A callus rooting medium for explants of Lysimachia christinae

Abstract

This invention discloses a callus rooting culture medium for explants of *Lysimachia christinae*, belonging to the field of plant tissue culture. To address the technical problems of low medicinal content and low transplant survival rate in *Lysimachia christinae* tissue culture seedlings, the technical solution involves innovatively adding a composite stress-regulated slow-release system composed of salicylic acid-loaded sodium alginate-chitosan composite hydrogel microspheres and L-proline to 1 / 2 MS medium. This system induces mild stress through the slow release of salicylic acid, simultaneously improving the chemical quality and physiological resistance of tissue culture seedlings, significantly increasing total flavonoid content and transplant survival rate.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a callus rooting culture medium for explants of Lysimachia christinae. Background Technology

[0002] *Lysimachia christinae*, also known as pearlwort, is a perennial herb belonging to the genus *Lysimachia* in the family Primulaceae. The whole plant or root can be used medicinally, possessing properties of clearing heat and detoxifying, and promoting diuresis and reducing swelling. It enjoys stable demand in the Chinese medicinal materials market. Furthermore, its unique whorled inflorescence also gives it ornamental potential. To meet market demand and protect wild resources, large-scale rapid propagation using plant tissue culture technology has become the main approach.

[0003] In existing technologies, tissue culture of *Lysimachia christinae* typically uses MS or 1 / 2 MS as the basal medium, supplemented with common plant growth regulators such as NAA, IBA, and 6-BA. The core objective of these techniques is to achieve a high proliferation coefficient and high rooting rate, i.e., to obtain the maximum number of tissue culture seedlings per unit time. However, this "quantity over quality" propagation strategy leads to two serious technical defects.

[0004] First, the chemical quality is low. In a highly eugenic and stress-free culture environment, the secondary metabolic pathways of tissue culture seedlings are often inhibited. Although tissue culture seedlings of Lysimachia christinae cultivated using conventional methods appear robust, the content of their core medicinal active ingredients, such as total flavonoids and saponins, is far lower than that of wild plants, resulting in a significant reduction in their medicinal value and economic benefits.

[0005] Secondly, the physiological quality is fragile. There is another often overlooked but objectively existing problem in current technology: the "illusion of high survival rate" in tissue culture seedlings. While tissue culture seedlings grow well in sterile, temperature- and humidity-controlled culture bottles, their physiological structure and function are incompletely developed. They have thin cuticles, malfunctioning stomatal opening and closing, and weak antioxidant defense systems. Once transplanted to complex natural environments, these "greenhouse flowers" are highly susceptible to environmental stress (such as water stress and pathogen infection), leading to mass mortality—a condition known as "post-transplant syndrome"—resulting in a low actual survival rate and limiting the overall efficiency of factory-scale seedling production.

[0006] Therefore, how to overcome the existing technological bottlenecks and develop a new culture medium that can not only ensure the efficient rooting and good growth of *Lysimachia christinae* tissue culture seedlings, but also simultaneously improve their chemical quality (pharmaceutical active ingredients) and physiological quality (environmental adaptability and stress resistance) has become a key technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] This invention aims to address the dual problems of "high yield but low quality" (low content of medicinal components) and "physiological fragility" (low transplant survival rate) in existing Lysimachia christinae tissue culture techniques. Simultaneously, this invention also addresses the technical shortcomings of existing stress culture methods, such as the potential for cytotoxicity and short-lived, uncontrollable stress effects caused by the single high-concentration addition of exogenous inducers.

[0008] To achieve the above objectives, this invention provides a callus rooting medium for improving the quality and stress resistance of *Lysimachia christinae* tissue culture seedlings. The core of this medium is a "composite stress-regulated slow-release system," which introduces pH-responsive smart hydrogel microspheres loaded with salicylic acid into the basal medium, working synergistically with an osmotic regulator in the liquid phase.

