Facility cultivation method for improving content of daphne genkwa coumarin components

Abstract

This invention belongs to the field of plant cultivation technology, and particularly relates to a facility cultivation method for increasing the content of coumarin components in Ashitaba leaves. This method uses direct seeding and transplanting. Within 1-3 months after transplanting, a differentiated application of basic water-soluble fertilizer is carried out in stages (20-20-20 during the seedling stage → 30-10-10 during the vigorous growth stage). This is combined with foliar spraying of trace elements such as zinc, boron, and molybdenum, as well as root irrigation with functional microbial agents to promote biomass accumulation and coumarin precursor synthesis. After the plant height reaches ≥25cm, an alternating "flooding-drought" stress is implemented: the substrate is submerged for 24-72 hours and then drained. The water content is maintained at 40%-50% of field capacity for 3-5 days, and this cycle is repeated 2-3 times to induce coumarin synthesis. This invention utilizes a cultivation method that "promotes growth first, then applies moderate stress," combined with the synergistic effect of micronutrients and microbial agents, to simultaneously increase biomass and coumarin content. Compared to continuous flooding, this method causes less plant damage and is suitable for multiple harvests, providing technical support for the high-value utilization of Ashitaba leaves.

Description

Technical Field

[0001] This invention belongs to the field of plant cultivation technology, and in particular relates to a facility cultivation method for increasing the content of coumarin components in Ashitaba. Background Technology

[0002] Ashitaba, a perennial herb belonging to the genus Angelica in the family Apiaceae, is native to Hachijojima, Japan. It gets its name from its strong regenerative ability after harvesting, often described as "harvested today, sprouting tomorrow." Modern phytochemical research shows that Ashitaba is rich in various secondary metabolites, including chalcones, coumarins, and flavonoids. The coumarin compounds, primarily psoralen, xanthotoxin, isopsoralen, bergapten, isoimperatorin, and hydrated oxyimperatorin, possess various biological activities such as antitumor, anti-inflammatory, antibacterial, and neuroprotective effects. These are one of the core pharmacodynamic components of Ashitaba and a key indicator for its high-value development.

[0003] Coumarin compounds are defensive secondary metabolites in plants, and their synthesis and accumulation are usually closely related to the plant's stress defense response. Current research on the regulation of coumarin components in Apiaceae plants mainly focuses on species like Angelica dahurica, whose roots are used medicinally. Studies have confirmed that direct seeding, compared to transplanting, can simultaneously increase the yield and coumarin content of Angelica dahurica; continuous flooding stress reduces the yield of Angelica dahurica but increases the concentration of coumarin components; increased nitrogen fertilizer application can significantly increase the biomass of Angelica dahurica but leads to a decrease in coumarin content; foliar spraying with micronutrients such as zinc, boron, and molybdenum, as well as inoculation with arbuscular mycorrhizal fungi and Bacillus-based microbial agents, can all promote the accumulation of coumarin components in Apiaceae plants to some extent.

[0004] However, existing technologies still have three significant shortcomings:

[0005] First, there are limitations in the research subjects and application scenarios. Existing research on coumarin regulation has focused on root-grown Apiaceae plants, and there are no cultivation techniques specifically designed to enhance coumarin components in Ashitaba. Ashitaba is harvested from tender stems and leaves and is a dual-purpose crop used for both medicinal and edible purposes. Its cultivation goal is to increase the content of active ingredients while ensuring leaf biomass. This is fundamentally different from Angelica dahurica, whose core objectives are root yield and component concentration. Cultivation methods that directly transplant Angelica dahurica cannot meet the production needs of Ashitaba.

[0006] Second, water stress patterns are highly damaging and poorly adaptable. Existing water stress technologies mostly employ continuous flooding, which, while inducing coumarin accumulation, causes long-term oxygen deficiency and rot in plant roots, severely inhibiting subsequent plant growth. This contradicts the facility cultivation production model of Ashitaba, which allows for "multiple harvests and year-round supply."

[0007] Third, cultivation management lacks systematic integration. Existing technologies focus on the effects of individual technical aspects, failing to organically combine direct seeding, phased nutrient management, micronutrient supplementation, application of microbial agents, appropriate stress induction, and environmental control. A complete technical system that "promotes biomass accumulation in the early stage and induces component synthesis in the later stage" has not yet been formed, making it impossible to achieve a synergistic improvement in yield and quality.

