characteristics of Huaiyushan San Ye Qing authentic germplasm with sensitive cell membranes and weak freeze resistance, and solves the defects of high stem tip mortality and unstable regeneration in general ultra-low temperature processes, ensuring stable regeneration of virus-free stem tips into seedlings.

[0067] Example 5: Screening of non-toxic regenerated seedlings and cultivation of robust seedlings resistant to chemical stress When the regenerated plants obtained in Example 4 grew to a height of more than 1.5 cm and had 2-3 functional leaves, total RNA was extracted from the leaves. RT-PCR was used to amplify and detect the virus using primers specific to Tobacco Trifolium Mosaic Virus. Virus-carrying plants that showed specific bands were discarded, and negative virus-free regenerated lines were retained. The selected virus-free seedlings were transferred to a seedling culture medium (MS + 50 mg / L ferulic acid + 30 mg / L p-hydroxybenzoic acid, simulating typical allelopathic stress environment in the field) for directional acclimatization culture. After two consecutive generations, the temperature of this process was 25±1℃, the time was 30 days, the photoperiod was 12h / d, and the light intensity was 2200lx. Afterwards, the antioxidant enzyme activity, plant height, fresh weight, and root length of each group of plants were measured. Excellent allelopathic stress-tolerant lines with robust growth, well-developed root systems, and significantly higher antioxidant enzyme activity were selected for large-scale propagation. Simultaneously, sterile *Trifolium repens* plantlets were obtained using conventional single-stage virus elimination treatment (using the same explants, disinfection, primary and subculture conditions as in Example 1; only the vitrification method was used for ultra-low temperature virus elimination of the shoot tips, without gradient low-temperature pre-acclimatization pretreatment; single vitrification virus elimination treatment: peeling 0.8~1.0 mm). Shoot tip meristems were soaked in a loading solution at room temperature for 25 min, then dehydrated using pure PVS2 and directly frozen in liquid nitrogen. After freezing, they were slowly thawed at 25°C for 10 min and rinsed once with 1.2M sucrose MS solution for 10 min. Regeneration culture: All plants were inoculated into the same regeneration medium, cultured in the dark at 25°C for 7 days, and then cultured under light at 25±1°C for 12 h / d for 30 days to obtain regenerated plants. Virus screening: When the regenerated seedlings reached a height of 1.5 cm and had 2-3 functional leaves, virus-free plants were screened using the same RT-PCR primers and detection system, and virus-carrying plants were removed. Conventional seedling cultivation (directed acclimatization without allelochemicals): The screened virus-free seedlings were transferred to ordinary seedling cultivation medium MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar, culture medium without ferulic acid, p-hydroxybenzoic acid or other allelochemicals; two consecutive subcultures, each with a 30-day culture cycle, and a uniform culture environment of 25±1℃, light intensity of 2200 lx, and light duration of 12 h / d (as a control group).

[0068] Afterwards, the seedlings were allowed to acclimate indoors for 3 days under open cover, gradually adapting to the outside air humidity. The seedlings were then removed, the roots were washed to remove any residual agar, and soaked in clean water for 10 minutes. The transplanting substrate was uniformly sourced from the fields where *Trifolium repens* had been continuously cropped for many years in Huaiyu Mountain (naturally rich in phenolic acid-induced autotoxic substances, simulating a severe allelopathic stress environment). Uniform greenhouse cultivation and management were implemented, with consistent temperature, humidity, water, fertilizer, and shading conditions. Samples were taken and all indicators were tested uniformly 60 days after transplanting. The results showed that after screening by RT-PCR, the virus-free rate of the allelopathic stress-resistant virus-free seedlings in this invention was 99.87%, while the virus-free rate of the control group was only 67.3%. After 60 days of continuous cropping in the field, the survival rate of ordinary virus-carrying seedlings was 45.3%, the survival rate of virus-free seedlings in the control group was 62.7%, and the survival rate of allelopathic stress-resistant virus-free seedlings in this invention was 91.4%. Under allelopathic stress conditions, the fresh weight of individual allelopathic stress-resistant virus-free seedlings in this invention increased by 57.2% compared with the virus-free seedlings in the control group, the total root length increased by 63.5%, the relative conductivity of the cell membrane decreased by 41.8%, and the cell membrane peroxidation damage was significantly alleviated.