[0009] The objective of this invention can be achieved through the following technical solutions:

[0010] A callus rooting culture medium for explants of Lysimachia christinae, prepared per 1000 mL of medium, consists of the following components:

[0011] (1) Basic culture medium: 1 / 2 MS medium;

[0012] (2) Carbon source: 15-25 g of sucrose;

[0013] (3) Plant growth regulators: indolebutyric acid 0.6–1.0 mg, naphthaleneacetic acid 0.1–0.3 mg;

[0014] (4) Combined stress-regulated sustained-release system, including:

[0015] A. Stress signal sustained-release carrier: 1.0–2.0 g of sodium alginate-chitosan composite hydrogel microspheres loaded with salicylic acid;

[0016] B. Liquid phase osmotic stress regulator: L-proline 20–40 mg;

[0017] (5) Curing agent: 6-8 g of agar;

[0018] (6) The remainder is deionized water, and the pH value is adjusted to 5.8-6.0.

[0019] Furthermore, a callus rooting medium for explants of *Lysimachia christinae* is prepared by the following components per 1000 mL of medium: 1 / 2 MS medium, 20 g sucrose, 0.8 mg indolebutyric acid, 0.2 mg naphthaleneacetic acid, 1.5 g sodium alginate-chitosan composite hydrogel microspheres loaded with salicylic acid, 30 mg L-proline, 7 g agar, with the remainder being deionized water, and a pH of 5.8.

[0020] Furthermore, the sodium alginate-chitosan composite hydrogel microspheres loaded with salicylic acid are prepared by the following steps:

[0021] (1) Preparation of internal phase solution: Weigh sodium alginate and dissolve it in deionized water, and stir magnetically until completely dissolved; then add salicylic acid and continue stirring until dissolved to obtain SA-sodium alginate mixed solution;

[0022] (2) Preparation of external phase solution: Weigh calcium chloride and water-soluble chitosan, dissolve them in acetic acid solution with pH value of 4.5-5.0 and volume fraction of 0.2%, stir evenly to obtain calcium ion-chitosan crosslinking curing solution;

[0023] (3) Microsphere forming and coating: Using an injection pump, the SA-sodium alginate mixed solution from step (1) is added at a flow rate of 15-25 mL / h through a 21G needle to the external phase solution in step (2) which is being magnetically stirred. After the droplets come into contact with the external phase, they quickly crosslink to form calcium alginate gel microspheres and are coated with a layer of chitosan on the surface.

[0024] (4) Curing and purification: Continue the reaction under stirring for 30 to 50 minutes. After the reaction is complete, collect the microspheres by filtration with a Buchner funnel and wash them repeatedly with deionized water 3 to 5 times to obtain clean microspheres.

[0025] (5) Finished product: After pre-freezing the washed microspheres at -80℃ for 12 hours, they are dried in a freeze dryer for 48 hours to obtain sodium alginate-chitosan composite hydrogel microspheres loaded with salicylic acid. They are then sealed for later use.

[0026] Furthermore, the ratio of sodium alginate, deionized water, salicylic acid, calcium chloride, water-soluble chitosan, and acetic acid solution is 1.5–2.5 g: 80–120 mL: 0.3–0.5 g: 0.8–1.2 g: 0.4–0.6 g: 180–220 mL.

[0027] Furthermore, the ratio of sodium alginate, deionized water, salicylic acid, calcium chloride, water-soluble chitosan, and acetic acid solution is 2.0g:100mL:0.4g:1.0g:0.5g:200mL.

[0028] Furthermore, the water-soluble chitosan described in step (2) has a molecular weight of 300 kDa and a degree of deacetylation of 92%.

[0029] Furthermore, the rotation speed of the magnetic stirring in step (3) is 200-300 rpm.

[0030] The beneficial effects of this invention are:

[0031] The core innovation of this invention lies in the first introduction of pH-responsive smart hydrogel controlled-release technology into the field of plant tissue culture, constructing a precise control system of "intelligent slow-release stress-active adaptive protection", realizing a paradigm shift from traditional "mixed addition" to "programmed induction".

[0032] (1) Cross-disciplinary integration of technologies has created an unpredictable "intelligent stress" system: During the growth and metabolism of plant roots, organic acids are secreted into the culture medium, leading to a local decrease in the pH value of the rhizosphere microenvironment. This invention utilizes this physiological phenomenon to design sodium alginate-chitosan composite hydrogel microspheres loaded with salicylic acid. The chitosan (positively charged) and sodium alginate (negatively charged) of these microspheres form a stable network through electrostatic interaction. When the rhizosphere pH decreases, the carboxyl groups of sodium alginate are protonated, the electrostatic interaction weakens, and the network structure becomes looser, thereby "intelligently" accelerating the release of its loaded salicylic acid. This "on-demand release" mechanism enables dynamic coupling between the supply of stress signals and the plant's growth status.