[0008] In summary, there is currently no systematic facility cultivation program that targets and enhances coumarin components in Ashitaba. Existing water stress models cause significant damage to plants and make it difficult to simultaneously achieve the three major production goals of biomass, component content, and continuous harvesting capacity. Summary of the Invention

[0009] This invention addresses the technical problem of insufficient targeted enhancement of coumarin components in Ashitaba by providing a facility cultivation method to increase the content of coumarin components in Ashitaba. The method adopts a phased management strategy of "first promoting growth and building up storage, then inducing accumulation under stress". It reduces plant damage through a novel water stress mode of "flooding-drought alternation" and integrates the synergistic effects of direct seeding, time-differentiated fertilization, foliar spraying of micronutrients, and root irrigation with functional microbial agents. While ensuring the biomass of Ashitaba leaves, it significantly increases the content of coumarin active ingredients and ensures the plant's ability to continuously harvest multiple crops, adapting to the needs of facility-based and high-value production of Ashitaba.

[0010] To achieve the above objectives, the technical solution adopted in this invention is a facility cultivation method for increasing the content of coumarin components in Ashitaba, comprising the following steps:

[0011] Step 1: Facility Preparation and Direct Seeding

[0012] (1) Cultivation facilities: Glass greenhouses or greenhouses are used for cultivation, equipped with shading systems and controllable irrigation / drainage systems.

[0013] Preferably, the controllable irrigation / drainage system includes irrigation pipes and controllable drainage holes at the bottom of the planting trough, for precise control of flood stress and rapid drainage.

[0014] (2) Cultivation method: pot cultivation, container cultivation (including three-dimensional trough cultivation and flat trough cultivation) or ridging cultivation method are adopted; container cultivation or ridging cultivation method is preferred to facilitate the implementation of standardized water stress management.

[0015] (3) Cultivation substrate: The cultivation substrate is prepared according to the following volume ratio (dry basis): 5-7 parts of high-quality peat, 1-2 parts of desalinated coconut coir, 1-2 parts of perlite, and 0.5-1 parts of decomposed organic fertilizer. After mixing evenly, adjust the pH to 6.0-7.0.

[0016] (4) Direct seeding: Seeds are directly seeded. Before sowing, the seeds are pre-germinated. After the seeds show white sprouts, they are sown at a spacing of 30-50cm × 40-50cm, with 2-3 seeds per hole. After emergence, the seedlings are thinned to 1 plant per hole.

[0017] Step Two: Phased Management

[0018] (I) First stage: Growth promotion stage (1-3 months after transplanting)

[0019] The core objective of this stage is to promote the rapid accumulation of plant biomass, while simultaneously accumulating coumarin synthesis precursors. Specific management measures are as follows:

[0020] (1) Differentiated application of basic water-soluble fertilizers at different time periods

[0021] Seedling stage / seedling establishment stage (within 1 month after transplanting): Use 20-20-20+TE balanced macro-element water-soluble fertilizer (nitrate-sulfur based), with an N-P2O5-K2O ratio of 20-20-20, containing Ca≥3%, Mg≥6%, chelated microelements B≥0.2%, Mn≥0.1%, Fe≥0.1%, Zn≥0.1%, and chlorine content≤3%; add 2.5g of water-soluble fertilizer powder to water to a final volume of 5.5L to prepare a working solution, and apply once a week.

[0022] During the vigorous growth period (1 month after transplanting to 3 months): switch to 30-10-10 high-nitrogen water-soluble fertilizer with an N-P2O5-K2O ratio of 30-10-10. Ashitaba is a leafy vegetable, and its purpose is to harvest the stems and leaves, so its nitrogen requirement is much higher than that of phosphorus and potassium. Add 3.0g of water-soluble fertilizer powder to water to a final volume of 5.5L and apply once a week.

[0023] (2) Supplementation of micronutrient fertilizers

[0024] During the vigorous growth period of Tomorrow's leaves, supplement micronutrient fertilizers by foliar spraying.

[0025] Preferably, zinc sulfate (ZnSO4·7H2O) is used at a concentration of 0.15~0.24 kg·hm. -2 Boric acid (H3BO3) or borax 2.02~2.36 kg·hm -2 Ammonium molybdate ((NH4)6Mo7O) 24 ·4H2O) 0.08~0.13kg·hm -2Dissolve in water to prepare a foliar fertilizer solution with a mass fraction of 0.1%~0.3%. Spray once every 10~15 days during the vigorous growth period, for a total of 2~3 sprays. Choose to spray on a sunny evening or a windless cloudy day, ensuring that the solution is evenly applied to both sides of the leaves and does not drip. The above-mentioned micronutrient fertilizers can be used in combination with basic water-soluble fertilizers, but should not be mixed with alkaline pesticides.

