Sterile rapid propagation method of elaeagnus pungens stem tip meristem

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a sterile rapid propagation method of elaeagnus pungens stem tip meristem. The method comprises the following steps: immersing a stem section in a treating agent, and then washing with sterile water; inoculating the stem tip meristem into a primary induction culture medium which does not contain an exogenous antibacterial agent, and culturing to obtain sterile cluster buds; the method comprises the following steps: selecting robust rootless seedlings, and soaking the base parts of the seedlings in a solution containing IBA for pretreatment; then, the seedlings are inoculated into a rooting culture medium without exogenous auxin to be cultured, and complete regenerated plants are obtained; and hardening seedlings and transplanting. According to the invention, the long-acting silver ion / nano-silver composite protective layer is constructed, and the functional synergistic application of the protective layer in the whole process of subsequent primary culture, proliferation and seedling strengthening and rooting induction is realized; technical contradictions between efficient sterilization and explant activity maintenance, long-term sterile culture and endophyte pollution control, and high-quality root system induction and culture link disjunction in a traditional rapid propagation technology are synergistically solved.

Description

Technical Field This invention relates to the field of plant tissue culture technology, and more specifically, to a method for the aseptic and rapid propagation of Elaeagnus pungens shoot tip meristem. Background Technology Elaeagnus pungens root is the dried root of the Elaeagnus pungens plant (family Elaeagnaceae). It has important medicinal and ecological value, and its large-scale propagation is a key foundation for ensuring the sustainable use of medicinal resources and meeting the needs of ecological restoration. Achieving efficient, stable, and sterile propagation of high-quality seedlings is of great significance for maintaining germplasm purity, improving planting efficiency, and protecting wild resources. Current aseptic rapid propagation techniques for woody plants such as Elaeagnus pungens, especially micropropagation techniques using shoot tip meristems as explants, while pursuing efficient cloning and virus-free seedling production, generally face prominent contradictions such as high contamination rates, difficulty in inhibiting browning, and instability of the regeneration system. Taking conventional shoot tip culture or axillary bud induction as examples, although genetically consistent plants can be obtained, the following technical bottlenecks exist in actual operation: Sterilization of explant surfaces is difficult, and endophytic bacterial contamination is hard to completely remove, resulting in a high failure rate in primary culture. Meristem and surrounding tissues are highly susceptible to oxidation and browning after cutting. Conventional antioxidant treatments have limited effectiveness, which seriously affects cell viability and subsequent germination. During the propagation stage, slow growth, low bud formation rate, or vitrification often occur, resulting in a low effective seedling coefficient. In addition, the rooting stage often relies on high concentrations of exogenous hormones, which can easily cause abnormal root morphology, poor transplant adaptability, and has a long cycle and high cost, making it difficult to meet the stringent requirements of uniformity and survival rate for factory seedling production. Summary of the Invention The purpose of this invention is to address the problems existing in the aseptic rapid propagation technology of Elaeagnus pungens stem tip meristem, such as severe contamination and browning in the primary culture, low efficiency and unstable quality in the proliferation and rooting stages, and poor synergy among various stages, which makes it difficult to meet the requirements of large-scale production. The purpose of this invention is to provide a method for the aseptic and rapid propagation of Elaeagnus pungens shoot apical meristems. By constructing a long-lasting silver ion / nano-silver composite protective layer in situ on the surface of the explant during the pretreatment stage, this protective layer is functionally and synergistically applied throughout the subsequent primary culture, propagation and seedling strengthening, and root induction processes. This method synergistically solves the technical contradictions in traditional rapid propagation techniques between efficient sterilization and explant viability maintenance, long-term aseptic culture and endophytic bacteria contamination control, and the disconnect between high-quality root induction and the culture process. To achieve the above objectives, the present invention aims to provide a method for the aseptic and rapid propagation of Elaeagnus pungens stem apical meristem, comprising the following steps: Step S1: Take stem segments with axillary buds, wash them, immerse them in a silver nitrate-sodium hypochlorite-antioxidant composite treatment agent and shake them, then rinse them with sterile water; During the treatment process, a nanoscale silver ion / nano-silver composite protective layer is formed in situ on the surface of the explant, and then the shoot tip meristem is peeled off; Step S2: The shoot tip meristem is inoculated into a primary induction medium without exogenous antibacterial agents and cultured. The silver protective layer continuously releases silver ions to provide an antibacterial environment, thereby obtaining sterile clustered shoots. The clustered shoots were transferred into a sequence of proliferation culture medium and seedling strengthening culture medium for propagation and robust cultivation; Step S3: Select robust rootless seedlings and pretreat their bases by soaking them in a solution containing IBA. The synergistic effect of trace silver ions from the silver protective layer and IBA induces the formation of root primordia. The seedlings were then inoculated into a rooting medium without exogenous auxin and cultured to obtain complete regenerated plants; Step S4: Hardening off and transplanting. As a further improvement to this technical solution, in step S1, the composition of the silver nitrate-sodium hypochlorite-antioxidant composite treatment agent is as follows: 0.1% (w / v) silver nitrate, 1.0% (v / v) sodium hypochlorite, 0.05% (v / v) Tween-80, 100 mg / L ascorbic