A method and apparatus for evaluating the drought resistance of bamboo based on the coupling of leaf anatomical structure dynamics and photosynthetic physiology.

Abstract

This invention relates to the field of analytical testing technology, and in particular to a method and apparatus for evaluating bamboo drought resistance based on the coupling of leaf anatomical structure dynamics and photosynthetic physiology. Under standardized sampling and measurement conditions, this invention focuses on repeatable dynamic characteristics of anatomical structure and couples them with photosynthetic physiological state, while simultaneously suppressing the risk of distortion in water use efficiency in the low-value range. This results in a method and apparatus for evaluating bamboo drought resistance that can be used to accurately and robustly determine the drought resistance level of bamboo under drought stress and rehydration conditions, and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of analytical testing technology, and in particular to a method and apparatus for evaluating the drought resistance of bamboo based on the coupling of dynamic leaf anatomy and photosynthetic physiology. Background Technology

[0002] Plant drought resistance is a crucial foundation for germplasm resource screening, breeding selection, and cultivation management. Currently, rapid evaluation methods for plant drought resistance typically rely on photosynthetic physiological indicators such as gas exchange and chlorophyll fluorescence, or on phenotypic indicators such as growth rate and survival rate.

[0003] The above methods have the following shortcomings in engineering applications: First, single physiological indicators are easily affected by the measurement window, the instantaneous state of the leaves, and environmental fluctuations, especially in the later stages of drought stress, the transpiration rate ( E ) or porosity ( Gs The value may be close to extremely low, resulting in low water use efficiency. WUE First, the effects of drought on leaf anatomy are often magnified mathematically, leading to the misjudgment of near-complete cessation of gas exchange as high drought resistance. Second, many studies rely on static observations or qualitative descriptions of leaf anatomy, lacking standardized target areas and normalized benchmarks. The overall leaf deformation caused by drought and rehydration significantly impacts the stability and repeatability of area measurements. Third, existing evaluation processes often fail to integrate drought and rehydration—two continuous and interconnected processes—within a single evaluation system, resulting in unstable grading and poor comparability across batches. In some cases, germplasm that performs well during drought may die during rehydration due to irreversible damage to its anatomical structure.

[0004] Therefore, there is an urgent need to develop a more accurate method for evaluating plant drought resistance. Summary of the Invention

[0005] To address the shortcomings of existing methods, this invention provides a method for evaluating bamboo drought resistance based on the coupling of leaf anatomical structure dynamics and photosynthetic physiology, comprising:

[0006] Bamboo was subjected to drought treatment and rehydration treatment sequentially to obtain drought-treated bamboo leaves and rehydrated bamboo leaves; photosynthetic gas exchange parameters were collected from untreated bamboo leaves, drought-treated bamboo leaves, and rehydrated bamboo leaves respectively to obtain the net photosynthetic rate (…). A ), transpiration rate ( E ) and porosity ( Gs The water use efficiency of bamboo leaves under drought treatment was calculated. WUE D );

[0007] Microscopic images of cross-sections of bamboo leaves were acquired for each group. Two adjacent bundles of vascular bundle sheaths were identified in the images, and the region between the two bundles of sheaths was defined as the inter-bundle region based on their inner boundaries.ROI ), calculate the area of ​​the inter-beam region ( A IBS ); Identify the cavities of vesicular and spindle-shaped cells within the interfascicular region, and calculate the area of ​​vesicular cells ( A BC ) and the area of ​​the cavity of the spindle cell ( A FCAV The area of ​​vesicular cells and the cavity area of ​​spindle cells were normalized using the area of ​​the interfascicular region as an internal benchmark to obtain the normalized proportion of vesicular cells. R BC ) and the normalized proportion of spindle cell cavities ( R FCAV );

[0008] The dynamic characteristics of the anatomical structure were calculated using the normalized proportions of vesicular cells and the normalized proportions of spindle-shaped cells with cavities; these dynamic characteristics included: the vesicular cell drought deformation index (…). DI BC ), spindle cell cavity drought deformation index ( DI FCAV ), vesicular cell rehydration index ( RI BC ) and the rehydration index of spindle cell cavity ( RI FCAV );

[0009] The dynamic characteristics of the anatomical structure were weighted and summed with the water use efficiency of bamboo leaves under drought treatment to obtain the structure-physiology coupling index (SPI). Coupling Index Among them, a threshold triggering rule is set to identify the water use efficiency distortion risk range. When the water use efficiency is entered into the water use efficiency distortion risk range, the water use efficiency is penalized and / or downweighted.

