Method for evaluating growth performance of mangrove forest seedlings in estuary high-salinity environment

Abstract

The invention discloses a method for evaluating the growth performance of mangrove forest seedlings in an estuary high-salinity environment, and the method comprises the steps: constructing an analysis framework in which a dynamic salinity stress system and a multi-dimensional monitoring technology are coupled, and achieving the precise evaluation of the environmental adaptability of the mangrove forest seedlings; the method comprises the following steps: firstly, carrying out sterile germination and matrix domestication on a target estuary provenance, then establishing a three-dimensional stress model containing basic salinity maintenance, sudden salinity impact and tidal cycle fluctuation, and strictly controlling the sudden salinity impact amplitude within 20% of the historical extreme value of the estuary; synchronously acquiring canopy volume and root architecture parameters of the seedlings by adopting a three-dimensional laser scanning technology, and constructing a morphological database in combination with leaf apparent characteristic records; subcellular structure indexes such as cell wall thickening, chloroplast envelope integrity and mitochondrial ridge density are observed and analyzed through a transmission electron microscope, the molecular mechanism of osmotic regulation and energy metabolism is disclosed, and the concentration of suspended solids in the water body, the oxidation-reduction potential of sediments and the functional diversity of microorganisms are synchronously monitored.

Description

Technical Field

[0001] The present invention relates to the field of ecology and environmental science, and in particular to a method for evaluating the growth performance of mangrove seedlings in a high-salinity environment in an estuary. Background Art

[0002] Mangrove seedlings are an important life stage in the mangrove ecosystem. As an important transition zone connecting land and sea, estuarine mangrove ecosystems play an irreplaceable role in coastal protection, biodiversity conservation and carbon sequestration. However, the high salinity environment poses a significant challenge to the growth of mangrove seedlings. Accurately assessing their growth performance is of vital importance for understanding the ecological adaptation mechanism of mangroves and formulating scientific protection strategies.

[0003] Static salinity culture models under traditional laboratory conditions are unable to truly reflect the comprehensive environmental stresses faced by seedlings, resulting in significant limitations in the analysis of growth response mechanisms. The existing assessment technology system has four major defects: First, salinity stress simulations mostly use linear gradient increases or fixed high-salinity treatments, which can neither reproduce the sudden rise and fall of natural salinity nor easily induce non-physiological mortality interference due to extreme parameter settings. Second, morphological monitoring relies on manual sampling measurements, which is limited by plant damage caused by contact sampling and the two-dimensional observation dimension, making it difficult to simultaneously obtain canopy volume dynamics and root architecture parameters. Third, physiological response analysis is limited to macroscopic indicators such as chlorophyll content and proline accumulation, and lacks analysis of microscopic mechanisms such as cell wall structure remodeling and chloroplast membrane system stability. Fourth, environmental factor research often focuses on a single salinity variable, ignoring the interactive effects of ecological factors such as suspended solids sedimentation, anaerobic state of sediments, and microbial community function, resulting in significant deviations between assessment results and actual field performance. To this end, a method for assessing the growth performance of mangrove seedlings in a high-salinity environment in an estuary is proposed. Summary of the Invention

[0004] The present invention provides the following technical solution: a method for evaluating the growth performance of mangrove seedlings in a high-salinity environment in an estuary, comprising the following steps: S1. Species adaptability pre-processing steps: Mature mangrove seed embryos were collected from the target estuary ecological zone. After germination in sterile culture medium, seedlings with uniform plant height and intact root systems were selected as experimental materials and subjected to substrate adaptation before transplantation. S2. Steps for dynamic simulation of salinity gradient: Based on the seasonal variation of salinity in the target estuary, a three-dimensional stress system was constructed, including a basic salinity maintenance group, a sudden salinity shock group, and a tidal cycle fluctuation group. The instantaneous increase in salinity in the sudden salinity shock group did not exceed 20% of the historical extreme value of the estuary. S3. Multi-dimensional morphology monitoring steps: Three-dimensional laser scanning technology was used to regularly obtain the aboveground canopy volume and underground root system configuration parameters of the seedlings, and simultaneously record the leaf surface characteristic parameters; S4. Physiological response analysis steps: Transmission electron microscopy was performed on leaf and root tissues collected during the experimental period, focusing on analyzing changes in cell wall thickening, chloroplast envelope integrity, and mitochondrial cristae density; S5. Ecological factor coupling analysis steps: The suspended solids concentration in the experimental container, the redox potential gradient of the sediment, and the functional diversity index of the microbial community were simultaneously monitored to construct an ecological factor-growth indicator correlation network; S6. Steps for building a comprehensive evaluation system: Integrate morphological monitoring data, physiological response indicators, and ecological factor parameters to establish an intelligent analysis platform that includes growth resilience index, physiological limit threshold, and ecological service efficiency evaluation modules; S7. Resilience Tracking Assessment Steps: During the experimental period, a period of salinity stress relief was set up, and the dynamic recovery trajectory of seedling growth parameters was continuously monitored to quantify the physiological compensation capacity after the relief of salinity stress. S8. Steps for outputting engineering repair solutions: Based on the comprehensive assessment results, an ecological restoration engineering design plan is automatically generated, which includes species configuration density gradient, salinity buffer zone spatial layout and micro-topography transformation parameters.

