Method for inducing flowering of davidia involucrata by simulating seasonal change

Abstract

The invention relates to the technical field of gardens, discloses a method for simulating seasonal change to induce flowering of davidia involucrata, and effectively solves the problem of flowering induction in non-original habitat cultivation of davidia involucrata. According to the scheme, a hormone-environment double-factor regulation model is established, so that the flowering induction success rate is remarkably increased. The whole induction period is shortened through the staged gradient treatment design, and plant physiological disorder is avoided. A dynamic monitoring and classification processing mechanism ensures synchronous development of plants in different physiological states, and reliable technical support is provided for large-scale production. The finally formed systematic treatment process enables the flowering process of davidia involucrata to be controllable and predictable, a new technical path is provided for protection of rare plants, and through combination of classification treatment and dynamic monitoring, different types of plants reach a unified nutrition reserve standard before entering a vernalization stage, and a reliable physiological foundation is laid for subsequent flowering induction.

Description

Technical Field

[0001] This invention relates to the field of landscape architecture, specifically to a method for inducing flowering of Davidia involucrata by simulating seasonal changes. Background Technology

[0002] Plant resource conservation and utilization is an important field of research in modern biology and ecology. Within the specific area of ​​rare tree species breeding technology, artificially controlled flowering is a core element for achieving germplasm preservation and large-scale production. Davidia involucrata, a Tertiary relict plant endemic to my country and a national first-class protected plant, possesses extremely high scientific research and ornamental value. However, natural flowering of Davidia involucrata requires strict seasonal environmental conditions, including a sufficient period of nutrient accumulation, a specific duration of low-temperature vernalization, and a suitable temperature and humidity recovery process. Under non-native cultivation conditions, flowering of Davidia involucrata is extremely unstable, with a natural flowering rate of less than 30%, severely restricting the conservation and breeding efforts of this species.

[0003] Current techniques for inducing flowering in *Davidia involucrata* primarily rely on temperature regulation to simulate seasonal changes, but they suffer from significant shortcomings in hormone regulation. Existing research lacks sufficient understanding of the dynamic changes of key hormones such as abscisic acid, gibberellin, cytokinin, and auxin throughout the induction process, and there is a lack of baseline data on hormone concentrations at each physiological stage. The application of exogenous hormones is often based on empirical treatments with single hormones, resulting in arbitrary timing of application and a lack of scientific basis for concentration ratios. More importantly, the synergistic or antagonistic effects between different hormones have not been systematically elucidated, leading to large fluctuations in hormone treatment effects and even the adverse effect of inhibiting flower bud differentiation. This "blind application" model makes it difficult to shorten the induction cycle and achieve a high flowering success rate.

[0004] The aforementioned deficiencies stem from a weak foundation in the study of the flowering physiological mechanisms of Davidia involucrata and limitations in technical means. Due to the lack of continuous hormone monitoring techniques and systematic multi-factor interaction studies, researchers are unable to accurately grasp the critical time window for hormone regulation. These shortcomings have led to a series of abnormal effects: First, the induction period has been forced to extend to 6-9 months, resulting in high time and energy costs; second, the flowering rate varies by more than 40% between different batches of plants, making commercial production difficult; third, some plants exhibit physiological disorders such as excessive vegetative growth and leaf drop after hormone treatment, and even lose their flowering ability; ultimately, this results in low efficiency in the artificial propagation of Davidia involucrata, failing to meet the dual goals of germplasm preservation and market demand.

[0005] Therefore, we propose a method to induce flowering of Davidia involucrata by simulating seasonal changes, in order to solve the problems mentioned above. Summary of the Invention

[0006] The purpose of this invention is to provide a method for inducing flowering of Davidia involucrata by simulating seasonal changes, in order to address the problem mentioned in the background art that the synergistic or antagonistic effects between different hormones have not been systematically elucidated, leading to large fluctuations in hormone treatment effects and even the adverse effect of inhibiting flower bud differentiation. This "blind application" mode makes it difficult to shorten the induction period and achieve a high flowering success rate.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for inducing flowering of Davidia involucrata by simulating seasonal changes, the specific steps of which are as follows: S1. Select 2-3 year old Davidia involucrata seedlings and pre-culture them for 14 days under the conditions of 22℃ temperature and 16 hours of light / 8 hours of darkness; collect leaf and shoot tip tissues, and detect the concentrations of endogenous abscisic acid, gibberellin, cytokinin and auxin. According to the concentration ratio of abscisic acid to gibberellin, the plants are divided into hormone-balanced type, dormant type and active growth type. S2. For dormant plants, spray the leaves with a mixture of 50 mg / L gibberellin and 20 mg / L cytokinin every 7 days for a total of 4 times; for active growth plants, spray the leaves with a 100 mg / L chlormequat chloride solution; for hormone-balanced plants, perform routine water and fertilizer management; cultivate for 45 days to achieve a soluble sugar content of over 8% in the plant leaves. S3. Perform low-temperature vernalization treatment. From day 1 to 14, lower the temperature from 22℃ to 15℃ and adjust the photoperiod to 10 hours of light / 14 hours of darkness. From day 15 to 35, maintain the temperature at 8-10℃. From day 36 to 49, lower the temperature to 4-6℃ and control the relative humidity at 65%. Detect the abscisic acid concentration every 7 days. Vernalization is complete when the abscisic acid concentration reaches 2.5-3.0 times the initial concentration. S4. Immerse the base of the plant in a 200 mg / L gibberellin solution for 4 hours, raise the temperature to 18°C ​​at a rate of 2°C every 2 days, and adjust the photoperiod to 14 hours of light / 10 hours of darkness. S5. After the temperature stabilizes at 18-20℃, spray the leaves with a mixture of 30mg / L gibberellin, 15mg / L cytokinin and 10mg / L auxin, once every 5 days, for a total of 3 times; adjust the formula according to the concentration ratio of cytokinin to auxin; treat for 15-20 days until flower bud primordia form. S6. Maintain the temperature at 20-22℃ and the relative humidity at 75%-80%; spray the leaves with a mixture containing 5 mg / L cytokinin and 0.1 mg / L brassinolide, once every 7 days, for a total of 2 sprays; treat for 20-25 days until the flower buds show color; S7. Adjust the temperature to 18-20℃ and the relative humidity to 65%-70%, stop the hormone treatment, and record the flowering data.

[0008] Preferably, step S1 is performed in the following manner: S1.1 Select 2-3 year old Davidia involucrata seedlings with a height of 30-50cm and a stem diameter of 0.8-1.2cm, and transplant them into cultivation containers with a volume of 3-5 liters. The cultivation substrate is a mixture of peat moss, perlite and vermiculite in a volume ratio of 2:1:1. Place them in an artificial climate chamber with a temperature of 22±1℃, a photoperiod of 16 hours of light / 8 hours of darkness, a light intensity of 10000-12000 lux, and a relative humidity of 60%-70%. Irrigate with a nutrient solution with an electrical conductivity of 1.5-2.0mS / cm and a pH of 5.5-6.5, applying 200ml per plant every 3 days, and pre-cultivate for 14 days. S1.2 On the morning of the day the pre-culture ended, from 9:00 to 10:00, collect 2-3 grams of the 3rd to 5th fully expanded leaves from the top of each plant and 1-2 grams of the 0.5-1.0 cm shoot tip tissue from each plant. After quick-freezing in liquid nitrogen, store at -80℃. The concentrations of abscisic acid (ABA), gibberellin (GA3), cytokinin (CTK), and auxin (IAA) were determined by enzyme-linked immunosorbent assay (ELISA). Each sample was measured three times and the average value was taken. The ABA / GA ratio was calculated. Plants with a ratio of 0.8-1.2 were marked as hormone-balanced, those with a ratio greater than 1.5 were marked as dormant, and those with a ratio less than 0.6 were marked as active growth.

[0009] Preferably, step S2 is performed in the following manner: S2.1 Based on the classification results of step S1.2, spray the leaves of dormant plants with a mixed solution containing 50 mg / L gibberellin GA3 and 20 mg / L cytokinin 6-BA, 30-50 ml per plant, once every 7 days, for a total of 4 times. Spray the leaves of active growth plants with a 100 mg / L chlormequat chloride (CCC) solution, using the same spraying method and frequency. Do not apply exogenous hormones to hormone-balanced plants, and continue irrigating with nutrient solution as in step S1-1. All three types of plants are cultured in an environment with a temperature of 22±1℃, a photoperiod of 16 hours of light / 8 hours of darkness, a light intensity of 10000-12000 lux, and a relative humidity of 60%-70%. S2.2 Maintain the culture conditions of step S2.1 and culture continuously for 45 days. On the 15th, 30th and 45th days after the start of culture, collect 1-2 grams of the 3rd to 5th fully unfolded leaves from the top of each plant. Use the anthrone colorimetric method to determine the soluble sugar content, use the Kjeldahl nitrogen determination method to determine the total nitrogen content and calculate the carbon-nitrogen ratio (C / N). When the test results on the 45th day show that the soluble sugar content of the leaves reaches more than 8% and the C / N ratio reaches more than 12, stop the culture. For plants that do not meet the standards, extend the culture for 7-14 days and retest.