[0010] The penalty correction is to multiply the measured water use efficiency of bamboo leaves under drought treatment by a penalty factor less than 1.

[0011] The weighting process involves making the weighting coefficient of the water use efficiency of bamboo leaves under drought treatment smaller than the sum of the weighting coefficients of the dynamic features of the anatomical structure.

[0012] The drought resistance level of plants is determined based on the structure-physiology coupling index.

[0013] The drought resistance evaluation method of this invention, under standardized sampling and measurement conditions, focuses on repeatable dynamic characteristics of anatomical structures and couples them with photosynthetic physiological state, while also addressing low-value ranges. WUE D The risk of distortion is suppressed, thereby improving the robustness, accuracy, and discriminative power of drought resistance assessment results under extreme stress conditions.

[0014] In this invention, the drought resistance evaluation method uses the region between the two bundle sheaths as the standardized target region, and uses... A IBS Normalize area-type features to internal benchmarks, reduce the impact of overall leaf deformation caused by drought and rehydration on the stability of anatomical structure measurements, and improve repeatability and cross-batch comparability.

[0015] Meanwhile, using the area / ratio of cavities of vesicular and spindle cells, as well as their drought deformation index and rehydration index, as structural dynamic criteria, can simultaneously characterize drought damage and rehydration recovery capacity, thereby improving the stability of the classification.

[0016] Moreover, through E / Gs Threshold triggering mechanism WUE D To mitigate the risk of distortion and avoid misjudging near-cessation of gas exchange as high drought resistance, thereby reducing... WUE D False high valuation leads to misjudgment.

[0017] Furthermore, coupling the dynamic characteristics of anatomical structures with photosynthetic physiological parameters to form a unified evaluation output can improve the robustness and interpretability of drought resistance assessment.

[0018] Preferably, the photosynthetic gas exchange parameters are measured under saturated light intensity conditions.

[0019] Preferably, the threshold triggering rule is as follows:

[0020] The area is considered to be in the risk zone of water use efficiency distortion when any of the following conditions are met:

[0021] (i) E ≤ E th (ii) Gs ≤ Gs th (iii) E and / or Gs The proportion is lower than the preset threshold of the average value of its corresponding untreated bamboo leaf group;

[0022] in, E For transpiration rate, E th The threshold for transpiration rate. Gs For porosity, Gs th This is the porosity conductance threshold.

[0023] Preferably, the E th , Gs thThe preset ratio threshold can be predetermined based on at least one or a combination of the following: distribution characteristics of untreated bamboo leaf group data, instrument detection limit, stability of low-value segment measurement, and variability index of repeated measurements.

[0024] Preferably, the threshold is determined using an absolute threshold or a relative threshold;

[0025] When using an absolute threshold E th Take 0.20~0.35 mmol·m -2 ·s -1 More preferably 0.25 mmol·m -2 ·s -1 ; Gs th Take 0.005~0.02 mol·m -2 ·s -1 More preferably 0.01 mol·m -2 ·s -1 Furthermore, the unit of the absolute threshold is consistent with the unit of the measured value;

[0026] Preferably, the threshold can be set such that E or Gs When it is below this threshold, WUE D Abnormal amplification phenomena and / or boundary points where repeated measurements show a significant increase in variability.

[0027] When a relative threshold is used, the preset ratio is 10% to 30%, preferably 20%.

[0028] In some implementation schemes, net photosynthetic rate, transpiration rate, and stomatal conductance are measured using a portable photosynthesis measurement system.

[0029] In some implementations, the unit of the absolute threshold is consistent with the unit output by the portable photosynthesis measurement system.

[0030] Preferably, the penalty factor is 0.01 to 0.20, more preferably 0.05;

[0031] And / or, the weighting factor for the water use efficiency of bamboo leaves under drought treatment is 0.3~0.45;

[0032] And / or, the weighted coefficients of the dynamic features of the anatomical structure are summed to 0.55~0.7.

[0033] Preferably, the sum of all weighting coefficients is 1.