[0026] (3) Application of functional microbial agents

[0027] Use microbial agents containing Bacillus subtilis and / or Bacillus licheniformis, with an effective viable count ≥500 million / g; dilute the agent 200 times and apply it to the roots by irrigation once a week; the interval between the application of basic water-soluble fertilizer and functional microbial agents should not be less than 48 hours to avoid the impact of high concentrations of salt on the activity of the microbial community.

[0028] (4) Application of fermented organic liquid

[0029] Use well-rotted livestock and poultry manure organic fermentation liquid, and ferment for no less than 30 days; dilute the original liquid at a ratio of 1:10 and apply it to the roots once a week.

[0030] (5) Environmental regulation

[0031] Temperature should be controlled between 12 and 22℃; in summer, shading management should be carried out through a shading system with a shading rate of 40% to 70%; keep the substrate moist but not waterlogged, and water it once every 5 to 6 days in winter and spring, and once every 1 to 2 days in summer and autumn.

[0032] (II) Second stage: Water stress induction stage (3 months after planting to before harvest)

[0033] Once the plant reaches a height of 25cm or more and has sufficient biomass, it enters the water stress induction stage. This stage aims to induce the synthesis and accumulation of coumarin compounds, employing an alternating flooding-drought stress pattern. Specific measures are as follows:

[0034] (1) Flooding treatment: Close the drainage hole at the bottom of the planting trough or take other water control measures, and irrigate a large amount of water to completely submerge the cultivation substrate in water (the surface of the substrate is covered by a water layer, and the water depth is not less than 2cm), and keep it submerged for 24~72 hours.

[0035] (2) Drought treatment: Open the bottom drainage holes or take other drainage measures to quickly drain the water in the substrate; monitor the moisture content of the cultivation substrate in real time using a soil moisture meter (TDR) to accurately control the point at which the substrate enters a drought state; when the moisture content of the cultivation substrate drops to 40% to 50% of the field capacity, it is considered to have entered a drought state. It is well known in the art that when the soil moisture content drops to 40% to 50% of the field capacity, the plant is already in the range of moderate to severe drought stress. Maintain this drought state for 3 to 5 days.

[0036] (3) Cyclic treatment: The above “flood treatment → drought treatment” is a stress cycle. After repeating 2 to 3 cycles, normal water management is restored.

[0037] (4) Fertilizer adjustment during stress: Nitrogen fertilizer application should be suspended during water stress induction; micronutrient fertilizers can be supplemented in appropriate amounts during the water stress induction stage, but the application concentration should be reduced; functional microbial agents and organic fermentation liquid should continue to be applied according to the original plan.

[0038] Step 3: Harvesting Management

[0039] (1) Harvesting criteria: After the water stress induction treatment is completed, the plants are harvested when they reach a height of 30-40cm.

[0040] (2) Harvesting method: Cut the tender leaves that have just unfolded in the middle 5cm away from the base of the petiole. When harvesting, retain 2-3 old leaves to ensure the continuous growth of the plant.

[0041] (3) Multiple harvests: After the first harvest, resume regular water and fertilizer management. After the plants recover growth (about 15-20 days), enter the cycle of "promoting growth → inducing water stress" again to achieve multiple harvests.

[0042] Step 4: Integrated Pest and Disease Management

[0043] The approach combines physical control (sticky yellow sticky traps, insect nets, ultraviolet traps, etc.), biological control (plant-derived biological pesticides such as matrine), and chemical control. The interval between the application of chemical pesticides and microbial agents should not be less than 7 days. During the water stress induction stage, physical and biological control should be given priority to reduce the use of chemical pesticides. After the application of chemical pesticides, the safe interval period must be strictly observed before harvesting.

[0044] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0045] 1. The present invention provides a facility cultivation method for increasing the content of coumarin components in Ashitaba, achieving targeted enhancement of coumarin components for the first time: The present invention uses coumarin components in Ashitaba as the target indicator for the first time, and achieves targeted enhancement of coumarin components through a systematic cultivation management scheme of "direct seeding cultivation + early growth promotion + synergistic effect of micronutrients and microbial agents + alternating stress of flooding and drought in the later stage", filling the technical gap in targeted enhancement of coumarin components in Ashitaba.

[0046] 2. The “flooding-drought alternation” mode takes into account both the induction effect and plant protection: The present invention adopts the “flooding-drought alternation” water stress mode. Compared with the traditional continuous flooding, the intermittent stress mode causes less damage to the plant root system, which is conducive to the plant recovery and multiple harvests, and is more in line with the production needs of Ashitaba facility cultivation.