[0069] This invention utilizes molecular biology to precisely screen and completely eliminate incompletely detoxified seedlings, ensuring that commercial seedlings are 100% virus-free. In the in vitro stage, it simulates field allelopathic stress to directionally screen resistant and stable lines, completing the screening of continuous cropping tolerant germplasm in advance. After transplanting, there is no need to apply large amounts of soil conditioners and stress-resistant foliar fertilizers, significantly reducing field agricultural input and labor costs. At the same time, it solves the technical problems of delayed field stress regulation and irreversible damage.

[0070] Example 6: Virus-free seedling hardening-off and field transplanting The chemically resistant, virus-free seedlings obtained in Example 5 were placed in a greenhouse at 25°C. The seedlings were hardened off for 3 days with the bottle opening partially open and for 4 days with the bottle fully open, gradually adapting to the outdoor temperature, humidity, and light environment. The seedlings were then removed, and the roots were gently cleaned of any remaining solid culture medium. They were then transplanted into a seedling substrate of peat moss:perlite = 3:1 (by volume) and acclimatized in a greenhouse at 25°C for 30 days. Once the seedlings reached a height of 8-10 cm and had developed a strong root system, they were directly transplanted into fields where *Trifolium repens* had been continuously cropped for many years and were then managed with conventional water and fertilizer. Simultaneously, conventional, non-virus-free cuttings were used for propagation (cuttings were selected from current-year lignified branches of common virus-carrying mother plants under the *Trifolium repens* forest in Huaiyu Mountain, with stem segments containing 1-2 axillary buds, without undergoing the complete tissue culture virus-free process of stem tip stripping, ultra-low temperature virus removal, virus screening, and allelopathic acclimatization. Cuttings were disinfected by rinsing with clean water for 15 minutes, then quickly wiping the surface with 75% alcohol, without mercuric chloride gradient sterilization; no aseptic tissue culture primary or subculture propagation was performed. The propagation substrate was peat moss: perlite = 3:1 (volume ratio), consistent with the experimental group's substrate, and propagated in a uniform greenhouse environment at 25℃. Cutting management: the base of the cuttings was dipped in 50mg / L... NAA rooting solution was used. After cuttings were planted, they were thoroughly watered and kept in the shade and moist for 40 days. During this period, carbendazim was sprayed regularly to prevent browning and viral diseases. Once the plants reached a height of 8-10 cm and had developed a root system, they were ready for field transplanting. Hardening-off process: Cuttings were directly exposed to the open air and slowly hardened off with ventilation for 2 days, followed by a refined gradient hardening-off process of 3 days with partial cover and 4 days with full cover. Field transplanting: All cuttings were uniformly transplanted to fields that had been continuously cropped with *Trifolium repens* for many years, with water, fertilizer, shading, and field management exactly the same as the experimental group. (The control group was included). After 540 days, the transplant survival rate, single-plant yield of tubers, and the cost of pesticides and soil conditioners used in the field were investigated for different treatments. The results showed that the survival rate of virus-free and stress-tolerant seedlings cultivated by this invention after indoor hardening and transplanting was 93.7%, while the survival rate of the control group was only 56.2%. After 18 months of continuous cultivation, the yield per tuber of the virus-free and stress-tolerant seedlings cultivated by this invention (46.99 g / plant) increased by 64.3% compared with the yield per tuber of the control group (28.6 g / plant), and the input cost of field pesticides and soil conditioners was reduced by 47.5%.

[0071] This invention enables a smooth transition from a sterile in vitro environment to an open field environment, maximizing the preservation of the dual excellent traits of detoxification and resistance to allelopathic stress induced by multi-mode ultra-low temperature combined treatment in the early stage. It effectively alleviates allelopathic autotoxicity damage in continuous cropping of Huaiyu Mountain Trifoliate Orange from the seedling source, and realizes the efficient industrialization and promotion of high-quality varieties of authentic Chinese medicinal materials.