[0010] Preferably, step S3 is implemented in the following manner: S3.1 Transfer the plants that have met the standards in step S2.2 to a low-temperature vernalization environment. From day 1 to 14, the temperature is reduced from 22℃ to 15℃ at a rate of 0.5℃ per day. The photoperiod is adjusted to 10 hours of light / 14 hours of darkness, the light intensity is 8000-10000 lux, and the relative humidity is 60%-65%. Apply 100-150 ml of nutrient solution to each plant every 5-7 days. From day 15 to 35, the temperature is maintained at 8-10℃. The temperature is raised to 12℃ for 2 days a week, with an interval of 3-4 days. Other conditions remain unchanged. From day 36 to 49, the temperature is reduced to 4-6℃, the relative humidity is 65%, the light intensity is 5000-8000 lux, and apply 50-100 ml of clean water to each plant every 10-14 days. Stop applying nutrient solution. S3.2 During step S3.1, on days 7, 14, 21, 28, 35, 42, and 49, collect 1-2 grams of the 3rd to 5th leaves from the top of each plant and 0.5-1.0 grams of the shoot tip tissue (0.5-1.0 cm from the top). After quick-freezing in liquid nitrogen, store at -80℃. Determine the abscisic acid (ABA) concentration using enzyme-linked immunosorbent assay (ELISA). Each sample is measured three times and the average value is taken. When the ABA concentration reaches 2.5-3.0 times the initial concentration in step S1-2 and the change from the next test value is less than 10%, proceed to step S4. Plants that do not meet the standard are vernalized for 7-14 days at 4-6℃ and monitored every 7 days.

[0011] Preferably, step S4 is performed in the following manner: S4.1 Take out the plants that have completed vernalization in step S3.2, prepare a 200 mg / L gibberellin GA3 solution, immerse the base of the plant from the root collar upwards in the solution for 4 hours, the temperature is 10-12℃ and the relative humidity is 65%-70%, stir the solution once every 1 hour, after soaking, rinse the base with running water for 3-5 minutes, replant back into the original cultivation container, or irrigate the roots with 100 mL of 200 mg / L gibberellin GA3 solution as an alternative treatment; S4.2 After completing step S4.1, transfer the cultivation container to an artificial climate chamber. The initial temperature is 10℃, which is increased to 18±1℃ at a rate of 2℃ every 2 days and stabilized. The photoperiod is adjusted to 14 hours of light / 10 hours of darkness. The light intensity is gradually increased from 8000 lux to 12000-15000 lux, and the relative humidity is 65%~70%. Apply 150~200ml of nutrient solution to each plant every 3~5 days. After the temperature reaches 18℃, spray the leaves with a 50mg / L gibberellin GA3 solution on the 4th to 7th day, with a spraying amount of 30~50ml per plant. Collect leaf samples every 3~4 days to detect the GA3 / ABA ratio. When the ratio reaches 3.0 or above and is stable for two consecutive tests, proceed to step S5.

[0012] Preferably, step S5 is performed in the following manner: S5.1 After the temperature stabilizes at 18-20℃ and the GA3 / ABA ratio reaches 3.0 or higher in step S4.2, prepare a compound hormone solution containing 30 mg / L gibberellin GA3, 15 mg / L cytokinin 6-BA, and 10 mg / L auxin NAA. Spray 30-50 ml per plant on the leaves every 5 days for 3 consecutive times. 1-2 days before each spraying, collect 1-2 grams of leaf and stem tip tissue to test the CTK / IAA ratio. If the ratio is less than 1.0, adjust the 6-BA concentration to 25 mg / L. If the ratio is greater than 2.0, reduce the NAA concentration to 5 mg / L or discontinue use. After each spraying, add 20-30 ml of 0.2% potassium dihydrogen phosphate solution at 2-4 hours intervals. S5.2 During step S5.1, maintain a temperature of 18-20℃, a photoperiod of 14 hours of light / 10 hours of darkness, a light intensity of 12000-15000 lux, and a relative humidity of 70%-75%. Apply 150-200 ml of nutrient solution to each plant every 3-4 days. The mass ratio of nitrogen, phosphorus, and potassium in the nutrient solution is N:P:K=1:2:2. From the start of spraying, collect 0.5-1.0 g of apical meristem every 3-4 days to prepare paraffin sections with a thickness of 8-10 μm. After safranin-fast green staining, observe under an optical microscope. Record the number of days when flower bud primordia formation is confirmed, usually 15-20 days. For plants that have not reached the standard, spray 1-2 more times and extend the treatment by 5-7 days. After confirming the formation of flower bud primordia, proceed to step S6.

[0013] Preferably, step S6 is performed in the following manner: S6.1 Transfer the plants confirmed to have formed flower bud primordia in step S5.2 to a culture environment with a temperature of 20-22℃, relative humidity of 75%-80%, a photoperiod of 14 hours of light / 10 hours of darkness, and a light intensity of 15000-20000 lux. Stop applying gibberellin GA3. Prepare a mixed solution containing 5 mg / L cytokinin 6-BA, 0.1 mg / L brassinolide BR, and 0.05% boron and molybdenum. Spray 30-50 ml per plant on the leaves every 7 days for 2 consecutive times. Irrigate every 5-7 days, applying 100-150 ml of nutrient solution per plant each time. Control the soil moisture content to 55%-60%. The nutrient solution should have a nitrogen-phosphorus-potassium ratio of N:P:K = 1:2:2, with 150-200 mg / L calcium and 50-80 mg / L magnesium. S6.2. During step S6.1, maintain the same culture conditions. Starting from the day this step begins, observe and measure the inflorescence length, inflorescence diameter, bract opening degree, and color change every 4-5 days. Every 5-7 days, collect 0.5-1.0 grams of inflorescence tissue and determine the soluble sugar content using the anthrone colorimetric method. When the bracts change from light green to milky white or pure white, with a bract length of 5-8 cm, a width of 3-5 cm, a smooth, leathery surface, and intact edges, the color development of the flower buds is considered complete. The treatment time is usually 20-25 days. For plants that do not meet the standard, extend the culture time by 3-5 days and spray the mixed solution once more. After confirming that the color development of the flower buds is complete, proceed to step S7.

[0014] Preferably, step S7 is implemented in the following manner: S7.1. Transfer the plants whose flower buds have been confirmed to have completed color development in step S6.2 to the flowering management environment. Adjust the temperature to 18-20℃, the relative humidity to 65%-70%, maintain a photoperiod of 14 hours of light / 10 hours of darkness, and a light intensity of 12000-15000 lux using diffused light. Stop all exogenous hormone treatments. Irrigate every 5-7 days. Apply 100-150 ml of nutrient solution with an electrical conductivity of 1.0-1.5 mS / cm and a pH of 5.5-6.5 to each plant each time. The N:P:K mass ratio of the nutrient solution is N:P:K=1:1:1. Ventilate 2-3 times a day for 30-60 minutes each time. S7.2 During step S7.1, starting from the day the flower buds fully open, record the total number of flowering days, the total number of inflorescences, the duration of flowering, the length, width, and thickness of bracts, the Lab color value of bracts, the diameter of inflorescences, the number of florets, pollen viability, and the pollination success rate. Take photos from the front, side, and top using a camera with at least 12 megapixels. Retrospectively analyze the ABA, GA3, CTK, and IAA concentration data at each key time point throughout the process and plot the dynamic changes in hormones. Calculate the changing trends of the ABA / GA, GA / ABA, and CTK / IAA ratios. Define plants with bract length ≥5cm, L value ≥80, flowering period ≥10 days, and inflorescence diameter ≥3cm as successful cases. Enter the hormone curve data, processing parameters, and flowering data of successful cases into the database.

[0015] The beneficial effects of this invention are: 1. This application effectively solves the problem of flowering induction in the non-native cultivation of Davidia involucrata. The scheme significantly improves the success rate of flowering induction by establishing a hormone-environment dual-factor regulation model. The phased gradient treatment design shortens the overall induction cycle and avoids physiological disorders in the plants. The dynamic monitoring and classification treatment mechanism ensures the synchronous development of plants in different physiological states, providing reliable technical support for large-scale production. The resulting systematic treatment process makes the flowering process of Davidia involucrata controllable and predictable, providing a new technical approach for the protection of rare plants.

[0016] 2. This application effectively solves the problem of unstable hormone treatment effects caused by individual plant differences. By combining classified treatment with dynamic monitoring, different types of plants reach a uniform nutrient reserve standard before entering the vernalization stage, laying a reliable physiological foundation for subsequent flowering induction. At the same time, through extended cultivation and re-inspection mechanisms, it ensures that all treated plants meet the standards, avoiding the impact of uneven individual development on the overall flowering success rate. Attached Figure Description

[0017] Figure 1 This is a diagram illustrating the method steps of the present invention. Detailed Implementation

[0018] The technical solutions of 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.