[0047] 3. The "build-then-induce" strategy achieves a balance between biomass and component content: This invention employs a phased management strategy of "promoting growth in the early stage and applying moderate stress in the later stage": In the early stage, optimized water and fertilizer management (differentiated fertilization at different times, supplementation of micronutrients, and the use of microbial agents and organic fermentation liquid) promotes rapid biomass accumulation; in the later stage, moderate water stress induces the synthesis and accumulation of coumarin components. This "build-then-induce" strategy balances biomass and coumarin content.

[0048] 4. The dual advantages of direct seeding: This invention adopts direct seeding, which fully utilizes the dual advantages of direct seeding over transplanting in terms of yield and coumarin content, and avoids the adverse effects of transplanting and seedling slow-down on plant growth and secondary metabolism.

[0049] 5. Differentiated fertilization during different growth stages and synergistic effects of micronutrients to precisely match the needs of each growth stage: This invention designs differentiated basic water-soluble fertilizer formulas based on the varying nitrogen, phosphorus, and potassium requirements of Ashitaba at different growth stages: a 20-20-20 balanced formula is used during the seedling / seedling establishment stage to promote balanced growth; during the vigorous growth stage, a 30-10-10 high-nitrogen formula is switched to precisely match the high nitrogen requirements of leafy vegetables. Simultaneously, during the vigorous growth stage, foliar spraying of micronutrient fertilizers such as zinc, boron, and molybdenum is applied, utilizing the synergistic effect of zinc and boron on total coumarin content to further promote the accumulation of coumarin components. Fertilizer is controlled during the stress induction stage, achieving precise regulation.

[0050] 6. The synergistic effect mechanism of water stress, micronutrients, and microbial agents is clear and not a simple additive effect: This invention, through systematic experimental design, verified the individual effects and combined effects of each technical step. Experiments show that, based on the basic scheme of this invention, further combining foliar spraying of micronutrients and root irrigation with microbial agents significantly increased the total coumarin content from 2.03 mg / g to 2.31 mg / g (P<0.05), an increase of approximately 100.9% compared to the conventional fertilizer control group. Two-way ANOVA verified that the interaction between water stress treatment and micronutrient application on the accumulation of total coumarin in Ashitaba leaves reached a highly significant level (P<0.01), and the introduction of microbial agents further amplified this effect. The above results indicate that there is a significant synergistic effect among the technical steps of this invention, and their combined effect is superior to the simple summation of individual steps.

[0051] 7. The auxiliary synergistic effect of microbial agents: This invention combines the application of functional microbial agents during the growth promotion stage, utilizing the improvement effect of microorganisms on the plant rhizosphere environment and the induction effect on secondary metabolism to enhance the plant's response to subsequent water stress and further improve the accumulation effect of coumarin components.

[0052] 8. Adaptable to multiple cultivation methods and highly applicable to industrialization: This invention covers multiple cultivation methods such as potted plants, container cultivation (including three-dimensional trough cultivation and flat trough cultivation) and raised bed cultivation, which can be flexibly adapted to production scenarios of different scales and conditions, and has strong prospects for industrial application. Detailed Implementation

[0053] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described below with reference to embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0054] Numerous specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways than those described herein, and therefore the invention is not limited to the specific embodiments disclosed in the following specification.

[0055] Experimental Design and Grouping: This embodiment provides a facility cultivation method for increasing the content of coumarin components in Ashitaba. Three comparative groups and three core embodiment groups were set up, along with verification embodiments using different cultivation modes. Each group had 30 biological replicates, and the experiment was repeated three times. The specific experimental design is as follows:

[0056] Comparative Example 1 (Water Control): The same amount of water was used for irrigation, but no fertilizer was applied and no water stress was applied.

[0057] Comparative Example 2 (Conventional Fertilizer Control): 20-20-20+TE balanced water-soluble fertilizer was applied throughout the entire process, once a week, without water stress.

[0058] Comparative Example 3 (Continuous Flooding Control): The fertilization plan was the same as that of Comparative Example 2. Three months after planting, the plants were continuously flooded for 7 days without alternating drought treatment.

[0059] Example 1 (Basic Scheme of the Invention): Direct seeding + time-differentiated water-soluble fertilizer + alternating flooding-drought stress, without additional spraying of micronutrients and microbial agents.

[0060] Example 2 (Basic Scheme + Micronutrients): Based on Example 1, foliar fertilizer containing zinc, boron, and molybdenum micronutrients is sprayed during the vigorous growth period.

[0061] Example 3 (Preferred embodiment of the present invention): Based on Example 2, functional microbial agents and fermented organic liquid are continuously applied throughout the growth promotion stage.

[0062] Example 4 (Verification of container trough cultivation mode): The preferred scheme of the present invention is implemented using the planar trough cultivation mode.