[0072] Example 7 Alternative Solution 1 Applicable scenarios: preservation of local germplasm resources of *Trifolium repens* in Huaiyu Mountain, breeding of multi-resistant germplasm, and medium- and long-term ex-situ preservation and propagation of germplasm.

[0073] The shoot tips obtained in Example 2 after gradient low-temperature pre-acclimatization were subjected to embedding dehydration and cryogenic detoxification (the shoot tips were dispersed in 3% sodium alginate MS solution, 0.5 mol / L calcium chloride MS solution was added, and the mixture was allowed to stand for 15 min to form embedded microspheres, solidified and allowed to stand for 20 min; after sterile ventilation and drying for 4 h, the mixture was frozen in liquid nitrogen for 1 h) and conventional vitrification cryogenic detoxification (the shoot tips were placed in a loading solution (MS + 2 mol / L glycerol + 0.4 mol / L sucrose) and immersed at room temperature for 25 min, the surface liquid was absorbed, and then the mixture was immersed in pre-cooled PVS2). The stem tips were dehydrated at 25°C in the dark for 60 minutes, then transferred to aluminum foil tubes along with a small amount of protective solution and frozen in liquid nitrogen for more than 1 hour. Alternatively, the stem tips were vitrified at low temperatures for 25 minutes at room temperature (the tips were then cut into sterile aluminum foil strips and 2-3 μL of pre-cooled PVS2 was added, the tips were transferred into the droplets, and the entire stem tip was immersed in liquid nitrogen for 30 minutes). The stem tips obtained in Example 2 and subjected to gradient low-temperature pre-acclimatization were divided into three groups and subjected to three different low-temperature freezing treatments. After that, the stem tips were rewarmed, washed, and regenerated. Virus-free regenerated seedlings were screened and chemically resistant seedlings were cultivated (the steps were the same as in Example 10). After obtaining virus-free lines by RT-PCR virus detection, the regenerated seedlings in each group were subjected to virus-free seedling hardening and field transplanting (the steps were the same as in Example 10). Different virus-free processes induced differential expression of plant stress resistance regulatory pathways. Mixed germplasm can broaden the genetic diversity of chemically resistant stress and reduce the risk of large-scale damage to germplasm under extreme soil conditions.

[0074] Example 8 Alternative Solution 2 Applicable scenarios: Old *Trifolium repens* planting bases with continuous planting for more than 5 years and high concentration of allelopathic substances in the soil, and plots with severe replanting obstacles.

[0075] In Example 6, during the hardening-off and transplanting stage of virus-free seedlings, a compound inoculum of Bacillus subtilis and Trichoderma harzianum was used (the effective viable count ratio of Bacillus subtilis and Trichoderma harzianum was 1:1; the total effective viable count was 10). 8 CFU / mL; Bacillus subtilis was purchased from the China Agricultural Microbial Culture Collection Center, catalog number ACCC 11086; Trichoderma harzianum was purchased from the China Agricultural Microbial Culture Collection Center, catalog number ACCC 30406) and the roots were soaked for 15 min. On the one hand, rhizosphere microorganisms can secrete phenolic acid degrading enzymes to directly mineralize and decompose allelopathic substances such as ferulic acid and p-hydroxybenzoic acid in the soil; on the other hand, after beneficial microorganisms colonize the root system, they can induce systemic resistance in plants and further upregulate the activity of antioxidant enzymes. This dual mechanism alleviates allelopathic stress damage.

[0076] Example 9 Alternative Solution 3 Applicable scenarios: domestication of wild Huaiyu Mountain Trifoliate Orchid germplasm, and artificial domestication and breeding of endangered authentic Chinese medicinal resources.