[0019] Example 1: Please refer to Figure 1 A method for inducing flowering of Davidia involucrata by simulating seasonal changes, the specific steps of which are as follows: S1. Select 2-3 year old Davidia involucrata seedlings and pre-culture them for 14 days under the conditions of 22℃ temperature and 16 hours of light / 8 hours of darkness; collect leaf and shoot tip tissues, and detect the concentrations of endogenous abscisic acid, gibberellin, cytokinin and auxin. According to the concentration ratio of abscisic acid to gibberellin, the plants are divided into hormone-balanced type, dormant type and active growth type. S2. For dormant plants, spray the leaves with a mixture of 50 mg / L gibberellin and 20 mg / L cytokinin every 7 days for a total of 4 times; for active growth plants, spray the leaves with a 100 mg / L chlormequat chloride solution; for hormone-balanced plants, perform routine water and fertilizer management; cultivate for 45 days to achieve a soluble sugar content of over 8% in the plant leaves. S3. Perform low-temperature vernalization treatment. From day 1 to 14, lower the temperature from 22℃ to 15℃ and adjust the photoperiod to 10 hours of light / 14 hours of darkness. From day 15 to 35, maintain the temperature at 8-10℃. From day 36 to 49, lower the temperature to 4-6℃ and control the relative humidity at 65%. Detect the abscisic acid concentration every 7 days. Vernalization is complete when the abscisic acid concentration reaches 2.5-3.0 times the initial concentration. S4. Immerse the base of the plant in a 200 mg / L gibberellin solution for 4 hours, raise the temperature to 18°C ​​at a rate of 2°C every 2 days, and adjust the photoperiod to 14 hours of light / 10 hours of darkness. S5. After the temperature stabilizes at 18-20℃, spray the leaves with a mixture of 30mg / L gibberellin, 15mg / L cytokinin and 10mg / L auxin, once every 5 days, for a total of 3 times; adjust the formula according to the concentration ratio of cytokinin to auxin; treat for 15-20 days until flower bud primordia form. S6. Maintain the temperature at 20-22℃ and the relative humidity at 75%-80%; spray the leaves with a mixture containing 5 mg / L cytokinin and 0.1 mg / L brassinolide, once every 7 days, for a total of 2 sprays; treat for 20-25 days until the flower buds show color; S7. Adjust the temperature to 18-20℃ and the relative humidity to 65%-70%, stop the hormone treatment, and record the flowering data.

[0020] In this embodiment: In existing technologies, plant flowering induction techniques often rely on the regulation of a single environmental factor, failing to systematically integrate the synergistic effects of dynamic changes in plant endogenous hormones and external environmental conditions. Traditional methods often overlook the crucial role of hormone balance at different growth stages, resulting in a lack of targeted treatment solutions. Especially in species like Davidia involucrata, which are sensitive to seasonal changes, existing technologies struggle to accurately simulate hormone response mechanisms under natural conditions, often leading to physiological disorders in plants due to improper hormone regulation, resulting in flowering induction failure or abnormal flowering period.

[0021] Based on this, the inventors proposed to establish a hormone-environment dual-factor regulation model in stages. The model establishes the basal metabolic level through pre-culture, simulates seasonal changes through gradient treatment, and implements targeted hormone intervention at key nodes.

[0022] This application proposes the following technical solution: selecting Davidia involucrata seedlings at specific growth stages for pre-culture, and classifying the plants by detecting the concentration of endogenous hormones; implementing differentiated hormone treatments for different categories, and then performing vernalization treatment with gradient temperature control after the plants reach predetermined physiological indicators; combining basal soaking and exogenous hormone spraying during the temperature recovery stage, and finally inducing flower bud formation and development through the synergistic effect of environmental parameters and hormone ratios.

[0023] Pre-culture conditions refer to establishing the plant's basal metabolic state by controlling temperature, photoperiod, and nutrient supply. This can be achieved using an artificial climate chamber to precisely regulate environmental parameters, establishing a unified physiological baseline for subsequent treatments. The hormone classification system refers to classifying plant physiological states based on the concentration ratio of abscisic acid to gibberellin. This can be achieved using enzyme-linked immunosorbent assay (ELISA) to detect hormone concentrations, ensuring the specificity of the treatment plan. Differentiated hormone treatment involves applying specific ratios of exogenous hormones to different plant types, which can be implemented through foliar spraying to correct imbalances in endogenous hormones. Gradual vernalization involves gradually lowering the temperature and adjusting the photoperiod, which can be implemented step-by-step using programmed temperature control equipment to simulate the low-temperature accumulation process under natural conditions. Synergistic regulation of hormones and the environment involves simultaneously applying gibberellin and adjusting light parameters during the temperature recovery phase. This can be implemented using a timed and quantitative spraying system combined with environmental control devices to promote the physiological transformations required for flower bud differentiation.

[0024] This program first eliminates individual plant differences through standardized pre-culture, establishing a comparable physiological baseline. Plants are then categorized into three groups based on endogenous hormone levels. For dormancy-prone plants, gibberellin and cytokinin are administered to break dormancy; for actively growing plants, chlormequat chloride is applied to inhibit excessive vegetative growth. Once the plants reach the predetermined carbon-to-nitrogen ratio, a phased vernalization treatment is implemented, triggering the accumulation of endogenous abscisic acid through temperature gradient changes. During the temperature recovery phase, basal gibberellin immersion promotes nutrient transport, while foliar hormone spraying adjusts the cytokinin-to-auxin ratio, ultimately leading to flower bud development under specific temperature and humidity conditions. Throughout the process, monitoring hormone concentrations at key points provides real-time feedback for dynamic adjustments to treatment parameters.

[0025] Existing methods often employ fixed-pattern hormone treatments, while this approach achieves precise intervention by establishing a hormone concentration classification system. Traditional techniques typically use single, continuous low-temperature treatments, while this approach effectively simulates the natural vernalization process through staged gradient cooling. Existing techniques neglect individual differences in plant physiological states, while this approach ensures the effectiveness of the treatment plan through pre-cultivation and classification treatments. Existing methods lack dynamic adjustment mechanisms, while this approach achieves real-time optimization of treatment parameters through hormone detection at key points.

[0026] Through the above technical solutions, this application effectively solves the problem of flowering induction in the non-native cultivation of Davidia involucrata. The solution significantly improves the success rate of flowering induction by establishing a hormone-environment dual-factor regulation model. The phased gradient treatment design shortens the overall induction cycle and avoids physiological disorders in the plants. The dynamic monitoring and classification treatment mechanism ensures the synchronous development of plants in different physiological states, providing reliable technical support for large-scale production. The resulting systematic treatment process makes the flowering process of Davidia involucrata controllable and predictable, providing a new technical approach for the protection of rare plants.

[0027] Example 2: Please refer to Figure 1 The specific method for step S1 is as follows: S1.1 Select 2-3 year old Davidia involucrata seedlings with a height of 30-50cm and a stem diameter of 0.8-1.2cm, and transplant them into cultivation containers with a volume of 3-5 liters. The cultivation substrate is a mixture of peat moss, perlite and vermiculite in a volume ratio of 2:1:1. Place them in an artificial climate chamber with a temperature of 22±1℃, a photoperiod of 16 hours of light / 8 hours of darkness, a light intensity of 10000-12000 lux, and a relative humidity of 60%-70%. Irrigate with a nutrient solution with an electrical conductivity of 1.5-2.0mS / cm and a pH of 5.5-6.5, applying 200ml per plant every 3 days, and pre-cultivate for 14 days. S1.2 On the morning of the day the pre-culture ended, from 9:00 to 10:00, collect 2-3 grams of the 3rd to 5th fully expanded leaves from the top of each plant and 1-2 grams of the 0.5-1.0 cm shoot tip tissue from each plant. After quick-freezing in liquid nitrogen, store at -80℃. The concentrations of abscisic acid (ABA), gibberellin (GA3), cytokinin (CTK), and auxin (IAA) were determined by enzyme-linked immunosorbent assay (ELISA). Each sample was measured three times and the average value was taken. The ABA / GA ratio was calculated. Plants with a ratio of 0.8-1.2 were marked as hormone-balanced, those with a ratio greater than 1.5 were marked as dormant, and those with a ratio less than 0.6 were marked as active growth.

[0028] In this embodiment, the specific method of step S1 is further described as follows: Select 2-3 year old Davidia involucrata seedlings with a height of 30-50cm and a stem diameter of 0.8-1.2cm, and transplant them into cultivation containers with a volume of 3-5 liters. The cultivation substrate is a mixture of peat moss, perlite, and vermiculite in a volume ratio of 2:1:1. Place the container in an artificial climate chamber with a temperature of 22±1℃, a photoperiod of 16 hours of light / 8 hours of darkness, a light intensity of 10000-12000 lux, and a relative humidity of 60%-70%. Irrigate the container with a nutrient solution with an electrical conductivity of 1.5-2.0 mS / cm and a pH of 5.5-6.5, applying 200ml of nutrient solution to each plant every 3 days. Pre-culture for 14 days; on the morning of the end of the pre-culture, from 9:00 to 10:00, collect 2-3 grams of the 3rd to 5th fully expanded leaves from the top of each plant and 1-2 grams of the 0.5-1.0 cm shoot tip tissue from each plant. After being flash-frozen in liquid nitrogen, the samples were stored at -80℃. The concentrations of abscisic acid (ABA), gibberellin (GA3), cytokinin (CTK), and auxin (IAA) were determined by enzyme-linked immunosorbent assay (ELISA). Each sample was measured three times and the average value was taken. The ABA / GA ratio was calculated. Plants with a ratio of 0.8-1.2 were labeled as hormone-balanced, those with a ratio greater than 1.5 were labeled as dormant, and those with a ratio less than 0.6 were labeled as actively growing.