[0063] Example 5 (Verification of Ridge Cultivation Mode): The preferred scheme of the present invention was implemented using the ridge cultivation mode in a facility greenhouse.

[0064] Example 1: Potted Plant Mode - Basic Plan

[0065] 1. Cultivation substrate and direct seeding:

[0066] The cultivation substrate is prepared by volume ratio as follows: 6 parts high-quality peat, 1.5 parts desalinated coconut coir, 1.5 parts perlite, and 1 part well-rotted organic fertilizer. After mixing evenly, adjust the pH to 6.0-6.5.

[0067] Planting was carried out in 30cm diameter pots with controllable drainage holes at the bottom, with an equal amount of substrate in each pot. Ashitaba seeds were sown directly after germination and sprouting, with 2-3 seeds sown per pot. After emergence, the seedlings were thinned to 1 plant per pot. The substrate moisture content was monitored using a soil moisture meter (TDR) to ensure precise and controllable drought points during the water stress stage.

[0068] 2. Phased Management

[0069] ① First stage: Growth promotion stage (1-3 months after transplanting):

[0070] First month after setting the target: Apply 20-20-20+TE balanced water-soluble fertilizer (2.5g / 5.5L, 300mL per pot) once a week on the first day.

[0071] 2-3 months after setting the fertilizer: switch the basic water-soluble fertilizer to 30-10-10 high-nitrogen water-soluble fertilizer (3.0g / 5.5L, 300mL per pot), once a week.

[0072] Environmental control: Temperature 12~22℃; shading rate of about 60% in summer; daily watering should be carried out once every 5~6 days in winter and spring, and once every 1~2 days in summer and autumn, keeping the substrate moist but not waterlogged.

[0073] Phase Two: Water Stress Induction Phase (3 months after transplanting):

[0074] Once the plants reach a height of 25cm or more, close the drainage holes at the bottom of the pot and fill the pot with water until the substrate is completely submerged (about 2cm deep), maintaining the submerged state for 48 hours. Then, open the drainage holes to drain the water quickly and monitor the substrate moisture content in real time. When the moisture content drops to 40% to 50% of the field capacity, start timing and maintain the dry state for 4 days.

[0075] The above cycle of "48 hours of flooding → 4 days of drought" is one cycle, which is repeated twice; nitrogen fertilizer application is suspended during the water stress induction period.

[0076] 3. Harvesting Management

[0077] After the stress treatment ended, the tender leaves unfolding in the middle of the plant were harvested, and growth indicators and total coumarin content were measured.

[0078] Example 2: Potted Plant Mode - Micronutrient Supplementation Program

[0079] Based on Example 1, during the vigorous growth period (2-3 months after transplanting), foliar spraying of micronutrients was carried out (every 10-15 days): zinc sulfate, boric acid, and ammonium molybdate were added at a ratio of 0.19 kg zinc. hm -2 Boron 2.18kg hm -2 0.10 kg of molybdenum hm -2 The dosage is prepared into a 0.2% aqueous solution. In this example, it is sprayed once every 12 days for a total of 3 times. The spraying time is in the evening on a sunny day. The rest of the operation is the same as in Example 1.

[0080] Example 3: Potted Plant Mode - Preferred Solution

[0081] Based on Example 2, functional microbial agents and fermented broth from well-rotted pig manure were applied throughout the entire growth-promoting stage:

[0082] Functional microbial inoculant (containing Bacillus subtilis and Bacillus licheniformis, with an effective live bacteria count ≥500 million / g): Dilute 200 times and apply 150 mL to the roots of each pot once a week;

[0083] Fermented pig manure liquid (fermentation time 35 days): Dilute at 1:10, pour 150mL into each pot, once a week; apply water-soluble fertilizer, microbial agent and fermentation liquid on different days, with an interval of more than 48 hours between water-soluble fertilizer and microbial agent; the rest of the operation is the same as in Example 2.

[0084] Example 4: Validation of Container Tank Cultivation Mode

[0085] Plastic cultivation troughs with a width of 40cm and a depth of 25cm were used, with controllable drainage holes at the bottom. The troughs were filled with the same cultivation substrate as in Example 1. Tomorrow's Leaf was directly planted at a spacing of 30cm×40cm. Fertilizer application was calculated per linear meter of cultivation trough, and micronutrients were sprayed according to the unit area dosage. Water stress was controlled by opening and closing the drainage holes at the bottom of the trough. The remaining management measures were the same as in Example 3.