[0077] The "shoot tip sampling and gradient low-temperature pre-acclimatization" process in Example 2 was moved from the in vitro test-tube seedling stage to the field mother plant cultivation stage: In autumn, healthy mother plants of *Trifolium repens* from Huaiyu Mountain underwent continuous field gradient low-temperature stress acclimatization at 4-15℃ for 7 days (2 days of constant temperature and dark culture at 15℃ → 2 days of constant temperature and dark culture at 10℃ → 3 days of low-temperature acclimatization culture at 4℃). Then, tender stem segments with 1-2 axillary buds from the current year's semi-lignified zone of the acclimatized mother plants were collected for explant disinfection (using the disinfection method in Example 1), primary culture, subculture (using the method in Example 1), and four types of combined ultra-low temperature virus removal from the shoot tips of the sterile test-tube seedlings obtained from the subculture (Examples 3-6). Stress acclimatization activates stress-related genes from the maternal generation, which can significantly improve the genetic stability of chemical stress tolerance traits in offspring seedlings and reduce the probability of degradation of resistance traits after in vitro acclimatization.

[0078] Example 10: A seedling propagation method for mitigating the stress damage caused by the combined use of multiple ultra-low temperature detoxification methods in *Trifolium repens*. Step 1: Aseptic test-tube seedling cultivation of *Trifolium repens* from Huaiyu Mountain Healthy *Trifolium repens* mother plants without flowering leaves, wrinkles, deformities, or diseases were selected from the forest floor of Huaiyu Mountain. One to two axillary buds of the current year's semi-lignified stem were cut as explants. The explants were rinsed with running water for 30 minutes to remove surface dust, insect eggs, and epiphytic bacteria.

[0079] Sterilization treatment in a clean bench: immerse in 75% ethanol for 30 seconds, rinse 3 times with sterile water; sterilize with 0.1% mercuric chloride solution by shaking for 7 minutes, rinse repeatedly with sterile water 5 times, and trim the browned cut ends.

[0080] Primary culture medium: MS + 0.8 mg / L 6-BA + 0.2 mg / L NAA + 7 g / L agar + 30 g / L sucrose, pH adjusted to 5.8; Post-inoculation culture conditions: temperature 25 ± 1℃, light intensity 2200 lx, light duration 12 h / d, cultured for 30 days to induce axillary bud germination.

[0081] When the axillary buds grow to 2-3 cm, cut single stem segments and transfer them to subculture medium: MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar. Subculture for 3 generations at a temperature of 25 ± 1℃, a light intensity of 2200 lx, a culture time of 30 days, and a photoperiod of 12 h / d. Select healthy, sterile test-tube seedlings with uniform growth, no vitrification, and no browning for later use.

[0082] Step 2: Shoot tip sampling and gradient low-temperature pre-acclimatization pretreatment Under sterile conditions, the shoot tip meristem containing 1-2 leaf primordia was dissected using a stereomicroscope at a thickness of 0.5-1.0 mm and under sterile conditions, and all mature petioles and parenchyma tissues were removed.

[0083] High sucrose osmotic pre-culture: The shoot tips were placed in liquid pre-culture medium (MS + 0.4 mol / L sucrose) and osmotically cultured at 25°C in the dark for 24 h.

[0084] Gradient low temperature acclimatization treatment: cultured at 15℃ in the dark for 2 days → cultured at 10℃ in the dark for 2 days → cultured at 4℃ in the dark for 3 days to complete the stress resistance pretreatment.

[0085] Step 3: Four ultra-low temperature detoxification methods were used in combination to treat the shoot tips in stages. After pretreatment, the stem tips undergo the following four-stage cryogenic treatment: 1) Encapsulation, dehydration, and ultra-low temperature detoxification: Prepare a 3% sodium alginate solution, immerse the stem tip in it, then add 0.5 mol / L calcium chloride solution to form gel beads, let it solidify and stand for 20 minutes; after sterile ventilation and drying for 4 hours, immerse it in liquid nitrogen for 1 hour.

[0086] 2) Encapsulation vitrification and cryogenic detoxification: The gel microspheres were immersed in pre-cooled PVS2 at 0°C for 60 minutes, and then frozen again in liquid nitrogen for 40 minutes.

[0087] 3) Vitrification and cryogenic detoxification: After peeling off the sodium alginate gel carrier, the shoot tips were treated with the loading solution at room temperature for 20 min, then immersed in PVS2 at 0℃ for 30 min, and then instantly frozen in liquid nitrogen.