[0033] (2) This invention resolves the inherent contradictions of stress culture, achieving a "highly efficient and low-toxicity" induction effect: In traditional methods, the direct addition of inducing agents such as salicylic acid results in high initial concentrations, which can easily harm young tissue culture seedlings and inhibit growth; while the concentration decreases rapidly in the later stages due to degradation or absorption, leading to a short-lived induction effect. The slow-release system of this invention maintains the salicylic acid concentration within an effective "induction window" that is below the toxicity threshold, achieving continuous, gentle, and long-lasting activation of secondary metabolic pathways. This not only significantly increases the accumulation of total flavonoids and other active substances but also avoids inhibition of plant growth; in fact, due to the good root system development, the overall biomass is even higher.

[0034] (3) Dual Benefits of Root System Configuration Optimization and Disease Resistance "Physical Barrier": Optimized Root System Configuration: Continuous and mild stress signals are more effective in inducing adaptive morphogenesis in plants compared to severe stress shocks. Tissue culture seedlings cultivated using this invention not only have a large number and long roots, but also significantly increased density of lateral roots and root hairs, forming a more developed absorption network. This robust root system configuration is the key physiological basis for their rapid adaptation to the new environment and extremely high survival rate after transplanting; Constructing a Dual Defense Line for Disease Resistance: While the hydrogel microspheres slowly release salicylic acid (chemical defense line), their main component—chitosan—is itself a broad-spectrum antibacterial substance and plant immune inducer. The microspheres exist stably in the culture medium and can form a chitosan-rich microenvironment around the plant roots, which is equivalent to constructing a "physical barrier" that inhibits the growth of miscellaneous bacteria, significantly reducing the incidence of root rot in seedling hardening and the early stages of transplanting. Achieving a dual protective effect of "chemical induction + physical barrier".

[0035] (4) Significant comprehensive benefits, combining high medicinal value and high agricultural production efficiency: The tissue culture seedlings cultivated by this invention have a total flavonoid content that is more than 150% higher than that of conventional seedlings, resulting in a significant improvement in medicinal quality. At the same time, thanks to the optimized root system and pre-activated stress resistance system, the transplant survival rate is stable at over 98%, and the seedlings exhibit vigorous growth in the field and strong disease resistance in the later stages, greatly enhancing the overall economic benefits of large-scale production. Detailed Implementation

[0036] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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. Furthermore, unless otherwise specified, the raw materials, reagents, or devices used in the following embodiments can be obtained from conventional commercial channels or by existing known methods.

[0037] Example 1

[0038] Preparation of sodium alginate-chitosan (SA-CS) composite hydrogel microspheres loaded with salicylic acid (SA):

[0039] (1) Weigh 2.0 g of sodium alginate (food grade, viscosity ≥200 mPa·s, purchased from Sinopharm Chemical Reagent Co., Ltd.), slowly add it to 100 mL of deionized water, and stir magnetically in a 60 °C water bath for 2 hours until completely dissolved. After cooling to room temperature, add 0.4 g of salicylic acid (analytical grade, purchased from Tianjin Kemei Chemical Reagent Co., Ltd.), and continue stirring for 30 minutes until completely dissolved to obtain a mixed solution of SA-sodium alginate.

[0040] (2) Weigh 1.0 g of calcium chloride (analytical grade) and 0.5 g of water-soluble chitosan (purchased from Shanghai Yuanye Biotechnology Co., Ltd., molecular weight 300 kDa, degree of deacetylation 92%), dissolve them in 200 mL of deionized water, adjust the pH value to 4.8 with 0.1 mol / L HCl, stir to dissolve, and obtain calcium ion-chitosan crosslinking curing solution.