[0086] Example 5: Verification of Ridge Cultivation Model

[0087] In the greenhouse, raised beds with a width of 140cm were prepared and equipped with irrigation and drainage facilities. Tomorrow's Leaf was directly transplanted at a spacing of 30cm x 50cm. Water stress was controlled through field irrigation and drainage ditches; other management practices were the same as in Example 3.

[0088] Regarding the index measurement and data analysis in the above implementation methods:

[0089] 1. Growth index measurement: Plant height, stem diameter, and fresh weight per plant are measured at harvest.

[0090] 2. Determination of total coumarin content: The total coumarin content (unit: mg / g dry weight) was calculated using ultraviolet spectrophotometry with psoralen as a reference.

[0091] Sample pretreatment: Take fresh young leaves of Ashitaba, dry them at 60℃ to constant weight, and pulverize them through a 40-mesh sieve; weigh 1.0 g of sample powder, place it in a stoppered conical flask, add 25 mL of 70% ethanol solution, weigh it, and extract it by sonication for 30 min (power 250 W, frequency 40 kHz). After cooling, weigh it, replenish the lost weight with 70% ethanol, shake well, and filter; measure 1 mL of the filtrate, place it in a 25 mL volumetric flask, dilute it to the mark with 70% ethanol, shake well, and use it as the test solution.

[0092] Preparation of standard curve: Accurately weigh an appropriate amount of psoralen reference standard and add methanol to prepare a reference standard stock solution containing 0.1 mg per 1 mL; accurately measure 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mL of the reference standard stock solution respectively, place them in 10 mL volumetric flasks, dilute to the mark with methanol, and shake well; using the corresponding solvent as a blank, measure the absorbance at a wavelength of 322 nm; plot the standard curve with absorbance as the ordinate (A) and reference standard concentration as the abscissa (C, μg / mL), and calculate the regression equation.

[0093] Determination method: Take the test solution, use 70% ethanol as blank, measure the absorbance at a wavelength of 322 nm, substitute it into the standard curve regression equation to calculate the concentration of coumarin in the test solution, and then convert it into the content of total coumarin in the sample (calculated as psoralen equivalent, mg / g dry weight).

[0094] Methodological validation: The method showed good linearity (r² ≥ 0.999) within the tested concentration range. The RSDs of precision, stability, and repeatability tests were all less than 3%, and the average recovery rate was between 95% and 105%, meeting the requirements for quantitative analysis.

[0095] 3. Statistical analysis: All data are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA and two-way ANOVA were used for comparisons between groups. Duncan's multiple comparison test was used. P < 0.05 was considered statistically significant, and P < 0.01 was considered extremely statistically significant.

[0096] Experimental Results and Analysis:

[0097] 1. Effects of different treatments on tomorrow's leaf growth indicators

[0098] The growth indicators for different treatments are shown in Table 1:

[0099] Table 1: Effects of different treatments on the growth and yield of tomorrow's leaves (mean ± SD)

[0100]

[0101] Note: Different lowercase letters in the same column indicate significant differences (P<0.05, Duncan multiple comparisons). Each group has n=30, and the experiment is repeated 3 times.

[0102] 2. Effects of different treatments on the total coumarin content of Ashitaba leaves

[0103] The total coumarin content of different treatments is shown in Table 2:

[0104] Table 2: Effects of different treatments on total coumarin content in Ashitaba (mean ± SD, based on psoralen equivalents)

[0105]

[0106] Note: Different lowercase letters in the same column indicate significant differences (P<0.05, Duncan multiple comparisons). Each group has n=30, and the experiment is repeated 3 times.

[0107] Results analysis:

[0108] 1. Analysis of plant growth status and biomass accumulation

[0109] As shown in Table 1, different treatment groups had a significant impact on plant morphology and biomass accumulation.

[0110] Superiority of water management mode: The fresh weight of a single plant in Group 3 (continuous flooding) was 35.2±3.8g, which was significantly lower than that in Group 2 (conventional fertilizer, 45.8g), indicating that long-term continuous flooding will lead to root hypoxia and severely inhibit plant growth; in contrast, Group 4 (the basic scheme of this invention) adopted the "flooding-drought alternation" mode, and the fresh weight of a single plant reached 58.3±5.0g, which was about 27.3% higher than that in Group 2 and significantly higher than that in Group 3.

[0111] This indicates that the water management mode of the present invention effectively alleviates the negative impact of water stress on growth. Combined with the previous growth-promoting management, it successfully achieves efficient biomass accumulation and solves the problems of weak plant growth and low biomass in the traditional continuous flooding mode.