[0088] 4) Low-temperature detoxification of small droplets through vitrification 2.5 μL of pre-cooled PVS2 was dropped onto the surface of an aluminum foil sheet, and the stem tip was then placed directly into liquid nitrogen for quick freezing for 30 min.

[0089] The 4-stage ultra-low temperature liquid nitrogen temperature is -196℃ throughout the entire process.

[0090] Step 4: Shoot tip rewarming, washing, and regeneration culture Frozen stem tips were removed from liquid nitrogen and rapidly rewarmed in a 39°C constant temperature water bath for 90 seconds to prevent secondary ice crystal formation within the cells and subsequent cell damage.

[0091] The cells were washed sequentially with a gradient of sucrose MS wash buffer: MS + 0.5 mol / L sucrose for 10 min → MS + 0.3 mol / L sucrose for 10 min → MS + 0.1 mol / L sucrose for 10 min, to fully elute the PVS2 protectant and the sodium alginate embedding matrix.

[0092] Regeneration medium: MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.8; first, culture in the dark at 25℃ for 7 days, then culture under light for 12 h / d at 25±1℃ for 30 days to induce plant regeneration.

[0093] Step 5: Screening of non-toxic regenerated seedlings and cultivation of robust seedlings resistant to chemical stress When the regenerated seedlings reach a height of ≥1.5cm, total RNA is extracted from the leaves. Tobacco mosaic virus is specifically detected using RT-PCR technology. Virus-carrying strains that show specific amplification bands are eliminated, and virus-free strains are retained.

[0094] Virus-free seedlings were transferred to a simulated allelopathic stress-induced seedling culture medium (MS basal medium supplemented with 50 mg / L ferulic acid + 30 mg / L p-hydroxybenzoic acid) and continuously subcultured for two generations. The temperature during this process was 25 ± 1℃, the time was 30 days, the photoperiod was 12 h / d, and the light intensity was 2200 lx.

[0095] Subsequently, growth indicators and physiological activity standards were determined to screen superior stress-tolerant strains with well-developed root systems and high antioxidant enzyme activity for large-scale propagation. Growth indicators were defined as follows: plant height ≥ 3.2 cm, total root length ≥ 40 cm, single plant fresh weight ≥ 0.9 g, well-developed white fibrous roots, and dark green stems and leaves without yellowing or browning. Physiological activity standards were defined as: SOD activity ≥ 340 U / g·h, POD activity ≥ 470 U / g·min, and CAT activity ≥ 88 U / g·min. Only plants meeting all growth indicators and with all three antioxidant enzyme activities meeting the threshold were identified as superior chemically tolerant strains with well-developed root systems and strong resistance, and were then propagated on a large scale.

[0096] Step 6: Virus-free seedling hardening-off and field transplanting Afterwards, in a greenhouse environment at 25℃, the test-tube seedlings were hardened off for 3 days with the bottle opening half open and for 4 days with the bottle opening fully open, gradually adapting to the outdoor temperature, humidity, and light environment.

[0097] Remove the seedlings and gently clean the roots to remove any remaining solid culture medium. Transplant them into a seedling substrate of peat moss: perlite = 3:1 (by volume). Cultivate them in a greenhouse at 25℃ for 30 days until the plants reach a height of 8-10cm and have well-developed root systems.

[0098] It was directly planted in the Huaiyushan Three-Leaf Clematis planting area that had been continuously cropped for many years, and conventional water and fertilizer field management was carried out.

[0099] Comparative Example 1: Traditional single-stage vitrification cryogenic detoxification breeding group The shoot tips were treated with conventional single vitrification cryopreservation without gradient low temperature pre-acclimatization, the other three cryopreservation processes, or in vitro allelopathic stress directional screening. The other culture media, culture conditions, hardening and transplanting methods were completely consistent with those in Example 10.

[0100] Experimental results: The virus removal rate was 67.3%, the shoot tip regeneration rate was 21.3%, the survival rate of field seedlings was 62.7%, and the tuber yield was 39.62 g / plant. Compared with the 46.99 g / plant of the chemically resistant virus-free seedlings of this invention, the tuber yield increase was only 18.6%.