acid, 50 mg / L polyvinylpyrrolidone, in sterile deionized water. As a further improvement to this technical solution, in step S1, the rotation speed of the oscillation process is 80-100 rpm, and the processing time is 12-15 min; The sterile water rinsing is performed 3 times. As a further improvement to this technical solution, in step S1, the length of the shoot apical meristem is 0.5 to 1.0 mm, and it carries 1 to 2 leaf primordia. As a further improvement to this technical solution, in step S2, the primary induction culture medium is: 1 / 2 MS + 0.5 mg / L 6-BA + 0.05 mg / L NAA + 30 g / L sucrose + 3.5 g / L plant gel, pH 5.8; The culture conditions were as follows: first, dark culture for 7 days, then culture for 5 to 6 weeks under a light intensity of 1500–2000 lux and a photoperiod of 12 h / d. As a further improvement to this technical solution, in step S2, The proliferation medium was: MS + 1.5 mg / L 6-BA + 0.1 mg / L NAA, cultured for 25 days; The seedling culture medium was: MS + 0.5 mg / L 6-BA + 0.2 mg / L GA3, cultured for 20 days. As a further improvement to this technical solution, in step S3, the concentration of IBA in the IBA-containing solution is 1.0 mg / L, and the soaking pretreatment time is 1 hour. As a further improvement to this technical solution, in step S3, the rooting culture medium is: 1 / 2 MS + 0.5 g / L activated carbon + 20 g / L sucrose + 3.0 g / L plant gel, pH 5.8. As a further improvement to this technical solution, in step S3, the culture time for inducing rooting is 3 to 4 weeks. As a further improvement to this technical solution, in step S4, the transplanting substrate is a mixture of sterilized leaf mold, vermiculite and perlite in a volume ratio of 4:3:3. After transplanting, the substrate is first acclimatized in an environment with a humidity of 85-95%, and then the humidity is gradually reduced over 7-10 days to adapt to the external environment. Compared with the prior art, the beneficial effects of the present invention are as follows: In this method for the aseptic and rapid propagation of Elaeagnus pungens stem apical meristems, the chemical residues that need to be thoroughly removed in traditional sterilization processes are transformed into a stable, attached nanoscale silver ion / nano-silver composite protective layer through an in-situ reaction between the treatment agent and the explant surface. This protective layer continuously and slowly releases extremely low concentrations of silver ions throughout the subsequent culture cycle, serving as an internal antibacterial source during the initial and proliferation stages, replacing the exogenous antibacterial agents added to the conventional culture medium, and creating a long-lasting and clean microenvironment for the culture. During the rooting induction stage, the trace amounts of silver ions released from the same protective layer act as inhibitors of ethylene action, generating a synergistic signal with the externally pulsed auxin (IBA), optimizing the initiation and development of root primordia, thereby inducing a more robust root system in the rooting medium without the need for exogenous auxin. In summary, this invention, through the construction of a silver protective layer and its cross-stage functional synergistic application, systematically integrates and deeply couples key aspects such as explant pretreatment, establishment of a sterile system, rapid proliferation, and high-quality rooting. This not only ensures the efficient establishment of sterile and rapid clonal proliferation of Elaeagnus pungens shoot apical meristems, but also simultaneously overcomes common industry problems in traditional technologies, such as difficulty in balancing sterilization and activity, high subculture contamination rates, unstable rooting quality, and dependence on exogenous hormones. This provides a stable, efficient, and highly operable new solution for the large-scale, standardized rapid propagation of woody plants through tissue culture. Attached Figure Description Figure 1 This is a schematic diagram of the process of the present invention; Figure 2 This is a schematic diagram showing the final effective seedling count of a single explant in each group of experimental examples of the present invention. Detailed Implementation The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Please see Figure 1 As shown, the purpose of this invention is to provide a method for the aseptic and rapid propagation of Elaeagnus pungens stem apical meristem, comprising the following steps: Step S1: Cut healthy, semi-lignified shoots from the current year's growth, collected in spring, into stem segments 1.5–2.0 cm long with 2–3 axillary buds. First, pre-treat and clean the stem segments: rinse them under running sterile water for 2 hours to remove most surface contaminants. Then proceed to the core sterilization process: in a clean bench, completely immerse the stem segments in a "silver nitrate-sodium hypochlorite-antioxidant composite treatment agent" and place them on a shaker at 80–100 rpm for 12–15 minutes. The treatment agent consists of 0.1% (w / v) silver nitrate (AgNO3) as a long-acting antibacterial component, 1.0% (v / v) sodium hypochlorite (NaClO) as an immediate broad-spectrum bactericide, 0.05% (v / v) Tween-80 as a surfactant, and is compounded with 100 mg / L ascorbic acid (Vc) and 50 mg / L polyvinylpyrrolidone (PVP) as an antioxidant stabilizing system. The solvent is sterile deionized water. In this step, sodium hypochlorite rapidly kills surface microorganisms; ascorbic acid and PVP immediately neutralize oxygen free radicals, preventing oxidative browning of the explant cut surface. In the alkaline oxidizing environment, some silver ions are reduced and interact with organic components on the plant tissue surface to form a nanoscale silver ion / nano-silver complex layer, stably attached to the epidermis and superficial intercellular spaces. Subsequently, the tissue is rinsed three times with sterile deionized water to remove residual strong oxidants and free silver ions, while deliberately retaining this firmly bound silver protective layer to provide continuous protection for subsequent culture. Under a stereomicroscope, 0.5–1.0 mm shoot apical meristems with 1–2 leaf primordia were aseptically excised as initial explants. Step S2: Inoculate the shoot tip meristem into the primary induction medium. Culture medium formulation: 1 / 2 MS basal salt + 0.5 mg / L 6-BA + 0.05 mg / L NAA + 30 g / L sucrose + 3.5 g / L plant gel, pH adjusted to 5.8. Synergistic mechanism of cultivation