[0029] A plant height of 30–50 cm refers to the vertical height of the above-ground portion of the seedling. This can be achieved by regularly measuring plant height to select individuals that meet the standard. This range ensures the plant has sufficient nutrient reserves and physiological maturity. A stem diameter of 0.8–1.2 cm refers to the diameter of the main stem at 5 cm above the ground. This can be measured using calipers. This parameter reflects the degree of lignification and stress resistance of the plant. The cultivation substrate should be a 2:1:1 mixture of peat moss, perlite, and vermiculite, uniformly mixed using mechanical stirring. This ratio simultaneously meets the requirements for water retention, aeration, and slow nutrient release. A nutrient solution with an electrical conductivity of 1.5–2.0 mS / cm refers to the total soluble salt content in the solution. This can be achieved by adjusting the nitrogen, phosphorus, and potassium element ratio. This conductivity range avoids root osmotic stress while ensuring nutrient supply. Enzyme-linked immunosorbent assay (ELISA) for hormone concentration determination refers to the detection of hormone content in plant tissue extracts using antigen-antibody specific reactions. This method can be performed using commercially available kits and has high sensitivity and reproducibility, accurately reflecting the endogenous hormone balance in plants.

[0030] Physiological baselines for the plants were established using standardized pre-culture conditions. Light intensity and photoperiod parameters simulated the natural growing season environment, while nutrient solution conductivity and pH maintained optimal root absorption. Simultaneous sampling of shoot tip tissue and specific leaf positions yielded hormone data representing the synergistic response of the plant's apical meristem and mature leaves. The method of averaging three repeated measurements effectively reduced the impact of sampling errors on the classification results. A threshold for the ABA / GA ratio was set based on the interaction relationships of key regulatory factors for Davidia involucrata flower bud differentiation, classifying plants into three types with clearly defined treatment directions. For example, a high ABA / GA ratio in dormant plants indicated growth inhibition, requiring exogenous hormone regulation to break dormancy; while a low ratio in active growth plants suggested the need to suppress vegetative growth to promote reproductive transformation.

[0031] Traditional methods typically employ uniform culture conditions and lack physiological indicator detection, resulting in insufficient targeting of subsequent treatment plans. Existing technologies rely heavily on morphological observation for plant classification, failing to accurately reflect endogenous hormone dynamics. This approach ensures balanced root development space and nutrient supply by precisely controlling the volume of cultivation containers and substrate ratios; eliminates interference from diurnal rhythms and tissue heterogeneity on test results by setting standardized collection times and tissue locations; and provides a reliable basis for differentiated treatment by establishing a quantitative grading standard for the ABA / GA ratio.

[0032] Through the above technical solution, this application achieves accurate diagnosis of the physiological state of Davidia involucrata seedlings. The standardized pre-culture system lays a consistent physiological foundation for subsequent treatments, and the combined detection and ratio analysis of multiple hormones can accurately identify the developmental stage of the plant. This solution solves the problem of blind treatment plans caused by the lack of physiological indicators in traditional methods, enabling different types of plants to receive targeted regulatory measures, thereby improving the overall flowering induction efficiency.

[0033] Example 3: Please refer to Figure 1 The specific method for step S2 is as follows: S2.1 Based on the classification results of step S1.2, spray the leaves of dormant plants with a mixed solution containing 50 mg / L gibberellin GA3 and 20 mg / L cytokinin 6-BA, 30-50 ml per plant, once every 7 days, for a total of 4 times. Spray the leaves of active growth plants with a 100 mg / L chlormequat chloride (CCC) solution, using the same spraying method and frequency. Do not apply exogenous hormones to hormone-balanced plants, and continue irrigating with nutrient solution as in step S1-1. All three types of plants are cultured in an environment with a temperature of 22±1℃, a photoperiod of 16 hours of light / 8 hours of darkness, a light intensity of 10000-12000 lux, and a relative humidity of 60%-70%. S2.2 Maintain the culture conditions of step S2.1 and culture continuously for 45 days. On the 15th, 30th and 45th days after the start of culture, collect 1-2 grams of the 3rd to 5th fully unfolded leaves from the top of each plant. Use the anthrone colorimetric method to determine the soluble sugar content, use the Kjeldahl nitrogen determination method to determine the total nitrogen content and calculate the carbon-nitrogen ratio (C / N). When the test results on the 45th day show that the soluble sugar content of the leaves reaches more than 8% and the C / N ratio reaches more than 12, stop the culture. For plants that do not meet the standards, extend the culture for 7-14 days and retest.

[0034] In this embodiment: This application further proposes a specific implementation method for step S2, including classification and treatment based on plant hormone status, spraying scheme of specific hormone solutions, and monitoring and adjustment of the cultivation process. Dormant plants are treated with a mixture of gibberellin and cytokinin, actively growing plants are treated with chlormequat chloride solution, and plants with balanced hormones are maintained under routine management. During cultivation, the cultivation period is determined by detecting soluble sugar content and carbon-nitrogen ratio; plants that do not meet the standards are cultivated for an extended period and re-tested.

[0035] Treatment with a mixture of gibberellin and cytokinin involves foliar spraying of a mixture of the two hormones in a specific ratio onto specific plant types. A suitable solution is a mixture of 50 mg / L gibberellin GA3 and 20 mg / L cytokinin 6-BA, which breaks dormancy by regulating endogenous hormone balance. Treatment with chlormequat chloride solution involves applying a 100 mg / L chlormequat chloride CCC solution to overly vigorous plants to control vegetative growth by inhibiting gibberellin synthesis. Soluble sugar content detection uses the anthrone colorimetric method to determine the degree of sugar accumulation in leaves. Specifically, the absorbance value is measured at a wavelength of 620 nm using a spectrophotometer to determine whether the plant has completed nutrient reserves. Carbon-to-nitrogen ratio detection involves determining the total nitrogen content using the Kjeldahl method and calculating the C / N ratio based on soluble sugar data. This is used to assess the physiological state of the plant transitioning from vegetative to reproductive growth.

[0036] After pre-culturing and classification, plants were treated differently based on their hormone type. Dormant-prone plants were supplemented with exogenous gibberellins and cytokinins to promote the balance of endogenous hormones towards growth; actively growing plants were treated with chlormequat chloride to inhibit excessive growth and promote nutrient accumulation; plants with balanced hormones were maintained under existing management to avoid interference. Leaf samples were collected periodically during cultivation, and soluble sugar content and carbon-nitrogen ratio were monitored using chemical analysis. The cultivation stage was considered complete when soluble sugar reached 8% and the carbon-nitrogen ratio exceeded 12. Plants that did not meet the standards were allowed to continue accumulating nutrients by extending the cultivation period until the test indicators met the requirements. The entire cultivation process was conducted under constant temperature, humidity, and photoperiod conditions to ensure that environmental variables were controllable.

[0037] Traditional methods apply uniform hormone treatment to all plants, failing to address the fluctuations in treatment effects caused by individual differences in physiological state. This approach establishes a differentiated treatment system through preliminary hormone testing and classification, ensuring precise matching of exogenous hormone application to the actual needs of the plants. Simultaneously, it introduces dual detection indicators of soluble sugars and carbon-nitrogen ratio to avoid errors from judging based on a single indicator, ensuring that plants reach their optimal physiological state before transitioning to the next stage.

[0038] Through the above technical solution, this application effectively solves the problem of unstable hormone treatment effects caused by individual plant differences. By combining classified treatment with dynamic monitoring, different types of plants reach a uniform nutrient reserve standard before entering the vernalization stage, laying a reliable physiological foundation for subsequent flowering induction. At the same time, through extended cultivation and re-inspection mechanisms, it ensures that all treated plants meet the standards, avoiding the impact of uneven individual development on the overall flowering success rate.

[0039] Example 4: Please refer to Figure 1 The specific method for step S3 is as follows: S3.1 Transfer the plants that have met the standards in step S2.2 to a low-temperature vernalization environment. From day 1 to 14, the temperature is reduced from 22℃ to 15℃ at a rate of 0.5℃ per day. The photoperiod is adjusted to 10 hours of light / 14 hours of darkness, the light intensity is 8000-10000 lux, and the relative humidity is 60%-65%. Apply 100-150 ml of nutrient solution to each plant every 5-7 days. From day 15 to 35, the temperature is maintained at 8-10℃. The temperature is raised to 12℃ for 2 days a week, with an interval of 3-4 days. Other conditions remain unchanged. From day 36 to 49, the temperature is reduced to 4-6℃, the relative humidity is 65%, the light intensity is 5000-8000 lux, and apply 50-100 ml of clean water to each plant every 10-14 days. Stop applying nutrient solution. S3.2 During step S3.1, on days 7, 14, 21, 28, 35, 42, and 49, collect 1-2 grams of the 3rd to 5th leaves from the top of each plant and 0.5-1.0 grams of the shoot tip tissue (0.5-1.0 cm from the top). After quick-freezing in liquid nitrogen, store at -80℃. Determine the abscisic acid (ABA) concentration using enzyme-linked immunosorbent assay (ELISA). Each sample is measured three times and the average value is taken. When the ABA concentration reaches 2.5-3.0 times the initial concentration in step S1-2 and the change from the next test value is less than 10%, proceed to step S4. Plants that do not meet the standard are vernalized for 7-14 days at 4-6℃ and monitored every 7 days.