[0005] Species adaptation pre-processing steps: Mature mangrove seed embryos were collected from the target estuarine ecological zone. After germination on sterile culture medium, seedlings with a height difference of no more than 5% and intact root systems were selected for use as experimental materials. Before transplantation, the seedlings were acclimated for no more than 14 days by gradually changing the substrate formula (a mixture of target estuarine sediment and artificially formulated substrate). During the acclimation period, the seedling survival rate and the number of new roots were monitored daily. Steps for dynamic simulation of salinity gradient: Based on the seasonal variation of salinity in the target estuary, a three-dimensional stress system was constructed: Basic salinity maintenance group: the error between the set salinity value and the historical monthly average salinity data of the estuary shall not exceed ±2‰; Sudden salinity shock group: the instantaneous increase in salinity does not exceed 20% of the historical extreme value of the estuary, and the shock duration does not exceed 48 hours; Tidal cycle fluctuation group: simulates daily salinity fluctuations, and the fluctuation amplitude is within ±15% of the measured tidal range data of the target estuary; Multi-dimensional morphology monitoring steps: Using 3D laser scanning technology, the aboveground canopy volume and underground root system architecture parameters of the seedlings were measured over a 72-hour period, including taproot length, number of lateral roots, root surface area, and fractal dimension. Leaf surface characteristic parameters, including the coefficient of variation of leaf inclination, leaf area index, and petiole curvature, were also recorded simultaneously. Physiological response analysis steps: The leaf and root tissues collected during the experimental period were observed under transmission electron microscopy, with the following key analyses: Cell wall thickening: measure the ratio of primary cell wall to secondary cell wall thickness; Chloroplast envelope integrity: statistical order of grana lamellae arrangement; Changes in mitochondrial cristae density: Calculate the number of cristae structures per unit area; Ecological factor coupling analysis steps: The suspended solids concentration in the experimental vessel (measured using a laser particle size analyzer), sediment redox potential gradient (using a microelectrode array with spacing ≤ 2 cm), and microbial community functional diversity index (based on the carbon source utilization spectrum of the Biolog ECO board) were simultaneously monitored. An ecological factor-growth indicator association network was constructed, with network nodes including 12 environmental parameters and 18 morphological and physiological indicators. Steps to build a comprehensive evaluation system: Integrate morphological monitoring data, physiological response indicators and ecological factor parameters to establish an intelligent analysis platform, including: Growth resilience index: calculated based on canopy volume recovery rate and root system architecture stability; Physiological limit threshold: determined by the mutation point of chloroplast membrane damage rate; Ecological service efficiency evaluation module: quantify carbon sequestration capacity and bank protection function maintenance; Resilience Tracking Assessment Steps During the experimental period, a period of salinity stress relief was set up. The dynamic recovery trajectory of seedling growth parameters was monitored for 72 consecutive hours to quantify the recovery rate of leaf surface characteristic parameters and the compensation rate of root architecture parameters after salinity stress relief. Steps for outputting engineering repair solutions: Based on the comprehensive assessment results, the ecological restoration project design plan is automatically generated, including: Species configuration density gradient: Divide the density range from 0 to 100 plants / m² according to the growth resilience index; Spatial layout of salinity buffer zone: Optimize buffer zone width based on tidal cycle fluctuation group data; Microtopography modification parameters: Design the sediment accumulation slope in combination with root system architecture parameters.

[0006] Preferably, in the species adaptation pre-treatment step, the substrate adaptation acclimation adopts a step-by-step salinity increase method, with the weekly salinity increase not exceeding 2‰ until it is consistent with the average salinity of the target estuary. During the substrate adaptation acclimation stage, a step-by-step salinity increase method is implemented, with the initial salinity as the benchmark, and the salinity is gradually increased by no more than 2‰ per week until the salinity value is consistent with the measured average salinity of the target estuary. During the process, an automatic control system is used to monitor salinity changes in real time, and gradual adjustments are made to ensure the stability of the acclimation environment.

[0007] Preferably, in the salinity gradient dynamic simulation step, the salinity change frequency of the tidal cycle fluctuation group is synchronized with the measured tidal cycle of the target estuary, and the daily salinity fluctuation amplitude does not exceed 5‰. In the salinity gradient dynamic simulation link, a tidal cycle fluctuation group is set, and its salinity change frequency is completely synchronized with the measured tidal cycle of the target estuary. A precision control device is used to ensure that the daily salinity fluctuation amplitude does not exceed 5‰. A multi-parameter sensor is used to collect salinity data in real time, and dynamic comparison and calibration are performed with the preset tidal model.