process: No exogenous antibacterial agents are added to the culture medium. The silver protective layer constructed in step S1 continuously releases extremely low concentrations of silver ions during this stage, forming a durable antibacterial microenvironment around the culture, effectively inhibiting the resurgence of endophytic bacteria and secondary contamination caused by handling, allowing the explants to start growth under highly clean conditions. The cultivation procedure is as follows: first, dark culture for 7 days, followed by culture in a light-controlled culture chamber (temperature 25±1℃, light intensity 1500-2000 lux, photoperiod 12 h / d) for 5-6 weeks to obtain sterile clustered shoots. Then, proliferation and seedling strengthening culture were carried out: the clustered buds were divided into single buds or small clusters and transferred into the proliferation / seedling strengthening culture medium sequence. Proliferation phase: The buds were cultured in MS medium containing 1.5 mg / L 6-BA and 0.1 mg / L NAA for 25 days to achieve rapid bud expansion. Seedling strengthening stage: Transfer to MS medium containing 0.5 mg / L 6-BA and 0.2 mg / L GA3, and culture for 20 days to promote shoot elongation and robustness. Throughout this process, the continuous effect of the "silver protective layer" significantly reduced the contamination rate during subculturing and improved biomass accumulation efficiency. Step S3: Select robust rootless seedlings with a height > 2.5cm for rooting induction. The base of the seedlings was immersed in a sterile aqueous solution containing 1.0 mg / L IBA for 1 hour. During this process, trace amounts of silver ions from the protective layer produced a synergistic physiological effect with auxin (IBA). As an effective inhibitor of ethylene, silver ions can locally block the synthesis and signal transduction of ethylene at the rooting site, thereby relieving the inhibition of root elongation by ethylene and allowing auxin signals to more concentratedly induce the synchronized initiation and development of root primordia. The pretreated seedlings were inoculated into a rooting medium that did not contain any exogenous auxin. Culture medium formulation: 1 / 2 MS + 0.5 g / L activated carbon + 20 g / L sucrose + 3.0 g / L plant gel, pH 5.8. In this formulation, activated carbon is used to adsorb trace amounts of harmful substances such as phenols generated during the cultivation process. At this point, the induction of root primordia is synergistically achieved by the silver protective layer and IBA pulses. The hormone-free culture medium environment promotes the autonomous development of the root system dependent on endogenous hormones, which is conducive to the formation of a robust root system with abundant root hairs and complete functions. After 3–4 weeks of cultivation, complete regenerated plants with well-developed root systems and dense root hairs are obtained. Step S4: Use the standard hardening-off procedure. Wash off the culture medium adhering to the plant roots and transplant into a sterilized mixed substrate (leaf mold:vermiculite:perlite = 4:3:3, v / v / v). After transplanting, place in an acclimatization environment with a humidity of 85-95%. Then, gradually reduce the humidity and increase ventilation over 7-10 days to allow the plant to gradually adapt to the external natural environmental conditions and complete the transition from heterotrophic to autotrophic. This invention systematically integrates three traditionally independent key stages—explant pretreatment, aseptic culture system construction, and rooting induction—to create a closed-loop rapid propagation process centered on the sustained-release delivery of functional components. The key lies in the innovative application of a silver nitrate-sodium hypochlorite-antioxidant composite treatment agent during the pretreatment stage. This agent, while achieving efficient surface sterilization, allows its core component (silver ions) to form a long-lasting, sustained-release protective layer on the plant tissue surface in a safe and controllable manner. This design cleverly transforms sterilizing agent residues, traditionally considered the end point of the process and requiring complete removal, into a functional carrier that serves the entire subsequent culture cycle. In terms of functional synergy, this process establishes a network for the switching and synergistic interaction of the protective layer across different stages of cultivation. During the initial and propagation stages, the protective layer continuously releases trace amounts of silver ions, acting as a built-in antibacterial agent to effectively inhibit endophytic resurgence and external microbial contamination, significantly improving the stability of the aseptic system. During the root induction stage, the trace amounts of silver ions in the same protective layer act as biochemical regulators, inhibiting the physiological activity of ethylene and forming a spatiotemporal synergy with externally pulsed auxin (IBA) to jointly optimize the initiation signal of root primordia, thereby inducing more robust root systems while simplifying the culture medium formulation. This mechanism transforms the system from a passive mode dependent on external additives at each stage to an active, synergistic mode driven by the built-in functional layer. In summary, this invention deeply couples pretreatment, proliferation culture, and rooting induction units through the construction of a long-lasting silver ion protective layer and its cross-stage functional applications. This integrated approach not only ensures the efficient and aseptic establishment and rapid proliferation of Elaeagnus pungens shoot apical meristems, but also simultaneously overcomes the industry pain points of traditional rapid propagation techniques, such as the contradiction between pollution control and growth induction, the disconnect between various stages, and unstable rooting quality. It provides a stable, efficient, and highly operable new technical path for the large-scale, factory-style seedling production of woody plants. The following specific embodiments will further illustrate the method for aseptic rapid propagation of Elaeagnus pungens stem apical meristems provided by the present invention. Example 1 Step S1: Cut healthy, semi-lignified shoots from the current year's growth, collected in spring, into 1.5cm long stem segments with 3 axillary buds. First, perform pretreatment cleaning: rinse under running sterile water for 2 hours to remove