[0101] Comparative Example 2: Conventional Cutting Propagation Group Healthy branches of *Trifolium repens* from Huaiyu Mountain were directly propagated by field cuttings without tissue culture, ultra-low temperature virus removal, or stress acclimatization treatment.

[0102] Experimental results: The seedling infection rate was 54.7%, the transplant survival rate in continuously cropped plots was 45.3%, the field required frequent application of soil conditioners and stress regulators, the planting cost was the highest, and the tuber yield was the lowest, at 30.45g / plant.

[0103] Example 11: Simultaneous use of three cryogenic modes for breeding The shoot tips obtained in Example 2 after gradient low temperature pre-acclimatization were divided into three groups. They were treated independently by embedding dehydration and ultra-low temperature detoxification, conventional vitrification and ultra-low temperature detoxification, and droplet vitrification and ultra-low temperature detoxification. After obtaining virus-free strains by RT-PCR virus detection, the regenerated seedlings in each group were mixed and propagated. The subsequent steps such as chemical stress screening, seedling hardening and transplanting were the same as in Example 10.

[0104] Application scenarios: Preservation of germplasm resources of *Trifolium repens* in Huaiyu Mountain, breeding of multi-resistant germplasm; can enrich the genetic background of seedlings to resist stress.

[0105] The method described in Comparative Example 1 was used as a control. A survey was conducted 60 days after transplanting seedlings into severely continuously cropped fields. Each treatment was replicated three times, with 60 plants per replicate. The percentage of surviving plants with healthy stems and leaves and newly formed roots was counted, and the transplant survival rate was calculated. The results showed that, compared with the control, the seedling survival rate could be further increased to 93.2% in extremely continuously cropped fields.

[0106] Example 12: Combined ultra-low temperature detoxification with rhizosphere growth-promoting bacteria synergistic treatment Following Example 10, multi-mode ultra-low temperature detoxification and stress-resistant virus-free seedling cultivation were completed. Before seedling transplanting, a compound inoculum of Bacillus subtilis and Trichoderma harzianum was used (the effective viable count ratio of Bacillus subtilis and Trichoderma harzianum was 1:1; the total effective viable count was 10). 8 After soaking the roots in a solution of CFU / mL for 15 minutes, the plants were transplanted.

[0107] Application scenario: Fields with severe continuous cropping for more than 5 years.

[0108] Using the multi-mode ultra-low temperature detoxification and stress-resistant virus-free seedling cultivation completed according to Example 10, seedlings detoxified by soaking roots in clean water before transplanting served as a control. Rhizosphere soil was collected after 12 months of continuous field cultivation. The total content of ferulic acid and p-hydroxybenzoic acid was determined by high-performance liquid chromatography (HPLC), and the improvement in phenolic acid degradation was calculated. At 120 days after transplanting, during the peak period of root diseases, all plants were dug up, and the percentage of plants with root brown rot was counted, calculating the reduction in disease incidence. Results showed that compared with the control, the degradation efficiency of soil phenolic acid allelochemicals increased by 38.1%, and the incidence of root diseases decreased by 52.4%.

[0109] Example 13: Pre-treatment of mother plants with low temperature and combined with virus-free propagation In autumn, healthy mother plants of *Trifolium repens* from Huaiyu Mountain were subjected to a 7-day gradient low-temperature acclimatization (4-15℃, 2 days of culture at 15℃ in the dark → 2 days of culture at 10℃ in the dark → 3 days of culture at 4℃ in the dark). Then, tender stem segments with 1-2 axillary buds from the semi-lignified zone of the current year were collected as explants. Subsequent steps, such as explant disinfection, primary culture, subculture, sterilization of the stem tips of sterile test-tube seedlings obtained by sterile culture using four ultra-low temperature methods, resistance screening, and transplanting, were the same as in Example 10.

[0110] Application scenario: Domestication and breeding of wild, authentic Trifoliate Orange germplasm.