[0034] Preferably, the formula for calculating the water use efficiency is as follows:

[0035]

[0036] in, WUE D Water use efficiency of bamboo leaves under drought conditions; A Net photosynthetic rate; E This refers to the transpiration rate;

[0037] The calculation formula for the normalization process is as follows:

[0038]

[0039]

[0040] in, R BC The normalized proportion of vesicular cells; A BC The area of ​​the vesicular cells; A IBS The area of ​​the inter-beam region; R FCAV The normalized proportion of spindle cell cavities; A FCAV The area of ​​the cavity of the spindle cell;

[0041] The formula for calculating the drought deformation index of vesicular cells is as follows:

[0042]

[0043] in, The drought deformation index of vesicular cells; The proportion of vesicular cells in bamboo leaves after drought treatment is normalized. The proportion of vesicular cells in untreated bamboo leaves after normalization; max (·, ε) is the lower limit of the denominator. It is a very small positive number, and its value ranges from 10. -8 ~10 -3 Preferably 10 -6 ;

[0044] The formula for calculating the cavity drought deformation index of spindle cells is as follows:

[0045]

[0046] in, The drought deformation index of spindle cell cavities; The proportion of spindle-shaped cells in bamboo leaves after drought treatment; The proportion of spindle-shaped cells in untreated bamboo leaves after normalization; max (·, ε) is the lower limit of the denominator; It is a very small positive number, and its value ranges from 10. -8 ~10-3 Preferably 10 -6 ;

[0047] The formula for calculating the vesicular cell rehydration index is as follows:

[0048]

[0049] in, The rehydration index of vesicular cells; The proportion of vesicular cells in bamboo leaves after rehydration treatment and normalization. The proportion of vesicular cells in bamboo leaves after drought treatment is normalized. The proportion of vesicular cells in untreated bamboo leaves after normalization; max (·, ε) is the lower limit of the denominator. It is a very small positive number, and its value ranges from 10. -8 ~10 -3 Preferably 10 -6 ;

[0050] The formula for calculating the rehydration index of spindle cell cavities is as follows:

[0051]

[0052] in, The rehydration index of spindle cell cavities; The proportion of spindle-shaped cells in bamboo leaves after rehydration treatment; The proportion of spindle-shaped cells in bamboo leaves after drought treatment; The proportion of spindle-shaped cells in untreated bamboo leaves after normalization; max (·, ε) is the lower limit of the denominator; It is a very small positive number, and its value ranges from 10. -8 ~10 -3 Preferably 10 -6 ;

[0053] By introducing a very small positive number ε and using max(·, ε) as the lower limit of the denominator when division operations are involved, we can avoid the instability caused by the denominator being zero or very small, and further enhance the robustness.

[0054] The formula for calculating the structure-physiology coupling index is as follows:

[0055]

[0056] in, Coupling Index The structure-physiology coupling index; Anatomy Dynamic i For the first i Dynamic characteristics of the anatomical structure Anatomy Dynamic 1 =1- DIBC , Anatomy Dynamic 2 =1- DI FCAV , Anatomy Dynamic 3 = RI BC , Anatomy Dynamic 4 = RI FCAV ; w i For the first i Weighting coefficients for the dynamic characteristics of the anatomical structure; WUE D The measured values ​​of water use efficiency of bamboo leaves under drought treatment; f penalty As a penalty factor, when entering the water use efficiency distortion risk zone, f penalty Take a value less than 1, when the water use efficiency distortion risk range has not been entered. f penalty Take 1; w j The weighting coefficient for water use efficiency of bamboo leaves under drought treatment.

[0057] Preferably, the method for determining the drought resistance level of a plant based on the structure-physiology coupling index is as follows:

[0058] The structure-physiology coupling index is ≥ 0.80, which is judged as extremely drought-resistant (Level 1).

[0059] If the structure-physiology coupling index is 0.60 ≤ and <0.80, it is judged as having strong drought resistance (level 2).

[0060] If the structure-physiology coupling index is 0.45 ≤ and <0.60, the drought resistance is judged to be moderate (level 3).

[0061] If the structure-physiology coupling index is 0.30 ≤ and <0.45, the drought resistance is judged to be weak (level 4).

[0062] The structure-physiology coupling index is <0.30, indicating extremely weak drought resistance (level 5).