[0008] Preferably, in the multi-dimensional morphological monitoring step, the analysis of underground root architecture parameters includes quantitative indicators of taproot curvature, lateral root branching angle, and root hair density. In the analysis of underground root architecture parameters, a three-dimensional root scanning system combined with image analysis software is used to quantitatively measure taproot curvature, lateral root branching angle, and root hair density. By setting a standard sampling frame and an automatic counting algorithm, standardized collection and statistical analysis of root morphological parameters are achieved.

[0009] Preferably, in the physiological response analysis step, the degree of cell wall thickening is characterized by the cellulose crystallinity index, and the integrity of the chloroplast envelope is observed using fluorescence staining. During the physiological response analysis stage, the cellulose crystallinity index is measured by X-ray diffraction to characterize the degree of cell wall thickening, and the integrity of the chloroplast envelope is simultaneously observed using fluorescence staining (such as neutral red staining). Images of the stained tissue sections are obtained using a microscopic imaging system and quantitatively evaluated using image analysis software.

[0010] Preferably, in the ecological factor coupling analysis step, the microbial community functional diversity index is determined using the Biolog EcoPlate™ method, with a focus on differences in carbon source utilization patterns. In determining microbial community functional diversity, the Biolog EcoPlate™ method is used for carbon source metabolic fingerprint analysis. Microbial utilization of 31 carbon sources is monitored through 96 hours of continuous culture, and principal component analysis (PCA) is used to analyze the differential characteristics of carbon source utilization patterns among different salinity treatment groups.

[0011] Preferably, in the resilience tracking and assessment step, the period of relief from staged salinity stress is set to no more than 72 hours, and key morphological indicators are recorded at a frequency of no less than 6 hours during the recovery period. In the resilience tracking and assessment, the period of relief from staged salinity stress is set to no more than 72 hours, and an automated monitoring system is used to record key morphological indicators such as plant height and leaf number every 6 hours. The recovery rate is quantified through time series analysis.

[0012] Preferably, in the resilience tracking and assessment step, the physiological compensation capacity is quantitatively characterized by the chlorophyll content recovery rate of new leaves and the proportion of root regeneration length. A portable chlorophyll meter is used to regularly monitor the chlorophyll content of new leaves. A root scanning system is used to measure the root regeneration length per unit time. The chlorophyll content recovery rate and the proportion of root regeneration length are calculated to establish a quantitative model for physiological compensation capacity.

[0013] Preferably, in the step of constructing the comprehensive evaluation system, the weights for calculating the growth resilience index are set to: morphological indicators account for 55%, physiological indicators account for 30%, and ecological factors account for 15%. In calculating the growth resilience index, the analytic hierarchy process (AHP) is used to set the weights: morphological indicators (55%), physiological indicators (30%), and ecological factors (15%). A comprehensive evaluation model is constructed using an expert scoring matrix and consistency test to achieve standardized integration of multi-dimensional data.

[0014] Preferably, in the step of outputting the engineering restoration plan, the spatial layout of the salinity buffer zone adopts a gradual salinity gradient design, and the micro-topography modification parameters include optimized values for tidal gully density and beach elevation. In the spatial layout design of the salinity buffer zone, the gradual salinity gradient design principle is adopted, and the beach elevation and tidal gully density parameters are optimized in combination with a hydrodynamic model. Spatial interpolation analysis is performed using a geographic information system (GIS) to generate a micro-topography modification parameter map.

[0015] In summary, compared with the prior art, the present invention provides a method for evaluating the growth performance of mangrove seedlings in a high-salinity estuary environment, which has the following beneficial effects: This study creatively constructed a three-dimensional stress model using a dynamic salinity gradient simulation system. The sudden salinity shock group's instantaneous increase amplitude control strategy realistically reproduces the natural scene of sudden salinity changes in the estuary while avoiding unnatural mortality caused by extreme parameters. The introduction of the tidal cycle fluctuation group allows for spatiotemporal coupling of hydrodynamic conditions and salinity changes. Compared to static salinity cultivation models, this model can more accurately analyze the response mechanism of mangrove seedlings to dynamic environments. The application of 3D laser scanning technology in morphological monitoring enables non-contact, full-dimensional data collection. Its centimeter-level spatial resolution can simultaneously capture aboveground canopy volume dynamics and underground root system architecture parameters, breaking through the subjective and destructive limitations of traditional manual measurement methods. Real-time recording of leaf surface characteristic parameters further enriches the morphological assessment dimension and provides a data foundation for establishing quantitative relationships between growth phenotypes and environmental factors. The physiological response analysis phase, using transmission electron microscopy, extended the assessment scale to the subcellular structure level for the first time. Analysis of cell wall thickening revealed the microscopic mechanism of osmotic regulation, assessment of chloroplast membrane integrity correlated with the damage threshold of the photosynthetic apparatus, and changes in mitochondrial cristae density reflected the remodeling of energy metabolism. The quantitative analysis of these indicators provided a molecular and cellular evidence chain for understanding the mechanisms of salt stress tolerance. The ecological factor coupling analysis method breaks through the limitations of single environmental factor research. By constructing a correlation network of water suspended matter concentration, sediment redox potential gradient and microbial functional diversity, it reveals the comprehensive regulatory path of seedling growth by multi-factor interactions. This evaluation framework from a systems biology perspective more truly reflects the synergistic mechanism of the complex estuarine ecosystem. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the evaluation method of the present invention. DETAILED DESCRIPTION