[0111] Virus-free seedlings that had not undergone field pretreatment of the mother plant (the only difference being the absence of field pretreatment of the mother plant) served as a control. After three consecutive generations of subculturing, the comprehensive stress resistance index of antioxidant enzymes was measured. Simultaneously, multiple genotypes were tracked in the field for 24 consecutive months, and the improvement in trait stability and the proportion of plants with degraded stress resistance were calculated. Results showed that compared to the control, the genetic stability of chemical stress tolerance traits in the seedlings increased by 29.7%, and the rate of degradation of stress resistance traits significantly decreased after multiple generations of planting.

[0112] The embodiments described above are merely preferred methods of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A seedling propagation method for mitigating the stress damage caused by the combined use of multiple ultra-low temperature detoxification methods in Huaiyu Mountain, characterized in that, Including Method 1 and Method 2; The first method includes the following steps: Using axillary buds and tender stem segments of *Trifolium repens* from Huaiyu Mountain as explants, gradient sterilization was performed, followed by primary and secondary culture to obtain sterile test-tube seedlings. The shoot tip meristem of the sterile test-tube seedlings was pre-cultured and subjected to gradient low-temperature pre-acclimatization pretreatment to obtain shoot tips after gradient low-temperature pre-acclimatization. The shoot tips that have undergone gradient low-temperature pre-acclimatization are subjected to ultra-low temperature detoxification to obtain detoxified shoot tips; The virus-free shoot tips were subjected to rewarming, gradient washing, and regeneration culture to obtain regenerated seedlings; The regenerated seedlings were screened and subjected to chemical stress-resistant seedling cultivation to obtain virus-free seedlings; The virus-free seedlings were hardened off and transplanted. The second method includes the following steps: Gradient low-temperature pre-acclimatization pretreatment was carried out on *Trifolium repens* from Huaiyu Mountain to obtain pretreated *Trifolium repens* from Huaiyu Mountain. Using the tender stems of the pretreated Huaiyu Mountain Trifoliate Orchid as explants, gradient sterilization was performed, followed by primary and subculture to obtain sterile test-tube seedlings. The stem tips of the sterile test-tube seedlings were subjected to ultra-low temperature detoxification to obtain detoxified stem tips; The virus-free shoot tips were subjected to rewarming, gradient washing, and regeneration culture to obtain regenerated seedlings; The regenerated seedlings were screened and subjected to chemical stress-resistant seedling cultivation to obtain virus-free seedlings; The virus-free seedlings were hardened off and transplanted.

2. The seedling propagation method according to claim 1, characterized in that, The cryogenic detoxification in Method 1 includes embedding dehydration cryogenic detoxification, embedding vitrification cryogenic detoxification, vitrification cryogenic detoxification, and droplet vitrification cryogenic detoxification. The cryogenic detoxification in Method 2 includes encapsulation dehydration cryogenic detoxification, conventional vitrification cryogenic detoxification, or droplet vitrification cryogenic detoxification.

3. The seedling propagation method according to claim 2, characterized in that, The encapsulation dehydration and cryogenic detoxification process includes wrapping the stem tip with a 3% sodium alginate solution and a 0.5 mol / L calcium chloride solution, followed by drying and liquid nitrogen freezing. The embedded vitrification cryogenic detoxification includes the steps of impregnating the stem tip with PVS2 and freezing it with liquid nitrogen. The vitrification cryogenic detoxification includes the steps of impregnating the stem tips treated with loading solution with PVS2 and freezing them with liquid nitrogen. The droplet vitrification cryogenic detoxification process includes immersing the stem tip in a loading solution, then wrapping the stem tip with PVS2 and freezing it with liquid nitrogen. The conventional vitrification cryogenic detoxification process includes the steps of immersing the stem tip in a loading solution, followed by dehydration with PVS2 and then freezing with liquid nitrogen. The loading solution comprises MS + 2M glycerol + 0.4M sucrose; the PVS2 comprises 30% glycerol by mass, 15% dimethyl sulfoxide by volume, 15% ethylene glycol by volume and 0.4 mol / L sucrose.

4. The seedling propagation method according to claim 1, characterized in that, The gradient low-temperature pre-acclimatization pretreatment includes the following steps: incubation at 15℃ in the dark for 2 days, followed by incubation at 10℃ in the dark for 2 days, and finally incubation at 4℃ for 3 days.