most surface contaminants. Then, in a laminar flow hood, completely immerse the stem segments in the treatment agent and place them on a shaker at 80 rpm for 15 minutes. The treatment agent consists of 0.1% (w / v) silver nitrate (AgNO3) as a long-acting antibacterial component, 1.0% (v / v) sodium hypochlorite (NaClO) as an immediate broad-spectrum bactericide, 0.05% (v / v) Tween-80 as a surfactant, and is compounded with 100 mg / L ascorbic acid (Vc) and 50 mg / L polyvinylpyrrolidone (PVP) as an antioxidant stabilizing system. The solvent is sterile deionized water. Afterward, rinse three times with sterile deionized water using a shaking motion. Under a stereomicroscope, 0.5 mm shoot tip meristems with two leaf primordia were aseptically excised as initial explants. Step S2: Inoculate the shoot tip meristem into the primary induction medium. Culture medium formulation: 1 / 2 MS basal salt + 0.5 mg / L 6-BA + 0.05 mg / L NAA + 30 g / L sucrose + 3.5 g / L plant gel, pH adjusted to 5.8. The cultivation procedure was to first culture in the dark for 7 days, and then transfer to a light culture room (temperature 25±1℃, light intensity 1500-2000 lux, photoperiod 12 h / d) for 5 weeks to obtain sterile clustered buds. Then, proliferation and seedling strengthening culture were carried out: the clustered buds were divided into single buds or small clusters and transferred into the proliferation / seedling strengthening culture medium sequence. Proliferation phase: The buds were cultured in MS medium containing 1.5 mg / L 6-BA and 0.1 mg / L NAA for 25 days to achieve rapid bud expansion. Seedling strengthening stage: Transfer to MS medium with 0.5 mg / L 6-BA and 0.2 mg / L GA3 and culture for 20 days to promote bud elongation and robustness. Step S3: Select robust rootless seedlings with a height > 2.5cm for rooting induction. Immerse the base of the seedling in a sterile aqueous solution containing 1.0 mg / L IBA for 1 hour. The pretreated seedlings were inoculated into a rooting medium that did not contain any exogenous auxin. Culture medium formulation: 1 / 2 MS + 0.5 g / L activated carbon + 20 g / L sucrose + 3.0 g / L plant gel, pH 5.8. After 4 weeks of cultivation, a complete regenerated plant with a well-developed root system and dense root hairs was obtained. Step S4: Wash off the culture medium adhering to the plant roots and transplant into a sterilized mixed substrate (leaf mold:vermiculite:perlite = 4:3:3, v / v / v). After transplanting, place in an acclimatization environment with 85% humidity. Then, gradually reduce the humidity and increase ventilation over 10 days to allow the plant to gradually adapt to the external natural environmental conditions and complete the transition from heterotrophic to autotrophic. Example 2 Step S1: Cut healthy, semi-lignified shoots from the current year's growth, collected in spring, into stem segments 1.8 cm long with two axillary buds. First, pre-treat and clean the stem segments: rinse them under running sterile water for 2 hours to remove most surface contaminants. Then, in a clean bench, completely immerse the stem segments in the treatment agent and place them on a shaker at 90 rpm for 12 minutes. The treatment agent consists of 0.1% (w / v) silver nitrate (AgNO3) as a long-acting antibacterial component, 1.0% (v / v) sodium hypochlorite (NaClO) as an immediate broad-spectrum bactericide, 0.05% (v / v) Tween-80 as a surfactant, and is compounded with 100 mg / L ascorbic acid (Vc) and 50 mg / L polyvinylpyrrolidone (PVP) as an antioxidant stabilizing system. The solvent is sterile deionized water. Afterward, rinse three times with sterile deionized water using a shaking motion. Under a stereomicroscope, 0.8 mm shoot tip meristems with one leaf primordium were aseptically excised as initial explants. Step S2: Inoculate the shoot tip meristem into the primary induction medium. Culture medium formulation: 1 / 2 MS basal salt + 0.5 mg / L 6-BA + 0.05 mg / L NAA + 30 g / L sucrose + 3.5 g / L plant gel, pH adjusted to 5.8. The cultivation procedure was to first culture in the dark for 7 days, and then transfer to a light culture room (temperature 25±1℃, light intensity 1500-2000 lux, photoperiod 12 h / d) for 5 weeks to obtain sterile clustered buds. Then, proliferation and seedling strengthening culture were carried out: the clustered buds were divided into single buds or small clusters and transferred into the proliferation / seedling strengthening culture medium sequence. Proliferation phase: The buds were cultured in MS medium containing 1.5 mg / L 6-BA and 0.1 mg / L NAA for 25 days to achieve rapid bud expansion. Seedling strengthening stage: Transfer to MS medium with 0.5 mg / L 6-BA and 0.2 mg / L GA3 and culture for 20 days to promote bud elongation and robustness. Step S3: Select robust rootless seedlings with a height > 2.5cm for rooting induction. Immerse the base of the seedling in a sterile aqueous solution containing 1.0 mg / L IBA for 1 hour. The pretreated seedlings were inoculated into a rooting medium that did not contain any exogenous auxin. Culture medium formulation: 1 / 2 MS + 0.5 g / L activated carbon + 20 g / L sucrose + 3.0 g / L plant gel, pH 5.8. After 4 weeks of cultivation, a complete regenerated plant with a well-developed root system and dense root hairs was obtained. Step S4: Wash off the culture medium adhering to the plant roots and transplant into a sterilized mixed substrate (leaf mold:vermiculite:perlite = 4:3:3, v / v / v). After transplanting, place in an acclimatization environment with 90% humidity. Then, gradually reduce the humidity and increase ventilation over 9 days to allow the plant to gradually adapt to the external natural environmental conditions and complete the transition from heterotrophic to autotrophic. Example 3 Step S1: Cut healthy, semi-lignified shoots from the current year's growth, collected in spring, into stem segments 2.0 cm long with two axillary buds. First, pre-treat and clean the stem segments: rinse them under running sterile water for 2 hours to remove most surface contaminants. Then, in a clean bench, completely immerse the stem segments in the treatment agent and place them on a shaker at 100 rpm for 12 minutes. The treatment agent consists of 0.1% (w / v) silver nitrate (AgNO3) as a long-acting antibacterial component, 1.0% (v / v) sodium hypochlorite (NaClO) as an immediate broad-spectrum bactericide, 0.05% (v / v) Tween-80 as a surfactant, and is compounded with 100 mg / L ascorbic acid (Vc) and 50 mg / L polyvinylpyrrolidone (PVP) as an antioxidant stabilizing