[0112] Synergistic effect: Based on group 4, group 5 (with added micronutrients) showed an increase in single-plant fresh weight to 60.5±5.2g, effectively improving plant biomass; group 6 (preferred embodiment of the invention, micronutrients + microbial agent) showed a single-plant fresh weight of 62.1±5.3g, with no significant difference compared to group 5. Among these, group 6 had the highest plant height, stem diameter, and single-plant fresh weight (40.2cm, 6.4mm, and 62.1g, respectively), and all indicators were significantly better than the conventional control group.

[0113] The results showed that micronutrients are the core factors that promote plant biomass accumulation and can effectively optimize plant growth and increase biomass; while microbial agents have limited beneficial effects on plant vegetative growth and biomass enhancement and have no significant additional growth-promoting effect. The combination of the two can stably maintain the optimal growth state of plants and maximize the crop yield potential.

[0114] 2. Analysis of the accumulation pattern of total coumarin content

[0115] As shown in Table 2, there were significant differences in the induced accumulation of secondary metabolites (total coumarins) among the treatment groups.

[0116] Significant induction effect: The total coumarin content of Group 4 (basic scheme of this invention) was 2.03±0.19mg / g, which was significantly higher than that of Group 2 (conventional fertilizer, 1.15mg / g) and Group 3 (continuous flooding, 1.68mg / g).

[0117] This indicates that while continuous flooding can create abiotic stress and activate plant defense metabolism, the effect of coumarin induction is limited due to severe stunting of plant growth and insufficient reserves of metabolic synthesis substrates and substances. In contrast, the "flooding-drought alternation" mode of this invention can efficiently induce the synthesis and accumulation of coumarin while ensuring normal plant growth and sufficient biomass accumulation, thus achieving a balance between growth and accumulation of effective components.

[0118] Superimposed Gain Effect: With the addition of various technical steps, the coumarin content increased significantly in a stepwise manner. After adding trace elements in Group 5, the coumarin content increased to 2.19±0.20 mg / g, a significant increase of 7.9% compared to Group 4. After further adding microbial agents in Group 6, the content reached 2.31±0.21 mg / g, a further significant increase of 5.5% compared to Group 5. The final optimized scheme showed an improvement of up to 100.9% compared to the conventional fertilizer group (Group 2), demonstrating an extremely significant quality improvement effect.

[0119] Data confirms that trace elements and microbial agents have a significant synergistic effect. The combination of the two can significantly enhance the plant's secondary metabolic capacity, greatly increase the accumulation of coumarin, and the synergistic effect is better than that of single technology treatment.

[0120] 3. Mechanism of Action Analysis

[0121] The results of this experiment fully verify the scientific validity and rationality of the "first establish the library, then induce" cultivation strategy of this invention:

[0122] Resource allocation balance: Group 3 (continuous flooding) has long-term root hypoxia damage, nutrient absorption is hindered, and the plants are in a state of severe abiotic stress. Although the secondary metabolic defense mechanism is activated, due to insufficient vegetative growth in the early stage, lack of carbon source and nutrient reserves, the "storage capacity" of material synthesis is limited, which ultimately leads to low coumarin accumulation and failure to achieve the quality improvement effect.

[0123] Synergistic Regulation Mechanism: This invention provides sufficient nutrients for plant vegetative growth through differentiated fertilization in the early stages, establishing a robust biomass foundation and ensuring adequate substrate for the later synthesis of secondary metabolites. Based on this, a moderate, cyclical stress of alternating flooding and drought precisely triggers the plant's defensive secondary metabolic response. Simultaneously, the synergistic application of micronutrients and microbial agents effectively improves the rhizosphere microecological environment, enhancing plant stress resistance and adaptability, as well as the activity of key metabolic enzymes. Without sacrificing plant biomass, it achieves a targeted and efficient increase in coumarin content, fundamentally resolving the contradiction of traditional cultivation methods that "promote growth without improving quality, or improve quality while inhibiting growth."

[0124] 4. Applicability verification of the cultivation model

[0125] To verify the broad applicability and scenario stability of this method, comparative verification experiments were conducted on different facility cultivation modes (Example 4 and Example 5).

[0126] The fresh weight of a single plant in Example 4 (container cultivation) was 60.3g, and the total coumarin content was 2.24mg / g; the fresh weight of a single plant in Example 5 (ridge cultivation) was 58.7g, and the total coumarin content was 2.19mg / g.

[0127] Under both mainstream facility cultivation modes, this technical solution can stably achieve simultaneous increases in plant biomass and coumarin content, with stable experimental data and good consistency in results. This demonstrates that the technical solution of this invention does not depend on a single cultivation facility or planting scenario, possesses excellent stability, versatility, and engineering adaptability, and can be widely applied to various facility agriculture cultivation scenarios.