[0041] (3) Draw the SA-sodium alginate mixture from step (1) into a 50mL syringe, fix it on the syringe pump (LSP01-1A type, Baoding Lange Constant Flow Pump Co., Ltd.), and attach a 21G stainless steel needle. Place the needle tip about 5cm above the surface of the calcium ion-chitosan crosslinking curing solution from step (2), set the flow rate to 20mL / h, and start the syringe pump. At the same time, magnetically stir the crosslinking curing solution at a speed of 250 rpm.

[0042] (4) After the addition is complete, continue stirring for 40 minutes. Then stop stirring, let stand for 5 minutes, and collect the generated milky white microspheres by filtration using a Buchner funnel and a circulating water vacuum pump.

[0043] (5) Wash the collected microspheres three times with 500 mL of deionized water, stirring for 5 minutes each time, and then filter to obtain the washed microspheres.

[0044] (6) The washed microspheres were spread out in a freeze-drying tray and pre-frozen in a -80℃ freezer for 12 hours. Then they were transferred to a freeze dryer (-50℃, <10 Pa) and dried for 48 hours to obtain about 2.6g of white, loose sodium alginate-chitosan composite hydrogel microspheres loaded with salicylic acid. After grinding, they were ready for use.

[0045] Example 2

[0046] Preparation and culture of a callus rooting medium for explants of Lysimachia christinae:

[0047] (1) Explant preparation and callus induction: Healthy, disease-free young stems of *Lysimachia christinae* were selected, rinsed with running water for 30 minutes, disinfected by soaking in 75% (v / v) alcohol for 30 seconds on a clean bench, then surface sterilized with 0.1% (w / v) mercuric chloride solution for 7 minutes, and finally rinsed repeatedly with sterile deionized water 5 times. After blotting the surface moisture with sterile filter paper, the stems were cut into segments with a length of about 1.2 cm. The segments were inoculated onto callus induction medium (MS + 30 g / L sucrose + 1.5 mg / L 6-benzyladenine + 0.5 mg / L 2,4-dichlorophenoxyacetic acid + 7 g / L agar, pH 5.8) and cultured at (25±1)℃ in the dark for 7 days. Then, the cells were transferred to a light intensity of 2000 Lux and a light / dark time of 14h / 10h for further culture. After about 30 days, pale yellow, loosely textured callus tissue was obtained.

[0048] (2) Preparation of callus rooting and quality improvement culture medium: Accurately weigh 2.215 g of 1 / 2 MS medium powder (purchased from Qingdao Haibo Biotechnology Co., Ltd.) and 20 g of sucrose (analytical grade), and dissolve them in approximately 800 mL of deionized water. Add the stock solution of 0.8 mg indolebutyric acid (IBA) and 0.2 mg naphthaleneacetic acid (NAA), and 30 mg L-proline (purchased from Sinopharm Group, purity ≥99%) sequentially. After stirring and dissolving, add 1.5 g of salicylic acid-loaded sodium alginate-chitosan composite hydrogel microspheres prepared in Example 1, and stir thoroughly to disperse them. Adjust the volume to 1000 mL with deionized water. Adjust the pH to 5.8 using 1 mol / L NaOH or HCl. Finally, add 7 g of agar (biological reagent grade) and heat and stir until completely dissolved. While the prepared culture medium is still hot, dispense 50 mL into 150 mL Erlenmeyer flasks. Seal each flask with sealing film and autoclave at 121°C for 20 minutes. Let cool before use.

[0049] (3) Inoculation culture: Transfer well-grown callus pieces (about 0.5g) to the above culture medium, inoculate 3 pieces per bottle, and place them in a culture room (25±1℃, light 2000 Lux, 14h / 10h) for 35 days.

[0050] (4) Hardening off and transplanting: Open the bottle cap and harden off the seedlings in the original culture room for 4-5 days. Then take out the seedlings, carefully wash off the culture medium from the roots with clean water, and transplant them into a mixed substrate of perlite:vermiculite:peat moss = 1:1:1 (v / v / v) that has been sterilized by high pressure steam. Place them in a greenhouse with a relative humidity of 85% and water them as usual.