[0017] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0018] See also Figure 1 The present invention provides a technical solution, a method for evaluating the growth performance of mangrove seedlings in a high-salinity environment in an estuary, comprising the following steps: S1. Species adaptability pre-processing steps: Mature mangrove seed embryos were collected from the target estuary ecological zone. After germination in sterile culture medium, seedlings with uniform plant height and intact root systems were selected as experimental materials and subjected to substrate adaptation before transplantation. S2. Steps for dynamic simulation of salinity gradient: Based on the seasonal variation of salinity in the target estuary, a three-dimensional stress system was constructed, including a basic salinity maintenance group, a sudden salinity shock group, and a tidal cycle fluctuation group. The instantaneous increase in salinity in the sudden salinity shock group did not exceed 20% of the historical extreme value of the estuary. S3. Multi-dimensional morphology monitoring steps: Three-dimensional laser scanning technology was used to regularly obtain the aboveground canopy volume and underground root system configuration parameters of the seedlings, and simultaneously record the leaf surface characteristic parameters; S4. Physiological response analysis steps: Transmission electron microscopy was performed on leaf and root tissues collected during the experimental period, focusing on analyzing changes in cell wall thickening, chloroplast envelope integrity, and mitochondrial cristae density; S5. Ecological factor coupling analysis steps: The suspended solids concentration in the experimental container, the redox potential gradient of the sediment, and the functional diversity index of the microbial community were simultaneously monitored to construct an ecological factor-growth indicator correlation network; S6. Steps for building a comprehensive evaluation system: Integrate morphological monitoring data, physiological response indicators, and ecological factor parameters to establish an intelligent analysis platform that includes growth resilience index, physiological limit threshold, and ecological service efficiency evaluation modules; S7. Resilience Tracking Assessment Steps: During the experimental period, a period of salinity stress relief was set up, and the dynamic recovery trajectory of seedling growth parameters was continuously monitored to quantify the physiological compensation capacity after the relief of salinity stress. S8. Steps for outputting engineering repair solutions: Based on the comprehensive assessment results, an ecological restoration engineering design plan is automatically generated, which includes species configuration density gradient, salinity buffer zone spatial layout and micro-topography transformation parameters.