[0063] Preferably, the evaluation method further includes: after determining the drought resistance level of the plant, using the data on differences in the expression of photosynthesis-related gene families and / or differences in the content of photosynthesis-related metabolites, comparing the results with the evaluation results or generating a matching index for consistency verification or confidence enhancement.

[0064] Introducing data on differences in the expression of photosynthesis-related gene families and / or differences in the content of photosynthesis-related metabolites as evidence for consistency verification can enhance the confidence of mechanistic explanations and judgments without affecting the implementation of basic procedures.

[0065] Preferably, the photosynthesis-related gene family includes at least one of ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit (RbcS), phosphoenolpyruvate carboxylase (PEPC), pyruvate phosphate dikinase (PPDK), β-carbonic anhydrase (BCA), and NADP-malicase (NADP-ME).

[0066] Preferably, the photosynthesis-related metabolites include at least one of ribulose 1,5-bisphosphate (RuBP), malic acid (Mal), and pyruvate (Pyr).

[0067] Furthermore, the present invention provides an apparatus for implementing the bamboo drought resistance evaluation method based on the coupling of leaf anatomical structure dynamics and photosynthetic physiology, the apparatus comprising:

[0068] Standardized sampling module; used to collect bamboo leaves and determine the sampling area;

[0069] Anatomical sampling and microscopic imaging module; used to acquire cross-sectional microscopic images of bamboo leaves;

[0070] Image analysis and anatomical feature extraction module; used to extract the cavity area of ​​vesicular and spindle cells in the interfascicular region and calculate and output dynamic features of anatomical structures;

[0071] Photosynthetic gas exchange parameter acquisition module; used to determine water use efficiency.

[0072] Low-value distortion range identification and correction module; used to determine whether water use efficiency has entered the distortion risk range, and to perform penalty correction and / or weight reduction processing on water use efficiency.

[0073] The structure-physiology coupling evaluation module is used to weight and sum the dynamic characteristics of anatomical structures with water use efficiency, and output the structure-physiology coupling index.

[0074] And a drought resistance level determination module; used to determine and output the drought resistance level of plants based on the structure-physiology coupling index.

[0075] Preferably, the device further includes:

[0076] Consistency verification module; used to include data on differences in the expression of photosynthesis-related gene families and / or differences in the content of photosynthesis-related metabolites, and output consistency comparison results or matching indexes of the evaluation results.

[0077] Furthermore, this invention provides the application of the bamboo drought resistance evaluation method based on the coupling of leaf anatomical structure dynamics and photosynthetic physiology, or the device described herein, in the screening of drought-resistant bamboo germplasm resources or the breeding of drought-resistant bamboo.

[0078] Preferably, the bamboo is a species of bamboo in the genus *Phyllostachys*, and more preferably, *Phyllostachys bournei*.

[0079] Preferably, the application is for rapid screening and germplasm identification of *Phyllostachys breviscapus* under drought stress and rehydration recovery conditions.

[0080] Preferably, the bamboo leaves are selected from the 3rd to 4th fully unfolded functional leaves in the upper part of the plant, avoiding the midrib, and the middle part of the leaf is used as the measurement and sampling area.

[0081] Preferably, gas exchange parameters are collected and dissected on the same leaf to ensure consistency between anatomical features and photosynthetic physiological parameters.

[0082] Preferably, the leaf tissue is fixed, embedded, and sectioned to obtain a cross-sectional microscopic image of the bamboo leaf.

[0083] Preferably, the photosynthetic gas exchange parameters are collected before the leaf tissue is fixed, embedded, and sliced.

[0084] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0085] This invention, under standardized sampling and measurement conditions, focuses on repeatable dynamic features of anatomical structures and couples them with photosynthetic physiological state. It also suppresses the risk of distortion in water use efficiency in the low value range, and develops a method and device for evaluating the drought resistance of bamboo. This method and device can be used to accurately and robustly determine the drought resistance level of bamboo under drought stress and rehydration conditions, and has broad application prospects. Attached Figure Description

[0086] Figure 1 This is a schematic diagram illustrating the area of ​​the interfascicular region and the identification of cavities between vesicular and spindle cells in this invention.