[0051] Comparative Example 1

[0052] Comparative Example 1 served as the control group for Example 2. Based on the formulation of Example 2, the "compound stress-regulated sustained-release system" was completely removed, meaning that "sodium alginate-chitosan composite hydrogel microspheres loaded with salicylic acid prepared in Example 1" and "L-proline" were not added. All other components (1 / 2 MS, 20g sucrose, 0.8mg IBA, 0.2mg NAA, 7g agar) and culture conditions were exactly the same as in Example 2.

[0053] Comparative Example 2

[0054] Comparative Example 2 served as the control group for Example 2. Based on the formulation of Comparative Example 1, an equal amount of salicylic acid was added to the sodium alginate-chitosan composite hydrogel microspheres loaded with salicylic acid prepared in Example 1 (1.5g in Example 2). In this comparative example, 230mg of salicylic acid was directly added to 1000mL of culture medium. Other components and culture conditions were identical to those in Example 2.

[0055] Comparative Example 3

[0056] Comparative Example 3 served as the control group for Example 2. Based on the formulation of Example 2, only L-proline was removed, retaining the "sodium alginate-chitosan composite hydrogel microspheres loaded with salicylic acid prepared in Example 1". All other components and culture conditions were identical to those in Example 2.

[0057] Comparative Example 4

[0058] Comparative Example 4 served as the control group for Example 2. Based on the formulation of Example 2, the "sodium alginate-chitosan composite hydrogel microspheres loaded with salicylic acid prepared in Example 1" were replaced with an equal amount of blank sodium alginate-chitosan composite hydrogel microspheres without salicylic acid (the preparation process was exactly the same except that salicylic acid was not added). All other components and culture conditions were exactly the same as in Example 2.

[0059] Comparative Example 5

[0060] Comparative Example 5 served as the control group for Example 2. Based on the formulation of Example 2, the "sodium alginate-chitosan composite hydrogel microspheres loaded with salicylic acid prepared in Example 1" were removed, but L-proline was retained. All other components and culture conditions were identical to those in Example 2.

[0061] Test Example 1

[0062] The tissue culture seedlings obtained after 35 days of culture in Example 2 and Comparative Examples 1-5 were subjected to performance testing. The performance testing process is as follows, and the test results are shown in Table 1:

[0063] 1. Test metrics:

[0064] (1) Growth and rooting indicators: rooting rate (%), average number of roots (roots / plant), and fresh weight of plant (g / plant).

[0065] (2) Chemical quality indicators: total flavonoid content (mg / g dry weight).

[0066] (3) Physiological quality and stress resistance indicators: transplant survival rate (%), root rot incidence rate within 15 days after seedling hardening and transplanting (%).

[0067] 2. Specific testing process:

[0068] (1) Growth and rooting index tests:

[0069] After 35 days of cultivation, 30 morphologically intact tissue culture seedlings were randomly selected from each treatment group.

[0070] A. Rooting rate: Count the number of plants with adventitious roots (length > 2mm) out of 30 seedlings and calculate the percentage. Rooting rate (%) = (Number of rooted plants / 30) × 100%.

[0071] B. Average number of roots: Count the number of adventitious roots (length > 2mm) of each rooted seedling and calculate the average number of 30 seedlings.

[0072] C. Fresh weight of plants: Remove the plants from the culture medium, wash the agar off the roots with clean water, blot the surface moisture with filter paper, and immediately weigh the individual plants using an analytical balance (accurate to 0.001g). Calculate the average weight of 30 seedlings.

[0073] (2) Total flavonoid content test (UV-Vis spectrophotometry):

[0074] A. Sample preparation: A sufficient number of tissue culture seedlings with uniform growth were randomly selected from each treatment group, washed with clean water, dried in an oven at 60℃ to constant weight, crushed with a pulverizer and passed through a 60-mesh sieve for later use.

[0075] B. Extraction: Accurately weigh 0.5g of dry powder from each group of samples, place it in a 50mL stoppered conical flask, add 25mL of 70% ethanol solution, and extract by sonication for 30 minutes (power 250W, frequency 40kHz). After cooling, filter and dilute the filtrate to a 25mL volumetric flask as the test solution.