[0019] Species adaptation pre-processing steps: Mature mangrove seed embryos were collected from the target estuarine ecological zone. After germination on sterile culture medium, seedlings with a height difference of no more than 5% and intact root systems were selected for use as experimental materials. Before transplantation, the seedlings were acclimated for no more than 14 days by gradually changing the substrate formula (a mixture of target estuarine sediment and artificially formulated substrate). During the acclimation period, the seedling survival rate and the number of new roots were monitored daily. Steps for dynamic simulation of salinity gradient: Based on the seasonal variation of salinity in the target estuary, a three-dimensional stress system was constructed: Basic salinity maintenance group: the error between the set salinity value and the historical monthly average salinity data of the estuary shall not exceed ±2‰; Sudden salinity shock group: the instantaneous increase in salinity does not exceed 20% of the historical extreme value of the estuary, and the shock duration does not exceed 48 hours; Tidal cycle fluctuation group: simulates daily salinity fluctuations, and the fluctuation amplitude is within ±15% of the measured tidal range data of the target estuary; Multi-dimensional morphology monitoring steps: Using 3D laser scanning technology, the aboveground canopy volume and underground root system architecture parameters of the seedlings were measured over a 72-hour period, including taproot length, number of lateral roots, root surface area, and fractal dimension. Leaf surface characteristic parameters, including the coefficient of variation of leaf inclination, leaf area index, and petiole curvature, were also recorded simultaneously. Physiological response analysis steps: The leaf and root tissues collected during the experimental period were observed under transmission electron microscopy, with the following key analyses: Cell wall thickening: measure the ratio of primary cell wall to secondary cell wall thickness; Chloroplast envelope integrity: statistical order of grana lamellae arrangement; Changes in mitochondrial cristae density: Calculate the number of cristae structures per unit area; Ecological factor coupling analysis steps: The suspended solids concentration in the experimental vessel (measured using a laser particle size analyzer), sediment redox potential gradient (using a microelectrode array with spacing ≤ 2 cm), and microbial community functional diversity index (based on the carbon source utilization spectrum of the Biolog ECO board) were simultaneously monitored. An ecological factor-growth indicator association network was constructed, with network nodes including 12 environmental parameters and 18 morphological and physiological indicators. Steps to build a comprehensive evaluation system: Integrate morphological monitoring data, physiological response indicators and ecological factor parameters to establish an intelligent analysis platform, including: Growth resilience index: calculated based on canopy volume recovery rate and root system architecture stability; Physiological limit threshold: determined by the mutation point of chloroplast membrane damage rate; Ecological service efficiency evaluation module: quantify carbon sequestration capacity and bank protection function maintenance; Resilience Tracking Assessment Steps During the experimental period, a period of salinity stress relief was set up. The dynamic recovery trajectory of seedling growth parameters was monitored for 72 consecutive hours to quantify the recovery rate of leaf surface characteristic parameters and the compensation rate of root architecture parameters after salinity stress relief. Steps for outputting engineering repair solutions: Based on the comprehensive assessment results, the ecological restoration project design plan is automatically generated, including: Species configuration density gradient: Divide the density range from 0 to 100 plants / m² according to the growth resilience index; Spatial layout of salinity buffer zone: Optimize buffer zone width based on tidal cycle fluctuation group data; Micro-topography modification parameters: design sediment accumulation slope in combination with root system architecture parameters; Through species adaptation pre-treatment steps, the genetic background of the experimental materials is ensured to be highly consistent with the target estuarine ecological zone, eliminating the impact of provenance differences on the evaluation results and improving experimental repeatability; The dynamic simulation system of salinity gradient truly reflects the temporal and spatial heterogeneity of estuarine salinity. The sudden salinity shock group is designed to take into account both ecological safety thresholds and the simulation requirements of extreme events, avoiding unnatural deaths caused by excessive stress. Multi-dimensional morphological monitoring technology enables simultaneous acquisition of above-ground and underground parameters, and the accuracy of three-dimensional laser scanning can capture early warning signals of root system architecture; Physiological response analysis focused on subcellular structural changes, and transmission electron microscopy revealed the cell wall thickening and energy metabolism regulation mechanism, providing a microscopic evidence chain for salt tolerance mechanism research; The ecological factor coupling analysis established a multi-parameter correlation network, breaking through the limitations of traditional single-factor experiments and clarifying the influence weight of the water-sediment-microorganism interface interaction on seedling growth; The comprehensive assessment system integrates multi-source heterogeneous data, with the growth resilience index quantifying the species' potential to resist stress, the physiological limit threshold defining the safe cultivation boundary, and the ecological service efficiency assessment directly connecting to engineering application needs; Resilience tracking assessment breaks through the traditional survival rate evaluation model, quantifies physiological compensation capacity through dynamic recovery trajectories, and provides resilience evaluation indicators for saline-alkali land vegetation reconstruction; The engineering restoration plan output module realizes the evaluation-design closed loop, the density gradient configuration and micro-topography parameter optimization improve the survival rate of the restoration project, and the salinity buffer zone design extends the maintenance period of ecological service functions.

[0020] During the species adaptation pretreatment step, a step-by-step salinity increase method is used for substrate adaptation, with the weekly salinity increase not exceeding 2‰ until it matches the average salinity of the target estuary. During the substrate adaptation acclimation stage, a step-by-step salinity increase method is implemented, using the initial salinity as a benchmark and gradually increasing the salinity by no more than 2‰ per week until the salinity value matches the measured average salinity of the target estuary. During the process, an automatic control system is used to monitor salinity changes in real time, and gradual adjustments are made to ensure the stability of the acclimation environment. The slow increase in salinity can effectively induce the plant to produce an osmotic adjustment adaptation mechanism, avoid physiological damage caused by sudden salinity changes, and significantly improve the survival rate and adaptability of the target species in the salinity environment of the target estuary.

[0021] In the dynamic simulation step of salinity gradient, the frequency of salinity change in the tidal cycle fluctuation group is synchronized with the measured tidal cycle of the target estuary, and the daily salinity fluctuation amplitude does not exceed 5‰. In the dynamic simulation step of salinity gradient, a tidal cycle fluctuation group is set up, and its salinity change frequency is completely synchronized with the measured tidal cycle of the target estuary. A precision control device is used to ensure that the daily salinity fluctuation amplitude does not exceed 5‰. A multi-parameter sensor is used to collect salinity data in real time, and dynamic comparison and calibration are performed with the preset tidal model. Synchronizing tidal cycles and salinity fluctuations can accurately reproduce natural hydrological conditions, providing a highly ecologically relevant experimental environment for studying plant salinity response mechanisms, and significantly improving the field applicability of simulation experimental results.