[0087] Figure 2 This is a schematic diagram illustrating the threshold triggering, penalty correction, and dynamic changes in anatomical structure characteristics of the present invention; where A represents the transpiration rate. E With porosity Gs Low value interval identification and threshold E th , Gs th The diagram shows that B represents the water use efficiency of bamboo leaves under drought treatment. WUE DA schematic diagram comparing the effects of penalty correction before and after, where C represents the phased trajectory of the dynamic characteristics of the anatomical structure. The control phase uses a baseline value of 1, and the drought phase uses (1- DI Characterizing structural steady state, the rehydration stage is based on RI Characterize the efficiency of structural recovery.

[0088] Figure 3 This is a schematic diagram of the apparatus for evaluating bamboo drought resistance based on the coupling of leaf anatomical structure dynamics and photosynthetic physiology according to the present invention. Detailed Implementation

[0089] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0090] In the embodiments provided in this specification, unless specific techniques or conditions are specified, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0091] In this invention, the area of ​​the inter-beam region ( A IBS The interfascicular region (IFR) refers to the area of ​​a complete cross-section of a leaf between two adjacent bundle sheaths, defined by the inner boundaries of the two bundle sheaths. This region includes mesophyll cells, epidermal cells, and other cell types. The interfascicular region is defined by the inner boundaries of the two bundle sheaths in the cross-sectional micrograph. ROI () Figure 1 ).

[0092] area of ​​spindle cell cavity ( A FCAV The area enclosed by the cavity outline formed by spindle cells within the region between the two bundle sheaths is called the cavity boundary. Since the outline of the spindle cell body in a cross-section is not easily and stably identified, this invention uses the cavity boundary as an objective and quantifiable object.

[0093] The following examples use potted plants of *Dracaena sanderiana* as test materials and are only used to illustrate the advantages of the present invention. They should not be construed as limiting the present invention.

[0094] Example 1

[0095] This embodiment provides a method for evaluating the drought resistance of bamboo based on the coupling of leaf anatomical structure dynamics and photosynthetic physiology. The steps are as follows:

[0096] 1. Material handling, sampling design, and data acquisition

[0097] Potted *Phyllostachys pubescens* plants of uniform age and growth status were used as experimental materials. The bamboo was subjected to drought treatment and rehydration treatment sequentially. The drought treatment involved natural drought for 30 days, reducing the soil moisture content to approximately 40%. The rehydration treatment involved restoring water supply after the drought ended. Plants that grew naturally without any treatment served as a control group. Measurements and samples were taken at the control group, at the end of the drought, and 24 hours after rehydration. Three groups were established: a control group (untreated bamboo leaves), a drought treatment group (drought-treated bamboo leaves), and a rehydration treatment group (rehydrated bamboo leaves). Under each treatment condition, the 3rd to 4th fully unfolded functional leaves from the upper part of the plant were selected, avoiding the midrib, and the middle of the leaf was used as the measurement and sampling area. Sampling time was uniformly set at 14:00. Three plants were set up for each treatment group as biological replicates, and three leaves were selected from each plant for measurement. To ensure consistency between the measurement of gas exchange parameters and the sampling of anatomical structures, this embodiment first measures the photosynthetic gas exchange parameters of each leaf, and then collects and fixes anatomical samples from adjacent locations on the same leaf for subsequent section imaging and extraction of dynamic features of anatomical structures. Optionally, data on differences in the expression of photosynthetic-related gene families and the content of photosynthetic-related metabolites are collected simultaneously at the above time points as evidence of consistency verification, but are not essential limitations for implementing this invention.

[0098] The photosynthesis-related gene family includes the small subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase (RbcS), phosphoenolpyruvate carboxylase (PEPC), pyruvate phosphate dikinase (PPDK), β-carbonic anhydrase (BCA), and NADP-malylase (NADP-ME). The photosynthesis-related metabolites include ribulose-1,5-bisphosphate (RuBP), malate (Mal), and pyruvate (Pyr).

[0099] 2. Measurement of photosynthetic gas exchange parameters WUE D Calculation and threshold triggering rule correction

[0100] Photosynthetic gas exchange parameters were measured under saturated light intensity using a portable photosynthesis measurement system (LI-6800). The saturated light intensity for different bamboo species was determined through preliminary experiments; in this embodiment, the saturated light intensity for *Phyllostachys breviscapus* was approximately 2000 μmol·m⁻¹. -2 ·s -1 Simultaneously, the CO2 concentration was set at 400 ppm, the leaf chamber temperature at 25 ℃, and the flow rate at 500 μmol·s. -1 The relative humidity was 70-75%; the net photosynthetic rate of the drought-treated group samples was measured ( A ), transpiration rate ( E ), porosity ( Gs) and intercellular CO2 concentration ( Ci Indicators such as ).