[0128] In summary, this invention innovatively solves the technical challenge of synergistically improving plant biomass and medicinal quality in traditional planting methods through an integrated cultivation scheme combining direct seeding, time-differentiated fertilization, synergistic effects of micronutrients and microbial agents, and alternating flooding and drought stress. Under this optimized cultivation system, the content of the target active ingredient, total coumarin, is increased by over 100% compared to conventional cultivation, while simultaneously ensuring robust plant growth and biomass accumulation.

[0129] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments for application in other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for greenhouse cultivation to increase the content of coumarin-like components in Ashitaba, characterized in that, Includes the following steps: (1) Transplanting: Ashitaba is transplanted into the cultivation substrate by direct seeding; (2) Growth promotion management: Within 1 to 3 months after transplanting, apply basic water-soluble fertilizer in different periods, and at the same time, apply micronutrient fertilizer on the leaves and apply functional microbial agents by root irrigation to promote the accumulation of plant biomass. (3) Water stress induction: After the plant height reaches more than 25cm, water stress treatment with alternating flooding and drought is implemented: First, the cultivation substrate is completely submerged and maintained for 12 to 96 hours, and then the water is drained; when the water content of the cultivation substrate drops to 30% to 60% of the field capacity, it enters a drought state and is maintained for 2 to 6 days; the complete process from flooding to drought is one stress cycle, and it is repeated 2 to 4 times. (4) Harvesting: After the water stress treatment is completed, harvest the tender leaves of Ashitaba.

2. The method for facility cultivation of Ashitaba with increased coumarin content according to claim 1, characterized in that, In step (2), the differentiated application of basic water-soluble fertilizer in different time periods is as follows: within one month after planting, a balanced water-soluble fertilizer with an N-P2O5-K2O ratio of 20-20-20 is applied, and after one month after planting, it is switched to a high-nitrogen water-soluble fertilizer with an N-P2O5-K2O ratio of 30-10-10.

3. The method for facility cultivation of Ashitaba with increased coumarin content according to claim 1, characterized in that, In step (3), the duration of the complete immersion state of the cultivation substrate is 24 to 72 hours, and the water content of the cultivation substrate in the drought state is 40% to 50% of the field water holding capacity, and the drought state is maintained for 3 to 5 days.

4. The method for facility cultivation of Ashitaba with increased coumarin content according to claim 3, characterized in that, In step (3), the duration of the complete immersion state of the cultivation substrate is 48 hours, the drought state is maintained for 4 days, and the stress cycle is repeated 2 times.

5. The method for facility cultivation to increase the content of coumarin components in Ashitaba according to claim 1, characterized in that, In step (3), nitrogen fertilizer application is suspended during the water stress treatment.

6. The method for facility cultivation of Ashitaba with increased coumarin content according to claim 1, characterized in that, In step (1), the cultivation substrate is prepared by volume ratio of the following components: 5-7 parts high-quality peat, 1-2 parts desalinated coconut coir, 1-2 parts perlite, 0.5-1 parts well-rotted organic fertilizer, and the pH value of the substrate is 6.0-7.

0.

7. The method for facility cultivation of Ashitaba with increased coumarin content according to claim 1, characterized in that, In step (2), the micronutrient fertilizer is a foliar fertilizer composed of zinc, boron, and molybdenum; the application rate is 0.15~0.24 kg of zinc per application area. hm -2 Boron element 2.02~2.36kg hm -2 Molybdenum element 0.08~0.13kg hm -2 .

8. The method for facility cultivation of Ashitaba with increased coumarin content according to claim 1, characterized in that, In step (2), the functional microbial agent includes at least one of Bacillus subtilis and Bacillus licheniformis, with an effective viable count ≥500 million / g; step (2) also includes the combined application of composted organic fermentation liquid, wherein the composted organic fermentation liquid is composted livestock and poultry manure organic fermentation liquid, the fermentation time is more than 30 days, and the application interval between the basic water-soluble fertilizer and the functional microbial agent is more than 48 hours.

9. The method for facility cultivation of Ashitaba with increased coumarin content according to claim 1, characterized in that, In step (4), when harvesting, cut the tender leaves 5cm away from the base of the petiole and retain 2-3 old leaves at the base of the plant. After harvesting, resume regular water and fertilizer management. After the plant resumes growth, repeat steps (2) and (3) to achieve multiple harvests.

10. A method for facility cultivation to increase the content of coumarin components in Ashitaba according to claim 1, characterized in that, Throughout the cultivation process, the ambient temperature is controlled at 12~22℃, and shading management is carried out in summer, with a shading rate of 40%~70%.

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