[0022] The multi-dimensional morphological monitoring step involves analyzing underground root architecture parameters, including quantitative indicators of taproot curvature, lateral root branching angles, and root hair density. A three-dimensional root scanning system combined with image analysis software was used to quantify taproot curvature, lateral root branching angles, and root hair density. By setting a standard sampling frame and an automatic counting algorithm, standardized collection and statistical analysis of root morphological parameters were achieved. Quantifying root architecture parameters can reveal the mechanisms by which salinity stress affects underground resource acquisition, providing a scientific basis for optimizing taproot growth direction and regulating lateral root development, and assisting in the development of efficient root improvement programs.

[0023] During the physiological response analysis step, the degree of cell wall thickening is characterized by the cellulose crystallinity index, and the integrity of the chloroplast envelope is observed using fluorescence staining. During the physiological response analysis stage, the cellulose crystallinity index is measured using X-ray diffraction to characterize the degree of cell wall thickening, while fluorescence staining (such as neutral red staining) is used to observe the integrity of the chloroplast envelope. Images of stained tissue sections are acquired using a microscopic imaging system and quantitatively evaluated using image analysis software. Multi-index physiological testing can systematically analyze the dynamic changes in cell structure and function under salinity stress, providing data support for the screening of physiological markers for stress resistance and the development of physiological enhancement technologies.

[0024] During the ecological factor coupling analysis step, the BiologEcoPlate™ method was used to determine the functional diversity index of the microbial community, focusing on differences in carbon source utilization patterns. Carbon source metabolic fingerprinting was performed using the BiologEcoPlate™ method. Microbial utilization of 31 carbon sources was monitored through 96 hours of continuous incubation. Principal component analysis (PCA) was then used to analyze the differences in carbon source utilization patterns between salinity treatments. Functional diversity index analysis can reveal the functional responses of microbial communities to salinity gradients, providing an ecological basis for the development of plant-microbe collaborative restoration systems.

[0025] During the resilience tracking assessment, the period of relief from salinity stress was set to no longer than 72 hours, and key morphological indicators were monitored at a frequency of no less than six hours during the recovery period. During the resilience tracking assessment, the period of relief from salinity stress was set to no longer than 72 hours. An automated monitoring system was used to record key morphological indicators, such as plant height and leaf count, every six hours. Time series analysis was used to quantify the recovery rate. High-frequency monitoring accurately captured the rapid recovery phase after stress relief, providing a time threshold reference for determining the optimal salinity pulse management strategy and optimizing the dynamic control plan for ecological restoration projects.

[0026] During the resilience tracking assessment, physiological compensation capacity is quantified by the chlorophyll recovery rate of new leaves and the proportion of root regeneration length. Chlorophyll content in new leaves is regularly measured using a portable chlorophyll meter. Root regeneration length per unit time is measured using a root scanning system. The chlorophyll recovery rate and the proportion of root regeneration length are then calculated to establish a quantitative model for physiological compensation capacity. Quantification of physiological indicators can intuitively reflect a plant's ability to reallocate resources in salinity-fluctuating environments, providing comparable biological parameters for assessing the resilience potential of different species.

[0027] During the construction of the comprehensive assessment system, the weighting for calculating the resilience index was set to 55% for morphological indicators, 30% for physiological indicators, and 15% for ecological factors. The analytic hierarchy process (AHP) was used to assign weights for morphological indicators (55%), physiological indicators (30%), and ecological factors (15%). A comprehensive assessment model was constructed using an expert scoring matrix and consistency testing, achieving standardized integration of multidimensional data. The weighting system balanced the contributions of different assessment dimensions, avoiding bias in single-indicator evaluations and significantly improving the accuracy of ecological restoration effect predictions.

[0028] During the engineering restoration plan output step, the spatial layout of the salinity buffer zone adopts a gradual salinity gradient design, and the microtopography modification parameters include optimized values for tidal gully density and beach elevation. In the spatial layout design of the salinity buffer zone, the gradual salinity gradient design principle is adopted, combined with a hydrodynamic model to optimize beach elevation and tidal gully density parameters. Spatial interpolation analysis is performed using a geographic information system (GIS) to generate a microtopography modification parameter map. The gradual design can simulate the ecological functions of the natural salinity transition zone. Microtopography optimization can significantly improve the hydrological connectivity and species diversity of the buffer zone, providing a feasible spatial solution for estuarine ecological restoration projects.