system. The solvent is sterile deionized water. Afterward, rinse three times with sterile deionized water using a shaking motion. Under a stereomicroscope, 1.0 mm shoot tip meristems with one leaf primordium were aseptically excised as initial explants. Step S2: Inoculate the shoot tip meristem into the primary induction medium. Culture medium formulation: 1 / 2 MS basal salt + 0.5 mg / L 6-BA + 0.05 mg / L NAA + 30 g / L sucrose + 3.5 g / L plant gel, pH adjusted to 5.8. The cultivation procedure was to first culture in the dark for 7 days, and then transfer to a light culture room (temperature 25±1℃, light intensity 1500-2000 lux, photoperiod 12 h / d) for 6 weeks to obtain sterile clustered buds. Then, proliferation and seedling strengthening culture were carried out: the clustered buds were divided into single buds or small clusters and transferred into the proliferation / seedling strengthening culture medium sequence. Proliferation phase: The buds were cultured in MS medium containing 1.5 mg / L 6-BA and 0.1 mg / L NAA for 25 days to achieve rapid bud expansion. Seedling strengthening stage: Transfer to MS medium with 0.5 mg / L 6-BA and 0.2 mg / L GA3 and culture for 20 days to promote bud elongation and robustness. Step S3: Select robust rootless seedlings with a height > 2.5cm for rooting induction. Immerse the base of the seedling in a sterile aqueous solution containing 1.0 mg / L IBA for 1 hour. The pretreated seedlings were inoculated into a rooting medium that did not contain any exogenous auxin. Culture medium formulation: 1 / 2 MS + 0.5 g / L activated carbon + 20 g / L sucrose + 3.0 g / L plant gel, pH 5.8. After 3 weeks of cultivation, a complete regenerated plant with a well-developed root system and dense root hairs was obtained. Step S4: Wash away the culture medium adhering to the plant roots and transplant into a sterilized mixed substrate (leaf mold:vermiculite:perlite = 4:3:3, v / v / v). After transplanting, place in an acclimatization environment with 95% humidity. Then, gradually reduce the humidity and increase ventilation over 7 days to allow the plant to gradually adapt to the external natural environmental conditions and complete the transition from heterotrophic to autotrophic. After running the aseptic rapid propagation method of Elaeagnus pungens stem apical meristem according to Examples 1-3, the core efficacy and key performance indicators of the method were tested and evaluated as follows: 1. Establishment of aseptic system and testing of contamination control effectiveness Testing items: Primary contamination rate of explants (%), cumulative contamination rate of subsequent generations. Detection and statistical methods: 100 explants were treated in each example and inoculated into the primary culture medium. The contamination caused by bacteria or fungi was observed and recorded daily, and the contamination rate was calculated after 2 weeks. For explants that successfully started growth, they were followed up to the 4th week. The obtained shoot clusters were subcultured three times consecutively (100 shoots were inoculated each time), and the cumulative contamination rate during the entire subculture cycle was calculated. Control group: A control group using the traditional treatment method was set up, which was soaked in 2% (v / v) sodium hypochlorite solution (containing 0.05% Tween-80) for 15 minutes, and the subsequent steps were the same. 2. Evaluation of growth and proliferation efficiency Test items: induction rate of clustered shoots (%), proliferation coefficient (times). Detection and statistical methods: The proportion of explants that can form effective shoot clusters (≥3 shoots) after 8 weeks of inoculation is calculated as the shoot cluster induction rate. Thirty single shoots or shoot clusters (initial biomass denoted as M0) are randomly selected from the shoot clusters of each embodiment and inoculated into the proliferation medium. After 25 days of culture, the fresh weight of the harvested shoots (M1) is measured, and the proliferation coefficient (M1 / M0) is calculated. Outcome metrics: Obtain quantitative data on the growth and propagation stages. 3. Rooting Induction Quality Assessment Test items: Rooting rate (%), average number of roots (roots / plant), average root length (cm), transplant survival rate (%). Detection and statistical methods: For each example, 100 healthy, rootless seedlings meeting the standards were selected for rooting induction. After 4 weeks of cultivation, the proportion of plants with at least one adventitious root longer than 0.5 cm at the base was counted, which was the rooting rate. The number of adventitious roots and the length of the longest root were randomly measured in 30 rooted plants, and the average value was calculated. After the fully regenerated plants were hardened off and transplanted, the proportion of plants still surviving in the 4th week was counted, which was the transplant survival rate. Control setting: A rooting method control group was set up, omitting the silver protective layer construction step (i.e., using the traditional sterilization method) and IBA pretreatment, and the rootless seedlings were directly inoculated into 1 / 2MS rooting medium containing 0.5 mg / L IBA. Results indicators: Quantitative data were recorded at the rooting and transplanting stages to evaluate the impact of the "silver protective layer" combined with IBA pretreatment on rooting quality and subsequent acclimatization adaptability. The test results are shown in Table 1. Table 1. Detection results of Examples 1-3 Testing items Example 1 Example 2 Example 3 Traditional method control group Primary contamination rate of explants (%) 3 2 4 24 Subgenerational cumulative pollution rate (%) 2 1 2 / Cluster bud induction rate (%) 92 94 90 / Proliferation coefficient (times) 8.5 9.0 8.0 / Rooting rate (%) 98 96 95 85 Average number of roots per plant 6.2 5.8 5.5 3.8 Average root length (cm) 4.5 4.2 4.0 2.9 Transplant survival rate (%) 94 92 90 82 As shown in Table 1, the three embodiments using the method of this invention all achieved excellent aseptic control with a primary contamination rate of less than 5% and a cumulative contamination rate of less than 3% in subsequent generations, proving that the silver protective layer provides a continuously effective antibacterial environment. In terms of growth and proliferation, the induction rate of clustered shoots remained stable at over 90%, and the proliferation coefficient reached 8.0-9.0 times, demonstrating highly efficient and stable propagation capabilities.