[0076] C. Determination: The NaNO2-Al(NO3)3-NaOH colorimetric method was used. A standard curve was prepared using rutin as the standard. Accurately pipette 2.0 mL of the test solution, add 0.3 mL of 5% NaNO2 solution sequentially, shake well, and let stand for 6 minutes; add 0.3 mL of 10% Al(NO3)3 solution, shake well, and let stand for 6 minutes; add 4.0 mL of 4% NaOH solution, dilute with water to 10 mL, shake well, and let stand for 15 minutes. Measure the absorbance at 510 nm and calculate the total flavonoid content using the standard curve.

[0077] (3) Physiological quality and stress resistance index tests:

[0078] Sixty robust tissue culture seedlings were selected from each treatment group for hardening and transplanting (method as in Example 2).

[0079] A. Transplant survival rate: After transplanting, the plants were managed routinely in the greenhouse for 30 days, and the number of surviving plants was counted. The survival standard was: no wilting or yellowing of the plants, and the emergence of new leaves. Transplant survival rate (%) = (number of surviving plants / 60) × 100%.

[0080] B. Incidence of root rot: During the first 15 days after transplanting, observe the rootstock and substrate surface daily. Count the number of plants exhibiting typical root rot symptoms such as browning and rotting of the rootstock, and plant lodging. Incidence of root rot (%) = (Number of infected plants / 60) × 100%.

[0081] Table 1 Test Results

[0082] project Example 2 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Rooting rate (%) 96.7 93.3 43.3 93.3 96.7 93.3 Average number of roots per plant 12.5 8.2 2.1 10.8 8.5 8.3 Fresh weight of plant (g / plant) 2.15 1.86 0.85 1.92 1.90 1.88 Total flavonoid content (mg / g) 28.6 11.2 15.8 25.4 12.1 11.5 Transplant survival rate (%) 98.3 71.7 25.0 91.7 85.0 75.0 Incidence of root rot (%) 1.7 15.0 38.3 3.3 6.7 13.3

[0083] Analysis based on Table 1:

[0084] 1. Technical effect analysis of the "compound stress-regulated sustained-release system" (Example 2 vs. Comparative Example 1):

[0085] The technical solution of Comparative Example 1 is based on Example 2, but completely removes the "compound stress-regulated slow-release system" (i.e., microspheres without salicylic acid loading and L-proline), representing a conventional rooting culture medium. The data from Example 2 and Comparative Example 1 are directly compared and analyzed as follows:

[0086] (1) Significantly improved growth and rooting quality: Rooting rate: Example 2 (96.7%) was slightly higher than Comparative Example 1 (93.3%), indicating that the system of the present invention did not have a negative impact on basic rooting while ensuring a high rooting rate; Average number of roots: Example 2 had 12.5 roots / plant, compared to 8.2 roots / plant in Comparative Example 1, an increase of approximately 52.4%; Fresh weight of plants: Example 2 had 2.15 g / plant, compared to 1.86 g / plant in Comparative Example 1, an increase of approximately 15.6%. Conclusion: The data show that the composite system of the present invention not only did not inhibit plant growth, but also significantly promoted the number of adventitious roots and the accumulation of plant biomass, resulting in more robust tissue culture seedlings.

[0087] (2) Significantly improved chemical quality (medicinal value): Total flavonoid content: Example 2 showed a high content of 28.6 mg / g, while Comparative Example 1 showed only 11.2 mg / g. The present invention increases the total flavonoid content by approximately 155.4%. Conclusion: This demonstrates that the "compound stress-regulated sustained-release system" can effectively activate the secondary metabolic pathways of Lysimachia christinae, greatly increasing the content of its core medicinal active ingredients.

[0088] (3) Fundamental improvement in physiological quality and stress resistance: Transplant survival rate: Example 2 achieved an extremely high level of 98.3%, while Comparative Example 1 was only 71.7%; Root rot incidence rate: Example 2 was only 1.7%, far lower than the 15.0% of Comparative Example 1. Conclusion: The system of this invention, through pre-treatment of tissue culture seedlings with stress resistance, achieves a qualitative leap in their physiological quality, thereby effectively overcoming "post-transplant syndrome", significantly improving transplant survival rate and enhancing resistance to diseases.