[0029] This plan involves collecting mature mangrove seed embryos from the target estuary ecological zone, germinating them in sterile culture medium, and selecting seedlings with a height difference of no more than 5% and intact root systems as experimental materials. Prior to transplantation, the seedlings are acclimated for no more than 14 days by gradually changing the substrate formula. The substrate formula consists of a gradient mixture of target estuary sediment and an artificially formulated substrate in a volume ratio of 3:7 to 7:3. During the acclimation period, the seedling survival rate and the number of new roots are monitored daily. During the substrate acclimation phase, a stepwise salinity increase method is implemented, using the initial salinity as the benchmark and gradually increasing the salinity by no more than 2‰ per week until the salinity value matches the measured average salinity of the target estuary. During the process, an automatic control system is used to monitor salinity changes in real time and ensure the stability of the acclimation environment through gradual adjustments. A three-dimensional stress system was constructed based on the seasonal variation of salinity in the target estuary, including a basic salinity maintenance group, a sudden salinity shock group, and a tidal cycle fluctuation group. The basic salinity maintenance group set the salinity value to an error of no more than ±2‰ from the historical monthly average salinity data of the estuary. The sudden salinity shock group set the instantaneous increase in salinity to no more than 20% of the historical extreme value of the estuary, and the shock duration did not exceed 48 hours. The tidal cycle fluctuation group simulated daily salinity fluctuations, with the fluctuation amplitude not exceeding ±15% of the measured tidal range data of the target estuary. The salinity change frequency was fully synchronized with the measured tidal cycle of the target estuary, and a precision control device was used to ensure that the daily salinity fluctuation amplitude did not exceed 5‰. Three-dimensional laser scanning technology was used to obtain the aboveground canopy volume and underground root configuration parameters of the seedlings in a 72-hour cycle. The aboveground parameters included canopy volume, leaf inclination variation coefficient, leaf area index, and petiole curvature. The underground parameters included taproot length, number of lateral roots, root surface area, fractal dimension, taproot curvature, lateral root branching angle, and root hair density. The leaf surface characteristic parameters were recorded simultaneously, and standardized collection and statistical analysis of root morphological parameters were achieved through a standard sampling frame and automatic counting algorithm. S4. Physiological response analysis steps: Leaf and root tissues collected during the experimental period were observed under transmission electron microscopy, focusing on the degree of cell wall thickening, chloroplast envelope integrity, and changes in mitochondrial cristae density. The degree of cell wall thickening was characterized by measuring the cellulose crystallinity index using X-ray diffraction. The integrity of the chloroplast envelope was assessed by observing the order of grana lamellae using neutral red fluorescence staining. Changes in mitochondrial cristae density were used to calculate the number of cristae per unit area. The suspended solids concentration, sediment redox potential gradient, and microbial community functional diversity index within the experimental vessel were simultaneously monitored. Suspended solids concentration was measured using a laser particle size analyzer, while sediment redox potential was measured using a microelectrode array with spacing ≤ 2 cm. Microbial community functional diversity was assessed using the Biolog EcoPlate™ method to determine carbon source metabolic fingerprints, focusing on differences in carbon source utilization patterns. An ecological factor-growth indicator correlation network was constructed, encompassing 12 environmental parameters and 18 morphological and physiological indicators. Morphological monitoring data, physiological response indicators, and ecological factor parameters were integrated to establish an intelligent analysis platform that includes a growth resilience index, physiological limit thresholds, and an ecological service efficiency evaluation module. The growth resilience index calculation weights were set to 55% for morphological indicators, 30% for physiological indicators, and 15% for ecological factors. The weights were set using the analytic hierarchy process, and a comprehensive evaluation model was constructed using an expert scoring matrix and consistency test. The physiological limit threshold was determined by the mutation point of the chloroplast membrane damage rate, and the ecological service efficiency evaluation module quantified the carbon sequestration capacity and the maintenance of the bank protection function. The experimental period included periodic salinity stress relief phases, which lasted no longer than 72 hours, and monitoring of key morphological indicators was increased to every six hours during the recovery period. The dynamic recovery trajectory of seedling growth parameters was monitored for 72 consecutive hours to quantify the recovery rate of leaf surface characteristic parameters and the compensation rate of root architecture parameters after salinity stress relief. The physiological compensation capacity was quantitatively characterized by the recovery rate of chlorophyll content in new leaves and the proportion of root regeneration length. Based on the comprehensive assessment results, an ecological restoration project design plan is automatically generated, including the species configuration density gradient, salinity buffer zone spatial layout, and micro-topography modification parameters. The species configuration density gradient is divided into a density range of 0-100 plants / m² based on the quantitative results of the growth resilience index. The salinity buffer zone spatial layout adopts a gradual salinity gradient design and optimizes the buffer zone width based on tidal cycle fluctuation group data. Micro-topography modification parameters are combined with root system architecture parameters to design the sediment accumulation slope and include optimized values for tidal gully density and beach elevation.