[0001] In the crucial rooting stage, the method of this invention induced a rooting rate of over 95%, with root systems significantly superior in both quantity and quality to the control group directly induced by traditional hormone-based culture media. This ultimately achieved a transplant survival rate exceeding 90%. This fully validates the core invention of constructing a long-lasting silver protective layer through pretreatment, which not only fundamentally improves the stability of the aseptic system but also significantly enhances propagation efficiency and seedling quality through cross-stage functional synergy.

[0002] Test case To verify that the construction of the silver protective layer and its synergistic application in the two stages of antibacterial and rooting induction are key to achieving efficient aseptic establishment, stable proliferation and high-quality rooting in the aseptic rapid propagation method of Elaeagnus pungens provided by this invention, this experimental example, based on the complete method parameters of Example 2, systematically changed or omitted specific core steps and designed the following three sets of comparative experiments to intuitively and quantitatively demonstrate the unexpected superiority of the overall scheme of this invention and its core technical features. Control group C1: Traditional two-step sterilization method (no silver protective layer construction, conventional antibacterial agent added to the culture medium) Protocol: The pretreatment and cleaning steps for explants were the same. Sterilization was changed to a conventional two-step method: first, soaking in 75% (v / v) ethanol for 30 seconds, followed by rinsing once with sterile water; then, soaking in 2% (v / v) sodium hypochlorite solution (containing 0.05% Tween-80) for 15 minutes, and finally rinsing three times with sterile water. In subsequent steps, 50 mg / L gentamicin was added as an exogenous antibacterial agent to the primary induction medium, proliferation medium, and seedling strengthening medium. Rooting induction was performed using the traditional method: rootless seedlings were directly inoculated into 1 / 2 MS rooting medium (containing 0.5 g / L activated charcoal) supplemented with 0.5 mg / L IBA. Objective: To serve as a baseline control closest to existing technologies, and to compare and verify the fundamental advantages of the strategy of constructing an internal protective layer to replace exogenous addition in terms of pollution control, explant activity, and rooting quality. Control group C2: Silver-treated but without subsequent synergistic design (the protective layer only serves an antibacterial function). The procedure was exactly the same as in Example 2: explant pretreatment and silver protective layer construction. However, the synergistic design of the protective layer and IBA was omitted during the rooting induction stage: that is, IBA solution soaking pretreatment was not performed, and the rootless seedlings were directly inoculated into the same 1 / 2 MS rooting medium as the control group C1, which was supplemented with 0.5 mg / L IBA. Objective: To isolate and verify whether the specific physiological regulatory function of the silver protective layer during the rooting stage (through inhibition of ethylene-synergistic IBA) is necessary for obtaining high-quality roots. This control aims to demonstrate that even with the same antibacterial protective layer, the optimal rooting effect of this invention cannot be achieved without utilizing its synergistic signaling effect during the rooting stage. Control group C3: Simplified composite treatment agent (lacking components, unable to form an effective protective layer) Protocol: The explant pretreatment and cleaning steps were the same. A simplified treatment agent was used for sterilization: 1.0% (v / v) sodium hypochlorite + 0.05% (v / v) Tween-80 + 100 mg / L ascorbic acid, with sterile deionized water as the solvent (i.e., silver nitrate and PVP were omitted). The treatment time and rinsing steps were the same as in Example 2. All subsequent culture steps (including IBA pretreatment for root induction) were strictly performed according to Example 2. Objective: To verify whether a specific combination of silver nitrate and PVP components in the composite treatment agent is indispensable for the successful construction of a silver protective layer. It is anticipated that the absence of the core components will lead to the inability to form a stable and long-lasting protective layer, resulting in performance degradation in both contamination control and rooting synergy, thus demonstrating the non-obviousness of the specific formulation of this invention. The propagation process of the control groups C1, C2, and C3 was tested on the same scale as in Example 2 (100 explants each), covering a complete culture cycle, and evaluated using the same detection and statistical methods as in Examples 1-3. Key propagation efficiency and quality data are compared in Table 2. Figure 2 As shown. Table 2 Comparison of reproductive efficiency and quality between Example 2 and control groups C1, C2, and C3 Testing items Example 2 Control group C1 Control group C2 Control group C3 Primary contamination rate (8) 2 24 3 18 Subgenerational cumulative pollution rate (%) 1 15 2 12 Cluster bud induction rate (%) 94 65 92 70 Proliferation coefficient (times) 9.0 5.5 8.8 6.0 Rooting rate (%) 96 85 88 82 Average number of roots per plant 5.8 3.8 4.0 3.5 Average root length (cm) 4.2 2.9 3.1 2.7 Transplant survival rate (%) 92 82 84 80 According to Table 2 and Figure 2 As shown: 1. The fundamental advantages of silver protective layer as a substitute for exogenous antibacterial agents (Example 2 vs. Control Group C1) Results: The primary contamination rate of the control group C1 (traditional method) was as high as 24%, and the contamination accumulation in subsequent generations was serious (15%). The induction rate of clustered shoots and the proliferation coefficient were significantly lower, and the final number of seedlings from a single explant was only about 28% of that of the present invention.