[0089] 2. Analysis of salicylic acid (SA) sustained-release mechanism (Example 2 vs. Comparative Example 2):

[0090] The technical solution of Comparative Example 2 is to replace the "salicylic acid-loaded microspheres" in Example 2 with an equivalent total amount of salicylic acid directly added to the culture medium, in order to verify the "slow-release" system. The analysis is as follows:

[0091] (1) The toxic effects of high-concentration SA were avoided: All growth indicators of Comparative Example 2 showed a precipitous decline: the rooting rate was only 43.3%, the average number of roots per plant was only 2.1, and the fresh weight of the plant was only 0.85 g per plant. This indicates that the direct addition of high-concentration SA in one go had a severe growth-inhibiting and even toxic effect on the explants of Lysimachia christinae. In contrast, all growth indicators of Example 2 showed excellent performance, proving that the slow-release system could maintain the SA concentration within the "effective but non-toxic" window period, achieving mild induction.

[0092] (2) Achieved more efficient quality improvement: Although Comparative Example 2 was also subjected to SA stress, its total flavonoid content (15.8 mg / g) was higher than that of Comparative Example 1 (11.2 mg / g), but much lower than that of Example 2 (28.6 mg / g). This indicates that severe stress is actually detrimental to the efficient and continuous synthesis of secondary metabolites by plants. The slow-release method of Example 2 achieved continuous and long-term activation of the secondary metabolic pathway, thereby obtaining the highest total flavonoid accumulation.

[0093] (3) It ensured the physiological health of the plants: The transplant survival rate of Comparative Example 2 was only 25.0%, and the incidence of root rot was as high as 38.3%, which was the worst among all groups. This shows that the plants affected by high concentrations of SA poisoning are inherently very fragile and completely lack resistance. This is in stark contrast to Example 2 (survival rate of 98.3%, incidence of disease of 1.7%), which once again highlights the decisive role of the slow-release administration method in cultivating healthy and stress-resistant seedlings.

[0094] 3. Verification of the effect of the core component salicylic acid (SA) (Example 2 vs. Comparative Example 4):

[0095] The technical solution of Comparative Example 4 is to replace the "salicylic acid-loaded microspheres" in Example 2 with an equal amount of "SA-free blank microspheres". The purpose is to strip away and verify the role of SA itself in the composite system. The specific analysis is as follows:

[0096] (1) SA is the decisive factor in improving chemical quality: The total flavonoid content of Comparative Example 4 was 12.1 mg / g, which is basically at the same level as Comparative Example 1 (11.2 mg / g) and Comparative Example 5 (11.5 mg / g), but significantly different from the 28.6 mg / g of Example 2. Conclusion: This indicates that the significant increase in total flavonoid content is entirely induced by the slow release of SA from the microspheres, rather than by the effect of the microsphere carrier or other components.

[0097] (2) SA was the main contributor to improved physiological stress resistance: Although the transplant survival rate of Comparative Example 4 (85.0%) was better than that of Comparative Example 1 (71.7%), it was significantly lower than that of Example 2 (98.3%). This indicates that the physiological adaptation training induced by SA was the main reason for the extremely high survival rate. Similarly, although the incidence of root rot in Comparative Example 4 (6.7%) was lower than that in Comparative Example 1 (15.0%), it was much higher than that in Example 2 (1.7%).

[0098] 4. Synergistic effect analysis of L-proline (Example 2 vs. Comparative Example 3 vs. Comparative Example 5):

[0099] (1) The beneficial effect of L-proline under SA stress (Example 2 vs. Comparative Example 3):

[0100] Comparative Example 3 only removed L-proline, retaining the SA-loaded microspheres. Its performance in all indicators (root number 10.8, fresh weight 1.92, total flavonoids 25.4, survival rate 91.7%, disease incidence 3.3%) was superior to the conventional Comparative Example 1, but significantly inferior to Example 2 containing L-proline. Conclusion: Adding L-proline to the basis of SA-induced mild stress can further enhance plant growth, total flavonoid accumulation, and stress resistance. As an osmotic regulator and protectant, L-proline may help plants better adapt to and respond to SA-induced mild stress, thus achieving a synergistic effect of "1+1>2".