[0030] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0031] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for evaluating the growth performance of mangrove seedlings in a high-salinity estuarine environment, characterized in that: The steps include: S1. Species adaptability pre-processing steps: Mature mangrove seed embryos were collected from the target estuary ecological zone. After germination in sterile culture medium, seedlings with uniform plant height and intact root systems were selected as experimental materials and subjected to substrate adaptation before transplantation. S2. Steps for dynamic simulation of salinity gradient: Based on the seasonal variation of salinity in the target estuary, a three-dimensional stress system was constructed, including a basic salinity maintenance group, a sudden salinity shock group, and a tidal cycle fluctuation group. The instantaneous increase in salinity in the sudden salinity shock group did not exceed 20% of the historical extreme value of the estuary. S3. Multi-dimensional morphology monitoring steps: Three-dimensional laser scanning technology was used to regularly obtain the aboveground canopy volume and underground root system configuration parameters of the seedlings, and simultaneously record the leaf surface characteristic parameters; S4. Physiological response analysis steps: Transmission electron microscopy was performed on leaf and root tissues collected during the experimental period, focusing on analyzing changes in cell wall thickening, chloroplast envelope integrity, and mitochondrial cristae density; S5. Ecological factor coupling analysis steps: Simultaneously monitor the suspended solids concentration in the experimental container, the redox potential gradient of the sediment, and the functional diversity index of the microbial community; S6. Steps for building a comprehensive evaluation system: Integrate morphological monitoring data, physiological response indicators, and ecological factor parameters to establish an intelligent analysis platform that includes growth resilience index, physiological limit threshold, and ecological service efficiency evaluation modules; S7. Resilience Tracking Assessment Steps: During the experimental period, a period of salinity stress relief was set up, and the dynamic recovery trajectory of seedling growth parameters was continuously monitored to quantify the physiological compensation capacity after the relief of salinity stress. S8. Steps for outputting engineering repair solutions: Based on the comprehensive assessment results, an ecological restoration engineering design plan is automatically generated, which includes species configuration density gradient, salinity buffer zone spatial layout and micro-topography transformation parameters.

2. The method for evaluating the growth performance of mangrove seedlings in a high-salinity estuarine environment according to claim 1, characterized in that: In the species adaptation pre-treatment step, the substrate adaptation acclimation adopts a step-by-step salinity increase method, with the weekly salinity increase not exceeding 2‰ until it is consistent with the average salinity of the target estuary.

3. The method for evaluating the growth performance of mangrove seedlings in a high-salinity estuarine environment according to claim 1, characterized in that: In the salinity gradient dynamic simulation step, the salinity change frequency of the tidal cycle fluctuation group is synchronized with the measured tidal cycle of the target estuary, and the daily salinity fluctuation amplitude does not exceed 5‰.

4. The method for evaluating the growth performance of mangrove seedlings in a high-salinity estuarine environment according to claim 1, characterized in that: In the multi-dimensional morphological monitoring step, the analysis of underground root system configuration parameters includes quantitative indicators of main root curvature, lateral root branching angle and root hair density.

5. The method for evaluating the growth performance of mangrove seedlings in a high-salinity estuarine environment according to claim 1, characterized in that: In the physiological response analysis step, the degree of cell wall thickening is characterized by the cellulose crystallinity index, and the integrity of the chloroplast envelope is observed by fluorescent staining.

6. The method for evaluating the growth performance of mangrove seedlings in a high-salinity estuarine environment according to claim 1, characterized in that: In the ecological factor coupling analysis step, the microbial community functional diversity index is determined using the BiologEcoPlate™ method, with a focus on differences in carbon source utilization patterns.

7. The method for evaluating the growth performance of mangrove seedlings in a high-salinity estuarine environment according to claim 1, characterized in that: In the resilience tracking and assessment step, the period of relief of staged salinity stress is set to no more than 72 hours, and the monitoring frequency during the recovery period is increased to record key morphological indicators no less than every 6 hours.

8. The method for evaluating the growth performance of mangrove seedlings in a high-salinity estuarine environment according to claim 1, characterized in that: In the resilience tracking and evaluation step, the physiological compensation capacity is quantitatively characterized by the recovery rate of chlorophyll content in new leaves and the proportion of root regeneration length.

9. The method for evaluating the growth performance of mangrove seedlings in a high-salinity estuarine environment according to claim 1, characterized in that: In the step of constructing the comprehensive evaluation system, the calculation weights of the growth resilience index are set as follows: morphological indicators account for 55%, physiological indicators account for 30%, and ecological factors account for 15%.

10. The method for evaluating the growth performance of mangrove seedlings in a high-salinity estuarine environment according to claim 1, characterized in that: In the step of outputting the engineering restoration plan, the spatial layout of the salinity buffer zone adopts a gradual salinity gradient design, and the micro-topography transformation parameters include the optimized values of tidal gully density and beach elevation.

Images