[0003] Mechanism Analysis: Traditional two-step sterilization methods cause significant damage to explants and fail to address endophytic bacteria issues. Added exogenous antibiotics (gentamicin) may have toxic side effects on plant cells and easily induce antibiotic resistance in strains, leading to uncontrolled contamination later. This invention, through a continuous, slow-release physicochemical antibacterial mechanism provided by a silver protective layer, establishes a milder and more durable sterile microenvironment at the source, significantly improving explant survival rate and subsequent proliferation activity. This comparison strongly demonstrates that the strategy of transforming sterilization residues into an internal functional layer is fundamentally superior to the traditional passive approach of strong sterilization + exogenous addition.

[0004] 2. The key role of the silver-IBA synergistic mechanism in the rooting stage (Example 2 vs. control group C2) Results: Control group C2 was similar to Example 2 in terms of pollution control and proliferation efficiency, but its rooting rate (88% vs 96%), number of roots (4.0 vs 5.8), and root length (3.1 cm vs 4.2 cm) all decreased significantly, resulting in a reduction in transplant survival rate and final number of seedlings.

[0005] Mechanism Analysis: The control group C2 possessed the same silver protective layer, but it only functioned as an antibacterial component during the rooting stage and did not synergize with IBA pretreatment. During rooting, ethylene induced by exogenous IBA inhibits root elongation. This invention, through the local inhibition of ethylene by the slow-release of trace amounts of Ag⁺ from the protective layer, relieves its inhibitory effect on rooting, maximizes the efficacy of auxin signaling, and thus induces more numerous and robust roots. This comparison demonstrates that the function of the silver protective layer is not solely antibacterial; its role as an ethylene inhibitor during the rooting stage, combined with the spatiotemporal synergy of external IBA signaling, is the core mechanism for achieving the unexpected effect of high-quality rooting.

[0006] 3. The Indispensability of Specific Formulations for Composite Treatment Agents (Example 2 vs. Control Group C3) Results: The indicators of the control group C3 (simplified treatment agent) were between those of the traditional method (C1) and the present invention (Example 2), but were significantly inferior to those of Example 2, especially in terms of pollution control (18%) and rooting quality.

[0007] Mechanism analysis: The absence of silver nitrate prevents the provision of the source material for forming the silver protective layer; the absence of PVP results in an incomplete antioxidant system, potentially affecting the stable reduction and binding of silver ions. The results of control group C3 indicate that sodium hypochlorite and ascorbic acid alone cannot achieve the construction and stable adhesion of a long-lasting protective layer. This demonstrates that the specific formulation combination of 0.1% AgNO3 + 1.0% NaClO + 0.05% Tween-80 + 100 mg / L Vc + 50 mg / L PVP, through the interaction of its components in an alkaline oscillating environment, is a necessary but not obvious condition for achieving the novel application of in-situ formation of a stable nanoscale silver complex layer.