[0101] In the later stages of drought stress, weak drought-resistant germplasm E and / or Gs It may drop to extremely low levels, often leading to WUE D The numerical value is mathematically amplified abnormally. Therefore, this embodiment sets a threshold trigger rule for judgment and penalty correction:

[0102] When any of the following conditions are met, the area is determined to be in the water use efficiency distortion risk zone, triggering a penalty correction:

[0103] (i) E ≤ E th (ii) Gs ≤ Gs th (iii) E and / or Gs The proportion is lower than the preset threshold of the mean of its corresponding control group;

[0104] in, E For transpiration rate, E th The threshold for transpiration rate. Gs For porosity, Gs th This is the porosity conductance threshold.

[0105] In this embodiment, an absolute threshold is used for determination. E th = 0.25mmol·m -2 ·s -1 ,Pick Gs th 0.01 mol·m -2 ·s -1 In another alternative implementation, a relative threshold determination can also be used, i.e. E and / or Gs A penalty correction is triggered when the percentage falls below a preset threshold (e.g., 20%) of the corresponding control group mean.

[0106] This embodiment observed in the drought treatment group data that weakly drought-resistant germplasm showed improvement at the end of the drought period. E As low as 0.09 mmol·m -2 ·s -1 , Gs It can be as low as 0.005 mol·m -2 ·s -1 After triggering the penalty correction, the measured water use efficiency of the drought treatment group is multiplied by a penalty factor less than 1. fpenalty This embodiment uses a penalty factor. f penalty = 0.05, thus effectively suppressing WUE D The abnormal amplification ensures the accuracy and discriminative power of the evaluation index under extreme stress.

[0107] 3. Leaf cross-sectional imaging and dynamic feature extraction of anatomical structures

[0108] After measuring photosynthetic gas exchange parameters, samples were taken from adjacent locations within the measurement area of ​​the same leaf. These samples were then fixed, dehydrated, embedded, and sliced ​​to prepare transverse leaf samples with a thickness of 8–12 μm. Microscopic images (≥3) of the cross-sections of each bamboo leaf group were acquired using a microscopic imaging system under the same magnification and scale conditions. Adjacent bundles of vascular bundles were identified within the images, and the region between the two bundles was defined as the interbract region (standardized target area) based on their inner boundaries. ROI ), and identify vesicular and spindle-shaped cell cavities within the interfascicular region; calculate the area of ​​the interfascicular region using image processing software (e.g., ImageJ). A IBS The area of ​​vesicular cells and the area of ​​spindle cell cavities were calculated, and the areas of vesicular cells and spindle cell cavities were normalized using the area of ​​the interfascicular region as an internal benchmark. The calculation method is as follows:

[0109]

[0110]

[0111] in, R BC The normalized proportion of vesicular cells; A BC The area of ​​the vesicular cells; A IBS The area of ​​the inter-beam region; R FCAV The normalized proportion of spindle cell cavities; A FCAV This represents the area of ​​the cavity in the spindle cell.

[0112] Based on this, the dynamic characteristics of the anatomical structure are calculated using the normalized proportion of vesicular cells and the normalized proportion of cavitary cells; the dynamic characteristics of the anatomical structure include: vesicular cell drought deformation index, cavitary cell drought deformation index, vesicular cell rehydration index, and cavitary cell rehydration index.

[0113] For the control group, drought-treated group, and rehydrated group, the degree of tissue damage under water stress was quantitatively characterized by calculating the drought deformation index. This index reflects the proportion of structural features shrinking relative to normal water conditions.

[0114] The formula for calculating the drought deformation index of vesicular cells is as follows:

[0115]

[0116] in, The drought deformation index of vesicular cells; The proportion of vesicular cells in bamboo leaves after drought treatment is normalized. The proportion of vesicular cells in untreated bamboo leaves after normalization; max (·, ε) is the lower limit of the denominator. It is a very small positive number, with a value of 10. -6 .