[0040] In this embodiment, this application further proposes a specific implementation method for low-temperature vernalization treatment, including staged temperature control, photoperiod adjustment, and hormone concentration monitoring. Vernalization treatment is divided into three stages: In the first stage, the temperature is reduced from 22°C to 15°C at a rate of 0.5°C per day over 14 days, with the photoperiod adjusted to 10 hours of light / 14 hours of darkness, light intensity of 8000-10000 lux, and relative humidity of 60%–65%; in the second stage, the temperature is maintained at 8-10°C for 21 days, with a 12°C temperature fluctuation set for 2 days per week; in the third stage, the temperature is reduced to 4-6°C over 14 days, with relative humidity of 65% and light intensity of 5000-8000 lux. During vernalization, plant tissue samples are collected every 7 days to detect abscisic acid (ABA) concentration. Vernalization is considered complete when the concentration reaches 2.5-3.0 times the initial value and the fluctuation between two tests is less than 10%. Plants that do not meet the standard have their vernalization extended by 7–14 days.

[0041] Phased temperature control refers to simulating natural seasonal changes by progressively lowering temperatures across three temperature ranges. This can be achieved using a programmable temperature control system in an artificial climate chamber. The first phase involves slow cooling to avoid stress on the plants; the second phase involves periodic temperature fluctuations to simulate natural diurnal temperature variations; and the third phase involves deep cooling to promote the accumulation of dormant substances.

[0042] Abscisic acid (ABA) concentration monitoring involves periodically collecting leaf and shoot tip tissue samples for hormone testing. Specifically, this can be achieved using enzyme-linked immunosorbent assay (ELISA), with samples taken three times every seven days and the average value calculated. The process of vernalization is determined by dynamically tracking changes in hormone levels.

[0043] Extended vernalization treatment refers to continuing low-temperature cultivation of plants that have not yet met the standards. Specifically, this can be achieved by extending the environmental maintenance time by 4-6℃, and by supplementing monitoring to ensure that all plants reach the hormone threshold before proceeding to the next stage.

[0044] Vernalization regulates plant physiological states through the synergistic effects of temperature gradient changes and photoperiod. The first stage involves slow cooling combined with short-day conditions to initiate dormancy; the second stage involves temperature fluctuations stimulating the expression of cold-resistance genes; and the third stage involves deep cooling to promote abscisic acid (ABA) synthesis. Hormone monitoring every 7 days establishes quantitative indicators. When the ABA concentration stably reaches 2.5-3.0 times the initial value, it indicates that effective accumulation of low-temperature signals has been completed. For plants that do not meet the criteria, the treatment time is extended to ensure population synchronization and avoid premature end of vernalization, which could lead to flower bud differentiation failure.

[0045] Existing vernalization treatments often employ a single low-temperature environment and lack hormone monitoring, easily leading to insufficient or excessive treatment time. This scheme simulates natural climate change through phased temperature control, enhances plant adaptability by combining periodic temperature fluctuations, and accurately determines the vernalization endpoint by dynamically monitoring abscisic acid concentration, thus solving the problem of unstable treatment effects caused by the reliance on experience in traditional methods.

[0046] Through the above technical solutions, this application achieves precise control of the vernalization process, ensuring that plants complete the necessary physiological preparations before entering subsequent growth stages. Phased temperature changes avoid physiological damage caused by sudden temperature drops, and hormone concentration monitoring provides objective criteria for judgment, effectively preventing flowering failure due to insufficient vernalization or energy waste due to excessive vernalization, significantly improving the synchronicity and success rate of flowering induction.

[0047] Example 5: Please refer to Figure 1 The specific method for step S4 is as follows: S4.1 Take out the plants that have completed vernalization in step S3.2, prepare a 200 mg / L gibberellin GA3 solution, immerse the base of the plant from the root collar upwards in the solution for 4 hours, the temperature is 10-12℃ and the relative humidity is 65%-70%, stir the solution once every 1 hour, after soaking, rinse the base with running water for 3-5 minutes, replant back into the original cultivation container, or irrigate the roots with 100 mL of 200 mg / L gibberellin GA3 solution as an alternative treatment; S4.2 After completing step S4.1, transfer the cultivation container to an artificial climate chamber. The initial temperature is 10℃, which is increased to 18±1℃ at a rate of 2℃ every 2 days and stabilized. The photoperiod is adjusted to 14 hours of light / 10 hours of darkness. The light intensity is gradually increased from 8000 lux to 12000-15000 lux, and the relative humidity is 65%~70%. Apply 150~200ml of nutrient solution to each plant every 3~5 days. After the temperature reaches 18℃, spray the leaves with a 50mg / L gibberellin GA3 solution on the 4th to 7th day, with a spraying amount of 30~50ml per plant. Collect leaves every 3~4 days to detect the GA3 / ABA ratio. When the ratio reaches 3.0 or above and is stable for two consecutive tests, proceed to step S.

[0048] In this embodiment: This application further proposes an implementation plan for treating Davidia involucrata plants with gibberellin after low-temperature vernalization. Specifically, the base of the plant is immersed in a gibberellin solution with a concentration of 200 mg / L for 4 hours. During the immersion, the temperature is controlled at 10-12°C and the solution is stirred every hour. After immersion, the base of the plant is rinsed and replanted. Subsequently, the ambient temperature is raised to 18°C ​​at a rate of 2°C every 2 days and stabilized. The photoperiod is adjusted to 14 hours of light / 10 hours of darkness, and the light intensity is gradually increased to 12000-15000 lux. After the temperature stabilizes, gibberellin is sprayed on the leaves from the 4th to the 7th day, and the timing for transitioning to the next stage is determined by periodically detecting the GA3 / ABA ratio.

[0049] Soaking the base of plants in gibberellin (GA3) solution refers to supplementing exogenous gibberellin through root absorption. Polyethylene containers can be used to hold the solution for root soaking, which effectively promotes the transport of gibberellin to the apical meristem. Temperature gradient control refers to the transition from a low-temperature vernalization environment to a suitable temperature environment using programmed temperature control equipment, such as setting a daily temperature rise curve using a programmable environmental controller. GA3 / ABA ratio monitoring involves periodically collecting leaf samples to detect hormone content, for example, using an enzyme-linked immunosorbent assay (ELISA) kit to determine the concentrations of the two hormones and then calculating their ratio.

[0050] After vernalization, soaking the base of the plant in gibberellin solution quickly breaks dormancy, and combined with a gradual increase in temperature, it simulates the natural seasonal transition. Adjusting the photoperiod to a long-day pattern activates the photoperiodic response mechanism, and gradually increasing light intensity promotes the accumulation of photosynthetic products. Foliar spraying with gibberellin solution as a secondary supplementary treatment ensures an adequate supply of gibberellin to the apical meristem. By dynamically monitoring the concentration ratio of GA3 to ABA, it is possible to accurately determine whether the plant has completed the physiological transition, avoiding developmental abnormalities caused by entering the next stage too early or too late.

[0051] Traditional methods, which rely solely on temperature increases while neglecting the synergistic effects of hormones, can easily lead to a reversal of vernalization. This approach establishes a system that links hormone balance with the external environment through a dual mechanism of gibberellin absorption via roots and foliar application, combined with the synergistic regulation of temperature and photoperiod. Existing technologies lack effective state monitoring indicators; however, this approach introduces the GA3 / ABA ratio as a key threshold parameter, providing a quantitative basis for stage transitions.

[0052] Through the above technical solutions, this application effectively solves the problem of inhibited flower bud differentiation caused by incomplete physiological state transition after vernalization, and avoids growth stagnation caused by sudden environmental changes. The synergistic effect of programmed temperature control and hormone treatment significantly shortens the time interval from the end of vernalization to the initiation of flower bud differentiation. The establishment of a dynamic monitoring mechanism makes the judgment of treatment timing more accurate, reduces the number of ineffective treatments, and improves the overall induction efficiency.

[0053] Example 6: Please refer to Figure 1 The specific method for step S5 is as follows: S5.1 After the temperature stabilizes at 18-20℃ and the GA3 / ABA ratio reaches 3.0 or higher in step S4.2, prepare a compound hormone solution containing 30 mg / L gibberellin GA3, 15 mg / L cytokinin 6-BA, and 10 mg / L auxin NAA. Spray 30-50 ml per plant on the leaves every 5 days for 3 consecutive times. 1-2 days before each spraying, collect 1-2 grams of leaf and stem tip tissue to test the CTK / IAA ratio. If the ratio is less than 1.0, adjust the 6-BA concentration to 25 mg / L. If the ratio is greater than 2.0, reduce the NAA concentration to 5 mg / L or discontinue use. After each spraying, add 20-30 ml of 0.2% potassium dihydrogen phosphate solution at 2-4 hours intervals. S5.2 During step S5.1, maintain a temperature of 18-20℃, a photoperiod of 14 hours of light / 10 hours of darkness, a light intensity of 12000-15000 lux, and a relative humidity of 70%-75%. Apply 150-200 ml of nutrient solution to each plant every 3-4 days. The mass ratio of nitrogen, phosphorus, and potassium in the nutrient solution is N:P:K=1:2:2. From the start of spraying, collect 0.5-1.0 g of apical meristem every 3-4 days to prepare paraffin sections with a thickness of 8-10 μm. After safranin-fast green staining, observe under an optical microscope. Record the number of days when flower bud primordia formation is confirmed, usually 15-20 days. For plants that have not reached the standard, spray 1-2 more times and extend the treatment by 5-7 days. After confirming the formation of flower bud primordia, proceed to step S6.