[0008] The comparative experiments of the above systems fully demonstrate that the silver nitrate-sodium hypochlorite-antioxidant composite treatment agent constructed in this invention and the resulting formation of a silver protective layer, together with the subsequent culture without exogenous antibacterial agents and the silver-IBA synergistic rooting induction, constitute a deeply coupled and functionally progressive organic whole. The absence or replacement of any core component will lead to significant and quantifiable degradation of the propagation system in terms of aseptic stability, proliferation efficiency, or rooting quality. This strongly demonstrates the non-obviousness of this invention, and the synergistic and unexpected technical effects it produces in simultaneously solving long-standing technical contradictions such as difficulty in pollution control, low proliferation efficiency, and poor rooting quality in the rapid propagation of woody plants through its unique built-in functional layer construction and cross-stage synergistic design.

[0009] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for aseptic and rapid propagation of Elaeagnus pungens stem tip meristem, characterized in that, Includes the following steps: Step S1: Take stem segments with axillary buds, wash them, immerse them in a silver nitrate-sodium hypochlorite-antioxidant composite treatment agent and shake them, then rinse them with sterile water; During the treatment process, a nanoscale silver ion / nano-silver composite protective layer is formed in situ on the surface of the explant, and then the shoot tip meristem is peeled off; Step S2: The shoot tip meristem is inoculated into a primary induction medium without exogenous antibacterial agents and cultured. The silver protective layer continuously releases silver ions to provide an antibacterial environment, thereby obtaining sterile clustered shoots. The clustered shoots were transferred into a sequence of proliferation culture medium and seedling strengthening culture medium for propagation and robust cultivation; Step S3: Select robust rootless seedlings and pretreat their bases by soaking them in a solution containing IBA. The synergistic effect of trace silver ions from the silver protective layer and IBA induces the formation of root primordia. The seedlings were then inoculated into a rooting medium without exogenous auxin and cultured to obtain complete regenerated plants; Step S4: Hardening off and transplanting.

2. The method for aseptic rapid propagation of Elaeagnus pungens stem apical meristem according to claim 1, characterized in that: In step S1, the composition of the silver nitrate-sodium hypochlorite-antioxidant composite treatment agent is as follows: 0.1% (w / v) silver nitrate, 1.0% (v / v) sodium hypochlorite, 0.05% (v / v) Tween-80, 100 mg / L ascorbic acid, 50 mg / L polyvinylpyrrolidone, in sterile deionized water.

3. The method for aseptic rapid propagation of Elaeagnus pungens stem apical meristem according to claim 1, characterized in that: In step S1, the oscillation speed is 80-100 rpm and the processing time is 12-15 min. The sterile water rinsing is performed 3 times.

4. The method for aseptic rapid propagation of Elaeagnus pungens stem apical meristem according to claim 1, characterized in that: In step S1, the length of the shoot apical meristem is 0.5–1.0 mm, and it carries 1–2 leaf primordia.

5. The method for aseptic rapid propagation of Elaeagnus pungens stem apical meristem according to claim 1, characterized in that: In step S2, the primary induction culture medium is: 1 / 2 MS + 0.5 mg / L 6-BA + 0.05 mg / L NAA + 30 g / L sucrose + 3.5 g / L plant gel, pH 5.8; The culture conditions were as follows: first, dark culture for 7 days, then culture for 5 to 6 weeks under a light intensity of 1500–2000 lux and a photoperiod of 12 h / d.

6. The method for aseptic rapid propagation of Elaeagnus pungens stem apical meristem according to claim 1, characterized in that: In step S2 The proliferation medium was: MS + 1.5 mg / L 6-BA + 0.1 mg / L NAA, cultured for 25 days; The seedling culture medium was: MS + 0.5 mg / L 6-BA + 0.2 mg / L GA3, cultured for 20 days.

7. The method for aseptic rapid propagation of Elaeagnus pungens stem apical meristem according to claim 1, characterized in that: In step S3, the concentration of IBA in the IBA-containing solution is 1.0 mg / L, and the soaking pretreatment time is 1 hour.

8. The method for aseptic rapid propagation of Elaeagnus pungens stem apical meristem according to claim 1, characterized in that: In step S3, the rooting culture medium is: 1 / 2 MS + 0.5 g / L activated carbon + 20 g / L sucrose + 3.0 g / L plant gel, pH 5.

8.

9. The method for aseptic rapid propagation of Elaeagnus pungens stem apical meristem according to claim 1, characterized in that: In step S3, the culture time for induced rooting is 3 to 4 weeks.

10. The method for aseptic rapid propagation of Elaeagnus pungens stem apical meristem according to claim 1, characterized in that: In step S4, the transplanting substrate is a mixture of sterilized leaf mold, vermiculite and perlite in a volume ratio of 4:3:

3. After transplanting, the plants are first acclimatized in an environment with a humidity of 85-95%, and then the humidity is gradually reduced over 7-10 days to adapt to the external environment.

Images