[0117] The formula for calculating the cavity drought deformation index of spindle cells is as follows:

[0118]

[0119] in, The drought deformation index of spindle cell cavities; The proportion of spindle-shaped cells in bamboo leaves after drought treatment; The proportion of spindle-shaped cells in untreated bamboo leaves after normalization; max (·, ε) is the lower limit of the denominator; It is a very small positive number, with a value of 10. -6 .

[0120] To further evaluate the structural resilience and reconstruction ability of plants after stress relief, this invention introduces a rehydration index to quantify the degree of regression of structural features from a drought state to a control state. The structural recovery efficiency of different germplasms is described by comparing the recovery amount after rehydration treatment with the damage amount during drought. When the rehydration index approaches 1.0, it indicates that the anatomical structure has achieved complete morphological regression. When the rehydration index is greater than 1.0, it indicates that the degree of structural recovery exceeds the control level; in a preferred embodiment, the rehydration index result can be truncated to [0, 1] to enhance robustness.

[0121] The formula for calculating the vesicular cell rehydration index is as follows:

[0122]

[0123] in, The rehydration index of vesicular cells; The proportion of vesicular cells in bamboo leaves after rehydration treatment and normalization. The proportion of vesicular cells in bamboo leaves after drought treatment is normalized. The proportion of vesicular cells in untreated bamboo leaves after normalization; max (·, ε) is the lower limit of the denominator. It is a very small positive number, with a value of 10. -6 .

[0124] The formula for calculating the rehydration index of spindle cell cavities is as follows:

[0125]

[0126] in, The rehydration index of spindle cell cavities; The proportion of spindle-shaped cells in bamboo leaves after rehydration treatment; The proportion of spindle-shaped cells in bamboo leaves after drought treatment; The proportion of spindle-shaped cells in untreated bamboo leaves after normalization; max (·, ε) is the lower limit of the denominator; It is a very small positive number, with a value of 10. -6 .

[0127] 4. Calculate the structure-physiology coupling index and determine the drought resistance level of the plant.

[0128] Calculate the water use efficiency of bamboo leaves under drought treatment:

[0129]

[0130] in, WUE D Water use efficiency of bamboo leaves under drought conditions; A Net photosynthetic rate; E This refers to the transpiration rate;

[0131] The above-extracted DI BC and DI FCAV and RI BC and RI FCAV The indicators together constitute the dynamic characteristic items of the anatomical structure ( Anatomy Dynamic i The input variables of ) (where i =1, 2, 3, 4). In this embodiment, it is defined as: Anatomy Dynamic 1 =1- DI BC ; Anatomy Dynamic 2 =1- DI FCAV ; Anatomy Dynamic 3 = RIBC ; Anatomy Dynamic 4 = RI FCAV .

[0132] in, Anatomy Dynamic 1 and Anatomy Dynamic 2 It is a reverse steady-state index of drought damage (the larger the value, the smaller the damage). Anatomy Dynamic 3 and Anatomy Dynamic 4 This is an indicator of rehydration capacity (the larger the value, the more complete the recovery). In this embodiment, the calculation results of the above indicator are truncated and limited to [0, 1].

[0133] The formula assigns different weighting coefficients to each indicator based on its sensitivity to changes in moisture content. w i and compared with the penalty factor f penalty Corrected water use efficiency of bamboo leaves under drought treatment ( WUE D The logic is fused together to output a coupling index used to determine the drought resistance level. The calculation formula is as follows:

[0134]

[0135] In this embodiment, the specific allocation of each weight is as follows: weighting coefficients for dynamic features of anatomical structures. w i The sum is set to 0.6: Drought Damage Index w 1 (right DI BC )and w 2 (right DI FCAV The values ​​were set to 0.2 for each; for the recovery capacity index w 3 (right RI BC )and w 4 (right RI FCAV The weighting coefficients for water use efficiency of bamboo leaves under drought treatment were set to 0.1 to highlight the core weight of structural steady-state in drought resistance evaluation. w j Set to 0.4.

[0136] The threshold triggering and penalty correction mechanism and the changes in dynamic characteristics of anatomical structures are illustrated as follows: Figure 2 As shown.