[0054] In this embodiment: This application further proposes a method of preparing a compound solution containing gibberellin, cytokinin and auxin for foliar spraying after the temperature is stabilized at 18-20℃ and the ratio of gibberellin to abscisic acid meets the standard, adjusting the formula according to the concentration ratio of cytokinin to auxin, and adding potassium dihydrogen phosphate solution after spraying.

[0055] A compound hormone solution containing 30 mg / L gibberellin GA3, 15 mg / L cytokinin 6-BA, and 10 mg / L auxin NAA refers to an aqueous solution in which three plant growth regulators are mixed in a specific ratio. It can be prepared by dissolving a standard in deionized water. This ratio can synergistically promote the division and differentiation of apical meristem cells.

[0056] The CTK / IAA ratio test refers to the determination of the concentration ratio of cytokinin to auxin in leaf and shoot tip tissues by enzyme-linked immunosorbent assay. When the ratio is less than 1.0, the cytokinin concentration is increased to 25 mg / L. When the ratio is greater than 2.0, the auxin concentration is decreased to 5 mg / L or discontinued. This dynamic adjustment mechanism can maintain the optimal hormone balance required for flower bud differentiation.

[0057] Additional spraying with 0.2% potassium dihydrogen phosphate solution refers to supplementing phosphorus and potassium elements during the hormone treatment interval. Analytical grade potassium dihydrogen phosphate can be dissolved, filtered, and applied. This measure can enhance cellular energy metabolism and promote the transport of carbohydrates to flower buds.

[0058] Once the temperature conditions are met and the gibberellin to abscisic acid ratio is within acceptable limits, the flower bud differentiation process is initiated by periodically spraying a compound hormone solution. Before each spraying, the concentration ratio of cytokinin to auxin is measured, and the formula ratio is adjusted in real time based on the results: when cytokinin is relatively insufficient, its concentration is increased to enhance cell division activity; when auxin is excessive, its concentration is decreased to avoid inhibiting flower bud formation. After spraying, potassium dihydrogen phosphate solution is added to provide the necessary phosphorus and osmotic regulators for flower bud morphogenesis. Changes in meristematic tissue morphology are observed through paraffin sections. Treatment is terminated after confirmation of flower bud primordia formation; for plants that do not meet the criteria, the treatment cycle is extended to ensure complete differentiation.

[0059] Existing techniques for inducing flowering in Davidia involucrata often employ fixed concentrations of a single hormone, lacking real-time monitoring and feedback regulation of the dynamic balance of multiple hormones. This can easily lead to inhibited flower bud differentiation or excessive vegetative growth. This proposed method establishes a hormone ratio detection-feedback-adjustment mechanism, achieving precise regulation of hormone levels during the critical stage of flower bud differentiation. Combined with phosphorus and potassium supplementation, it effectively avoids physiological disorders caused by hormonal imbalances.

[0060] Through the above technical solution, this application can adjust the hormone ratio according to the real-time physiological state of the plant, ensuring that cytokinin and auxin are maintained in the optimal ratio range for promoting flower bud differentiation. At the same time, timely supplementation of phosphorus and potassium elements enhances the material basis required for flower bud development, significantly improving the synchronicity and success rate of flower bud primordia formation, and shortening the induction period to a predictable range of 15 to 20 days.

[0061] Example 7: Please refer to Figure 1 The specific method for step S6 is as follows: S6.1 Transfer the plants confirmed to have formed flower bud primordia in step S5.2 to a culture environment with a temperature of 20-22℃, relative humidity of 75%-80%, a photoperiod of 14 hours of light / 10 hours of darkness, and a light intensity of 15000-20000 lux. Stop applying gibberellin GA3. Prepare a mixed solution containing 5 mg / L cytokinin 6-BA, 0.1 mg / L brassinolide BR, and 0.05% boron and molybdenum. Spray 30-50 ml per plant on the leaves every 7 days for 2 consecutive times. Irrigate every 5-7 days, applying 100-150 ml of nutrient solution per plant each time. Control the soil moisture content to 55%-60%. The nutrient solution should have a nitrogen-phosphorus-potassium ratio of N:P:K = 1:2:2, with 150-200 mg / L calcium and 50-80 mg / L magnesium. S6.2. During step S6.1, maintain the same culture conditions. Starting from the day this step begins, observe and measure the inflorescence length, inflorescence diameter, bract opening degree, and color change every 4-5 days. Every 5-7 days, collect 0.5-1.0 grams of inflorescence tissue and determine the soluble sugar content using the anthrone colorimetric method. When the bracts change from light green to milky white or pure white, with a bract length of 5-8 cm, a width of 3-5 cm, a smooth, leathery surface, and intact edges, the color development of the flower buds is considered complete. The treatment time is usually 20-25 days. For plants that do not meet the standard, extend the culture time by 3-5 days and spray the mixed solution once more. After confirming that the color development of the flower buds is complete, proceed to step S7.

[0062] In this embodiment: This application further proposes transferring plants with completed flower bud color development to a flowering management environment, adjusting the temperature to 18-20℃, the relative humidity to 65%-70%, maintaining a photoperiod of 14 hours light / 10 hours darkness, a light intensity of 12000-15000 lux, using diffused light, stopping all exogenous hormone treatments, irrigating every 5-7 days, applying 100-150 ml of nutrient solution per plant each time with a conductivity of 1.0-1.5 mS / cm and a pH of 5.5-6.5, and a nitrogen:p:k mass ratio of N:P:K = 1:1:1, ventilating 2-3 times a day for 30-60 minutes each time; recording the condition from the day the flower buds fully open. Record the total number of flowering days, total number of inflorescences, duration of flowering days, bract length, width and thickness, bract Lab color value, inflorescence diameter, number of florets, pollen viability, and pollination success rate. Take photos from the front, side and top using a camera with at least 12 megapixels. Retrospectively analyze the ABA, GA3, CTK and IAA concentration data at each key time point throughout the process and plot the dynamic changes of hormones. Calculate the changing trends of ABA / GA, GA / ABA, and CTK / IAA ratios. Define plants with bract length ≥5cm, L value ≥80, flowering period ≥10 days and inflorescence diameter ≥3cm as successful cases. Enter the hormone curve data, processing parameters and flowering data of successful cases into the database.

[0063] Scattered light irradiation refers to filtering direct sunlight through a shade net with a shading rate of 30%–40%, which can be achieved by using a double-layered shade net. This method avoids scorching the epidermal cells of flower buds by strong sunlight. Ceasing exogenous hormone treatment means stopping the application of all synthetic plant growth regulators. This can be achieved by setting management procedures to automatically stop the operation of spraying equipment. This measure prevents hormone residues from interfering with the natural development process of flowers. Lab colorimetric measurement refers to measuring the luminance (L) value and color (a, b) values ​​of bracts using a spectrophotometer. This can be calibrated using a CIE standard D65 illuminant. This parameter can objectively quantify the color change process of bracts. Hormone dynamic change curves visualize hormone concentration data at different time points in the form of a line graph. This can be achieved by automatically generating trend lines using data processing software. This graph can reveal the waxing and waning patterns of key hormones during flowering induction.

[0064] After the flower buds have developed color, maintaining an ambient temperature between 18-20℃ allows the plant to smoothly transition from the reproductive growth stage to the flowering stage. Maintaining relative humidity between 65% and 70% ensures proper petal expansion while reducing the risk of pathogen growth. Irrigation with a specifically formulated nutrient solution, such as a 1:1:1 NPK solution, provides a balanced supply of nutrients needed for flowering. Regular ventilation, such as 2-3 times a day for 30-60 minutes each time, maintains air circulation and promotes pollen dispersal. By systematically recording bract morphology indicators and Lab color values, such as length ≥5cm and L value ≥80, objective evaluation standards for flowering quality can be established. Retrospective analysis of hormone concentration data, plotting ABA / GA ratio curves, can reveal the correlation between flower bud differentiation and hormone balance.

[0065] Current flowering management methods typically maintain a constant temperature and humidity environment, lacking precise control over the flowering period, which can easily lead to premature flower aging or deformities. Traditional techniques neglect data collection and analysis, relying solely on experience to judge flowering quality, making it impossible to establish replicable technical parameters. Conventional methods continue to apply hormones during the flowering period, which may cause abnormal flower development. This solution combines multi-parameter environmental control with data-driven monitoring to construct a complete flowering quality evaluation system, providing data support for subsequent technology optimization.