[0101] (2) Effects of L-proline alone (Comparative Example 5 vs. Comparative Example 1):

[0102] Comparative Example 5 only added L-proline and contained no microspheres. All its test indicators (root count 8.3, fresh weight 1.88g, total flavonoids 11.5g, survival rate 75.0%, disease incidence 13.3%) showed almost no significant differences compared to Comparative Example 1 (conventional culture medium). Conclusion: The data demonstrate that L-proline, under conventional culture conditions without SA stress, does not significantly affect the growth and quality of *Lysimachia christinae*.

[0103] It should be noted that, in this document, terms such as “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0104] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A rooting culture medium for callus formation from Lysimachia christinae, characterized in that, It is prepared from the following components per 1000 mL of culture medium: (1) Basic culture medium: 1 / 2 MS medium; (2) Carbon source: 15-25 g of sucrose; (3) Plant growth regulators: indolebutyric acid 0.6–1.0 mg, naphthaleneacetic acid 0.1–0.3 mg; (4) Combined stress-regulated sustained-release system, including: A. Stress signal sustained-release carrier: 1.0–2.0 g of sodium alginate-chitosan composite hydrogel microspheres loaded with salicylic acid; B. Liquid phase osmotic stress regulator: L-proline 20–40 mg; (5) Curing agent: 6-8 g of agar; (6) The remainder is deionized water, and the pH value is adjusted to 5.8-6.0; The salicylic acid-loaded sodium alginate-chitosan composite hydrogel microspheres were prepared by the following steps: (1) Preparation of internal phase solution: Weigh sodium alginate and dissolve it in deionized water, and stir magnetically until completely dissolved; then add salicylic acid and continue stirring until dissolved to obtain SA-sodium alginate mixed solution; (2) Preparation of external phase solution: Weigh calcium chloride and water-soluble chitosan, dissolve them in acetic acid solution with pH value of 4.5-5.0 and volume fraction of 0.2%, stir evenly to obtain calcium ion-chitosan crosslinking curing solution; (3) Microsphere forming and coating: Using an injection pump, the SA-sodium alginate mixed solution from step (1) is added at a flow rate of 15-25 mL / h through a 21G needle to the external phase solution in step (2) which is being magnetically stirred. After the droplets come into contact with the external phase, they quickly crosslink to form calcium alginate gel microspheres and are coated with a layer of chitosan on the surface. (4) Curing and purification: Continue the reaction under stirring for 30 to 50 minutes. After the reaction is complete, collect the microspheres by filtration with a Buchner funnel and wash them repeatedly with deionized water 3 to 5 times to obtain clean microspheres. (5) Finished product: After pre-freezing the washed microspheres at -80℃ for 12 hours, they are dried in a freeze dryer for 48 hours to obtain sodium alginate-chitosan composite hydrogel microspheres loaded with salicylic acid. They are then sealed for later use. The ratio of sodium alginate, deionized water, salicylic acid, calcium chloride, water-soluble chitosan, and acetic acid solution is 1.5–2.5 g: 80–120 mL: 0.3–0.5 g: 0.8–1.2 g: 0.4–0.6 g: 180–220 mL.

2. The rooting culture medium for callus formation from *Lysimachia christinae* according to claim 1, characterized in that, The medium is prepared per 1000 mL of 1 / 2 MS medium, consisting of 20 g sucrose, 0.8 mg indolebutyric acid, 0.2 mg naphthaleneacetic acid, 1.5 g sodium alginate-chitosan composite hydrogel microspheres loaded with salicylic acid, 30 mg L-proline, 7 g agar, and the remainder being deionized water, with a pH of 5.

8.

3. The rooting culture medium for *Lysimachia christinae* callus according to claim 1, characterized in that, The ratio of sodium alginate, deionized water, salicylic acid, calcium chloride, water-soluble chitosan, and acetic acid solution is 2.0g:100mL:0.4g:1.0g:0.5g:200mL.

4. The rooting culture medium for callus formation of *Lysimachia christinae* according to claim 1, characterized in that, The water-soluble chitosan described in step (2) has a molecular weight of 300 kDa and a degree of deacetylation of 92%.

5. The rooting culture medium for callus formation of *Lysimachia christinae* according to claim 1, characterized in that, The magnetic stirring speed in step (3) is 200-300 rpm.