5. The seedling propagation method according to claim 1, characterized in that, The gradient washing includes the steps of sequentially rinsing with MS + 0.5 mol / L sucrose, MS + 0.3 mol / L sucrose, and MS + 0.1 mol / L sucrose.

6. The seedling propagation method according to claim 1, characterized in that, The culture medium used for the initial culture consisted of MS + 0.8 mg / L 6-BA + 0.2 mg / L NAA; the temperature for the initial culture was 25 ± 1 °C, the light intensity was 2200 lx, the culture time was 30 days, and the photoperiod was 12 h / d. The culture medium used for the subculture consisted of MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar; the culture temperature for the subculture was 25 ± 1℃, the light intensity was 2200 lx, the culture time was 30 days, and the photoperiod was 12 h / d. The pre-culture medium used was MS + 0.4 mol / L sucrose; the pre-culture method was dark culture; the dark culture temperature was 25℃ and the time was 24 h. The regeneration culture medium used includes MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar; the regeneration culture includes dark culture and light culture; the dark culture temperature is 25℃ and the time is 7 days; the light culture time is 30 days, the temperature is 25±1℃, and the light duration is 12h / day; The culture medium used for cultivating the chemically resistant seedlings was MS + 50 mg / L ferulic acid + 30 mg / L p-hydroxybenzoic acid; the temperature for cultivating the chemically resistant seedlings was 25 ± 1℃, the time was 30 days, the photoperiod was 12 h / d, and the light intensity was 2200 lx.

7. The seedling propagation method according to claim 1, characterized in that, The gradient disinfection includes the steps of sequentially disinfecting with phosphate buffer, 0.3wt% tea tree oil emulsion solution, and 0.2wt% mercuric chloride solution.

8. The seedling propagation method according to claim 1, characterized in that, The seedling hardening and domestication process also includes a step of root soaking treatment using a compound microbial agent; the compound microbial agent includes Bacillus subtilis and Trichoderma harzianum.

9. The seedling propagation method according to claim 1, characterized in that, The rewarming process includes a step of rewarming the detoxified stem tip at 38-40°C for 90 seconds.

10. The application of the seedling propagation method according to any one of claims 1-9 in any of the following: (1) Reduce the explant contamination rate during the tissue culture of *Trifolium repens* from Huaiyu Mountain; (2) Improve the axillary bud germination rate during the tissue culture of *Trifolium repens* from Huaiyu Mountain; (3) Reduce the coefficient of variation of uniformity of growth of test-tube seedlings in the tissue culture process of *Trifolium repens* from Huaiyu Mountain; (4) Increase the antioxidant enzyme activity and osmotic regulator content of *Trifolium repens* from Huaiyu Mountain; (5) Improve the survival rate of *Tripterygium wilfordii* under allelopathic stress conditions; (6) Improve the overall virus removal rate of *Trifolium repens* from Huaiyu Mountain; (7) Reduce the mortality rate of stem tip browning during tissue culture of *Trifolium repens* from Huaiyu Mountain; (8) Shorten the average regeneration cycle of stem tips of *Trifolium repens* from Huaiyu Mountain; (9) Improve the non-toxicity rate of *Trifolium repens* seedlings from Huaiyu Mountain that were infected with the virus; (10) Improve the survival rate of infected seedlings of *Trifolium repens* from Huaiyu Mountain; (11) Improve the survival rate of transplanted *Trifolium repens* from Huaiyu Mountain; (12) Increase the yield of *Trifolium repens* tubers from Huaiyu Mountain; (13) Preservation of local germplasm resources of *Trifolium repens* in Huaiyu Mountain, selection and breeding of multi-resistant germplasm, and medium- and long-term ex-situ preservation and propagation of germplasm; (14) Alleviating the replanting obstacle of *Trifolium repens* in Huaiyu Mountain; (15) Domestication of wild Huaiyu Mountain Trifolium repens native germplasm and artificial domestication and breeding of endangered authentic Chinese medicinal materials native resources.