[0066] Through the above technical solutions, this application enables precise control of the flowering environment, avoiding petal drop caused by temperature fluctuations. The data recording system accurately identifies key parameter combinations from successful cases, providing a basis for establishing standardized production processes. Stopping exogenous hormone treatment eliminates the impact of artificial intervention on the natural development of flowers, improving the integrity of flower morphology. Systematic data collection and analysis reveals the quantitative relationship between hormone dynamics and flowering quality, providing a scientific basis for optimizing hormone application programs.

[0067] Example 8: Please refer to Figure 1 The specific method for step S7 is as follows: S7.1. Transfer the plants whose flower buds have been confirmed to have completed color development in step S6.2 to the flowering management environment. Adjust the temperature to 18-20℃, the relative humidity to 65%-70%, maintain a photoperiod of 14 hours of light / 10 hours of darkness, and a light intensity of 12000-15000 lux using diffused light. Stop all exogenous hormone treatments. Irrigate every 5-7 days. Apply 100-150 ml of nutrient solution with an electrical conductivity of 1.0-1.5 mS / cm and a pH of 5.5-6.5 to each plant each time. The N:P:K mass ratio of the nutrient solution is N:P:K=1:1:1. Ventilate 2-3 times a day for 30-60 minutes each time. S7.2 During step S7.1, starting from the day the flower buds fully open, record the total number of flowering days, the total number of inflorescences, the duration of flowering, the length, width, and thickness of bracts, the Lab color value of bracts, the diameter of inflorescences, the number of florets, pollen viability, and the pollination success rate. Take photos from the front, side, and top using a camera with at least 12 megapixels. Retrospectively analyze the ABA, GA3, CTK, and IAA concentration data at each key time point throughout the process and plot the dynamic changes in hormones. Calculate the changing trends of the ABA / GA, GA / ABA, and CTK / IAA ratios. Define plants with bract length ≥5cm, L value ≥80, flowering period ≥10 days, and inflorescence diameter ≥3cm as successful cases. Enter the hormone curve data, processing parameters, and flowering data of successful cases into the database.

[0068] In this embodiment: This application further proposes a technical solution to transfer the plant to a flowering management environment with a temperature of 18-20℃ and a relative humidity of 65%-70% after the flower buds have developed color, maintain a 14-hour light cycle and diffused light conditions, stop exogenous hormone treatment and adjust the nutrient solution formula to a balanced ratio of nitrogen, phosphorus and potassium, and simultaneously implement flowering data collection and dynamic hormone retrospective analysis.

[0069] Adjusting the environmental temperature during the flowering period refers to controlling the cultivation temperature within a specific range, such as 18-20℃, and slowing down the metabolic rate through temperature gradient control. This can be achieved by using a phased cooling program, which can prolong the duration of the flowering period.

[0070] Adjusting the nitrogen, phosphorus, and potassium mass ratio in the nutrient solution refers to bringing the element ratio to a balanced state, such as N:P:K=1:1:1. This is achieved by changing the conductivity and ion concentration of the nutrient solution. This adjustment can prevent flower drop caused by nutrient imbalance.

[0071] Hormone dynamic retrospective analysis refers to the curve processing of hormone concentration data throughout the entire cycle, and the correlation analysis between the measured values ​​and the time series by enzyme-linked immunosorbent assay (ELISA) can reveal the hormone interaction patterns at each stage.

[0072] The determination of bract colorimetric values ​​refers to the quantification of color characteristics using the Lab color space, achieved by measuring the L, a, and b values ​​using a spectrophotometer. This determination can establish a quantitative relationship between flowering quality and treatment parameters.

[0073] After the flower buds have developed color, the plants are transferred to a temperature-controlled, sealed cultivation space, where environmental parameters are adjusted according to a pre-set program. The nitrogen content in the nutrient solution is appropriately reduced, while the phosphorus and potassium content is increased accordingly; for example, a balanced solution with a conductivity of 1.0-1.5 mS / cm is used. The data acquisition system is activated after the flower buds open, recording morphological characteristics through image acquisition equipment. Simultaneously, the retrospective analysis system retrieves stored hormone detection data to generate concentration change curves. The drip irrigation system equipped with the cultivation containers supplies nutrient solution at a set frequency, and the ventilation system performs periodic air exchange.

[0074] Existing Davidia involucrata flowering management techniques lack a systematic data collection and traceability mechanism, making it impossible to establish a correlation model between treatment parameters and flowering quality. This solution implements full-cycle hormone monitoring and morphological data collection, constructing a time-series database, such as recording the correspondence between bract Lab chromaticity values ​​and GA3 concentrations. Compared to traditional methods that only record basic data such as flowering rate, this solution enables traceable analysis of the treatment process.

[0075] Through the above technical solution, this application solves the problem of the lack of a flowering quality evaluation system in the prior art, and realizes a quantitative correlation between treatment parameters and flowering traits. Specifically, it can accurately determine key time nodes of hormone treatment, such as optimizing the timing of gibberellin application by tracing back the time of GA3 peak occurrence. At the same time, the established database provides data support for the optimization of subsequent treatment schemes, such as adjusting the vernalization period of new batches based on hormone curves from historical successful cases.

[0076] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

[0077] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for inducing flowering of Davidia involucrata by simulating seasonal changes, characterized in that: The specific steps are as follows: S1. Select 2-3 year old Davidia involucrata seedlings and pre-culture them for 14 days under the conditions of 22℃ temperature and 16 hours of light / 8 hours of darkness; collect leaf and shoot tip tissues, and detect the concentrations of endogenous abscisic acid, gibberellin, cytokinin and auxin. According to the concentration ratio of abscisic acid to gibberellin, the plants are divided into hormone-balanced type, dormant type and active growth type. S2. For dormant plants, spray the leaves with a mixture of 50 mg / L gibberellin and 20 mg / L cytokinin every 7 days for a total of 4 times; for active growth plants, spray the leaves with a 100 mg / L chlormequat chloride solution; for hormone-balanced plants, perform routine water and fertilizer management; cultivate for 45 days to achieve a soluble sugar content of over 8% in the plant leaves. S3. Perform low-temperature vernalization treatment. From day 1 to 14, lower the temperature from 22℃ to 15℃ and adjust the photoperiod to 10 hours of light / 14 hours of darkness. From day 15 to 35, maintain the temperature at 8-10℃. From day 36 to 49, lower the temperature to 4-6℃ and control the relative humidity at 65%. Detect the abscisic acid concentration every 7 days. Vernalization is complete when the abscisic acid concentration reaches 2.5-3.0 times the initial concentration. S4. Immerse the base of the plant in a 200 mg / L gibberellin solution for 4 hours, raise the temperature to 18°C ​​at a rate of 2°C every 2 days, and adjust the photoperiod to 14 hours of light / 10 hours of darkness. S5. After the temperature stabilizes at 18-20℃, spray the leaves with a mixture of 30mg / L gibberellin, 15mg / L cytokinin and 10mg / L auxin, once every 5 days, for a total of 3 times; adjust the formula according to the concentration ratio of cytokinin to auxin; treat for 15-20 days until flower bud primordia form. S6. Maintain the temperature at 20-22℃ and the relative humidity at 75%-80%; spray the leaves with a mixture containing 5 mg / L cytokinin and 0.1 mg / L brassinolide, once every 7 days, for a total of 2 sprays; treat for 20-25 days until the flower buds show color; S7. Adjust the temperature to 18-20℃ and the relative humidity to 65%-70%, stop the hormone treatment, and record the flowering data.

2. The method for inducing flowering of Davidia involucrata by simulating seasonal changes according to claim 1, characterized in that: The specific method of step S1 is as follows: S1.1 Select 2-3 year old Davidia involucrata seedlings with a height of 30-50cm and a stem diameter of 0.8-1.2cm, and transplant them into cultivation containers with a volume of 3-5 liters. The cultivation substrate is a mixture of peat moss, perlite and vermiculite in a volume ratio of 2:1:

1. Place them in an artificial climate chamber with a temperature of 22±1℃, a photoperiod of 16 hours of light / 8 hours of darkness, a light intensity of 10000-12000 lux, and a relative humidity of 60%-70%. Irrigate with a nutrient solution with an electrical conductivity of 1.5-2.0mS / cm and a pH of 5.5-6.5, applying 200ml per plant every 3 days, and pre-cultivate for 14 days. S1.2 On the morning of the day the pre-culture ended, from 9:00 to 10:00, collect 2-3 grams of the 3rd to 5th fully expanded leaves from the top of each plant and 1-2 grams of the 0.5-1.0 cm shoot tip tissue from each plant. After quick-freezing in liquid nitrogen, store at -80℃. The concentrations of abscisic acid (ABA), gibberellin (GA3), cytokinin (CTK), and auxin (IAA) were determined by enzyme-linked immunosorbent assay (ELISA). Each sample was measured three times and the average value was taken. The ABA / GA ratio was calculated. Plants with a ratio of 0.8-1.2 were marked as hormone-balanced, those with a ratio greater than 1.5 were marked as dormant, and those with a ratio less than 0.6 were marked as active growth.

3. The method for inducing flowering of Davidia involucrata by simulating seasonal changes according to claim 2, characterized in that: The specific method of step S2 is as follows: S2.1 Based on the classification results of step S1.2, spray the leaves of dormant plants with a mixed solution containing 50 mg / L gibberellin GA3 and 20 mg / L cytokinin 6-BA, 30-50 ml per plant, once every 7 days, for a total of 4 times. Spray the leaves of active growth plants with a 100 mg / L chlormequat chloride (CCC) solution, using the same spraying method and frequency. Do not apply exogenous hormones to hormone-balanced plants, and continue irrigating with nutrient solution as in step S1-1. All three types of plants are cultured in an environment with a temperature of 22±1℃, a photoperiod of 16 hours of light / 8 hours of darkness, a light intensity of 10000-12000 lux, and a relative humidity of 60%-70%. S2.2 Maintain the culture conditions of step S2.1 and culture continuously for 45 days. On the 15th, 30th and 45th days after the start of culture, collect 1-2 grams of the 3rd to 5th fully unfolded leaves from the top of each plant. Use the anthrone colorimetric method to determine the soluble sugar content, use the Kjeldahl nitrogen determination method to determine the total nitrogen content and calculate the carbon-nitrogen ratio (C / N). When the test results on the 45th day show that the soluble sugar content of the leaves reaches more than 8% and the C / N ratio reaches more than 12, stop the culture. For plants that do not meet the standards, extend the culture for 7-14 days and retest.

4. The method for inducing flowering of Davidia involucrata by simulating seasonal changes according to claim 3, characterized in that: The specific method of step S3 is as follows: S3.1 Transfer the plants that have met the standards in step S2.2 to a low-temperature vernalization environment. From day 1 to 14, the temperature is reduced from 22℃ to 15℃ at a rate of 0.5℃ per day. The photoperiod is adjusted to 10 hours of light / 14 hours of darkness, the light intensity is 8000-10000 lux, and the relative humidity is 60%-65%. Apply 100-150 ml of nutrient solution to each plant every 5-7 days. From day 15 to 35, the temperature is maintained at 8-10℃. The temperature is raised to 12℃ for 2 days a week, with an interval of 3-4 days. Other conditions remain unchanged. From day 36 to 49, the temperature is reduced to 4-6℃, the relative humidity is 65%, the light intensity is 5000-8000 lux, and apply 50-100 ml of clean water to each plant every 10-14 days. Stop applying nutrient solution. S3.2 During step S3.1, on days 7, 14, 21, 28, 35, 42, and 49, collect 1-2 grams of the 3rd to 5th leaves from the top of each plant and 0.5-1.0 grams of the shoot tip tissue (0.5-1.0 cm from the top). After quick-freezing in liquid nitrogen, store at -80℃. Determine the abscisic acid (ABA) concentration using enzyme-linked immunosorbent assay (ELISA). Each sample is measured three times and the average value is taken. When the ABA concentration reaches 2.5-3.0 times the initial concentration in step S1-2 and the change from the next test value is less than 10%, proceed to step S4. Plants that do not meet the standard are vernalized for 7-14 days at 4-6℃ and monitored every 7 days.

5. The method for inducing flowering of Davidia involucrata by simulating seasonal changes according to claim 4, characterized in that: The specific method of step S4 is as follows: S4.1 Take out the plants that have completed vernalization in step S3.2, prepare a 200 mg / L gibberellin GA3 solution, immerse the base of the plant from the root collar upwards in the solution for 4 hours, the temperature is 10-12℃ and the relative humidity is 65%-70%, stir the solution once every 1 hour, after soaking, rinse the base with running water for 3-5 minutes, replant back into the original cultivation container, or irrigate the roots with 100 mL of 200 mg / L gibberellin GA3 solution as an alternative treatment; S4.2 After completing step S4.1, transfer the cultivation container to an artificial climate chamber. The initial temperature is 10℃, which is increased to 18±1℃ at a rate of 2℃ every 2 days and stabilized. The photoperiod is adjusted to 14 hours of light / 10 hours of darkness. The light intensity is gradually increased from 8000 lux to 12000-15000 lux, and the relative humidity is 65%~70%. Apply 150~200ml of nutrient solution to each plant every 3~5 days. After the temperature reaches 18℃, spray the leaves with a 50mg / L gibberellin GA3 solution on the 4th to 7th day, with a spraying amount of 30~50ml per plant. Collect leaf samples every 3~4 days to detect the GA3 / ABA ratio. When the ratio reaches 3.0 or above and is stable for two consecutive tests, proceed to step S5.

6. The method for inducing flowering of Davidia involucrata by simulating seasonal changes according to claim 5, characterized in that: The specific method of step S5 is as follows: S5.1 After the temperature stabilizes at 18-20℃ and the GA3 / ABA ratio reaches 3.0 or higher in step S4.2, prepare a compound hormone solution containing 30 mg / L gibberellin GA3, 15 mg / L cytokinin 6-BA, and 10 mg / L auxin NAA. Spray 30-50 ml per plant on the leaves every 5 days for 3 consecutive times. 1-2 days before each spraying, collect 1-2 grams of leaf and stem tip tissue to test the CTK / IAA ratio. If the ratio is less than 1.0, adjust the 6-BA concentration to 25 mg / L. If the ratio is greater than 2.0, reduce the NAA concentration to 5 mg / L or discontinue use. After each spraying, add 20-30 ml of 0.2% potassium dihydrogen phosphate solution at 2-4 hours intervals. S5.2 During step S5.1, maintain a temperature of 18-20℃, a photoperiod of 14 hours of light / 10 hours of darkness, a light intensity of 12000-15000 lux, and a relative humidity of 70%-75%. Apply 150-200 ml of nutrient solution to each plant every 3-4 days. The mass ratio of nitrogen, phosphorus, and potassium in the nutrient solution is N:P:K=1:2:

2. From the start of spraying, collect 0.5-1.0 g of apical meristem every 3-4 days to prepare paraffin sections with a thickness of 8-10 μm. After safranin-fast green staining, observe under an optical microscope. Record the number of days when flower bud primordia formation is confirmed, usually 15-20 days. For plants that have not reached the standard, spray 1-2 more times and extend the treatment by 5-7 days. After confirming the formation of flower bud primordia, proceed to step S6.

7. The method for inducing flowering of Davidia involucrata by simulating seasonal changes according to claim 6, characterized in that: The specific method of step S6 is as follows: S6.1 Transfer the plants confirmed to have formed flower bud primordia in step S5.2 to a culture environment with a temperature of 20-22℃, relative humidity of 75%-80%, a photoperiod of 14 hours of light / 10 hours of darkness, and a light intensity of 15000-20000 lux. Stop applying gibberellin GA3. Prepare a mixed solution containing 5 mg / L cytokinin 6-BA, 0.1 mg / L brassinolide BR, and 0.05% boron and molybdenum. Spray 30-50 ml per plant on the leaves every 7 days for 2 consecutive times. Irrigate every 5-7 days, applying 100-150 ml of nutrient solution per plant each time. Control the soil moisture content to 55%-60%. The nutrient solution should have a nitrogen-phosphorus-potassium ratio of N:P:K = 1:2:2, with 150-200 mg / L calcium and 50-80 mg / L magnesium. S6.

2. During step S6.1, maintain the same culture conditions. Starting from the day this step begins, observe and measure the inflorescence length, inflorescence diameter, bract opening degree, and color change every 4-5 days. Every 5-7 days, collect 0.5-1.0 grams of inflorescence tissue and determine the soluble sugar content using the anthrone colorimetric method. When the bracts change from light green to milky white or pure white, with a bract length of 5-8 cm, a width of 3-5 cm, a smooth, leathery surface, and intact edges, the color development of the flower buds is considered complete. The treatment time is usually 20-25 days. For plants that do not meet the standard, extend the culture time by 3-5 days and spray the mixed solution once more. After confirming that the color development of the flower buds is complete, proceed to step S7.

8. The method for inducing flowering of Davidia involucrata by simulating seasonal changes according to claim 7, characterized in that: The specific method of step S7 is as follows: S7.

1. Transfer the plants whose flower buds have been confirmed to have completed color development in step S6.2 to the flowering management environment. Adjust the temperature to 18-20℃, the relative humidity to 65%-70%, maintain a photoperiod of 14 hours of light / 10 hours of darkness, and a light intensity of 12000-15000 lux using diffused light. Stop all exogenous hormone treatments. Irrigate every 5-7 days. Apply 100-150 ml of nutrient solution with an electrical conductivity of 1.0-1.5 mS / cm and a pH of 5.5-6.5 to each plant each time. The N:P:K mass ratio of the nutrient solution is N:P:K=1:1:

1. Ventilate 2-3 times a day for 30-60 minutes each time. S7.2 During step S7.1, starting from the day the flower buds fully open, record the total number of flowering days, the total number of inflorescences, the duration of flowering, the length, width, and thickness of bracts, the Lab color value of bracts, the diameter of inflorescences, the number of florets, pollen viability, and the pollination success rate. Take photos from the front, side, and top using a camera with at least 12 megapixels. Retrospectively analyze the ABA, GA3, CTK, and IAA concentration data at each key time point throughout the process and plot the dynamic changes in hormones. Calculate the changing trends of the ABA / GA, GA / ABA, and CTK / IAA ratios. Define plants with bract length ≥5cm, L value ≥80, flowering period ≥10 days, and inflorescence diameter ≥3cm as successful cases. Enter the hormone curve data, processing parameters, and flowering data of successful cases into the database.

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