[0137] Figure 2 A in the figure represents the transpiration rate. E With porosity Gs Low value range identification and threshold E th , Gs th ; Figure 2 B in the figure shows the water use efficiency of bamboo leaves under drought treatment. WUE D The comparison before and after punishment correction is used to inhibit... E / Gs Extremely low WUE D Abnormal magnification; Figure 2 C in the figure represents the staged trajectory of the structural dynamics, where the control stage takes a baseline value of 1, and the drought stage takes a value of (1- DI The reversed steady state characterizing drought damage, with the rehydration stage as... RI Characterize the efficiency of structural recovery.

[0138] Based on the above structure-physiology coupling index ( Coupling Index The calculation formula combines the dynamic characteristic terms of the anatomical structure with the penalized and corrected values. WUE D Performing coupled calculations yields Coupling Index The drought resistance level is determined based on the structure-physiology coupling index (SPI). A SPI ≥ 0.80 indicates extremely high drought resistance (Level 1); 0.60 ≤ SPI < 0.80 indicates high drought resistance (Level 2); 0.45 ≤ SPI < 0.60 indicates moderate drought resistance (Level 3); 0.30 ≤ SPI < 0.45 indicates low drought resistance (Level 4); and SPI < 0.30 indicates extremely low drought resistance (Level 5).

[0139] Optional step: Perform consistency verification using data on differences in the expression of photosynthesis-related gene families and the content of photosynthesis-related metabolites.

[0140] After determining the drought resistance level of the plants, the data on the differences in expression of photosynthesis-related gene families and the differences in the content of photosynthesis-related metabolites are used to compare the consistency with the evaluation results or generate matching indexes for consistency verification or confidence enhancement.

[0141] For example, compared with the control group, the representative gene of RbcS showed a downregulation trend, while some genes of the NADP-ME, PEPC, PPDK, and BCA families related to photosynthesis showed an upregulation trend; compared with the drought group, the representative gene of RbcS showed a rebound in the rehydration group. Regarding photosynthesis-related metabolites, RuBP was significantly downregulated in the drought group and rebounded after rehydration, while Mal and Pyr were significantly upregulated in the drought group.

[0142] When the evaluation result is "extremely drought-resistant," the plant typically exhibits a higher rehydration index due to the dynamic characteristics of bubble-like cells and spindle-shaped cavity structures. Furthermore, the evaluation results regarding gene expression and metabolite changes are consistent with the evaluation results of the coupling between anatomical structure dynamics and photosynthetic physiology. Conversely, when the evaluation result is "extremely weak drought resistance," the plant's structural damage is more pronounced, and... WUE D Lower value ranges trigger events more frequently. The above consistency information can be used to improve interpretability and confidence, but is not a necessary limitation for implementing this invention.

[0143] Example 2

[0144] This embodiment provides an apparatus for implementing the bamboo drought resistance evaluation method based on the coupling of leaf anatomical structure dynamics and photosynthetic physiology in Embodiment 1 (such as...). Figure 3 As shown), the device includes:

[0145] Standardized sampling module; used to collect bamboo leaves and determine the sampling area;

[0146] Anatomical sampling and microscopic imaging module; used to acquire cross-sectional microscopic images of bamboo leaves;

[0147] Image analysis and anatomical feature extraction module; used to extract the cavity area of ​​vesicular and spindle cells in the interfascicular region and calculate and output dynamic features of anatomical structures;

[0148] Photosynthetic gas exchange parameter acquisition module; used to determine water use efficiency.

[0149] Low-value distortion range identification and correction module; used to determine whether water use efficiency has entered the distortion risk range, and to perform penalty correction and / or weight reduction processing on water use efficiency.

[0150] The structure-physiology coupling evaluation module is used to weight and sum the dynamic characteristics of anatomical structures with water use efficiency, and output the structure-physiology coupling index.

[0151] And a drought resistance level determination module; used to determine and output the drought resistance level of plants based on the structure-physiology coupling index;

[0152] Optionally, the device further includes:

[0153] Consistency verification module; used to include data on differences in expression of photosynthesis-related gene families and differences in the content of photosynthesis-related metabolites, and output consistency comparison results or matching index of evaluation results.

[0154] Test case

[0155] This experiment used the bamboo drought resistance evaluation method based on the coupling of leaf anatomical structure dynamics and photosynthetic physiology as described in Example 1, as well as the rapid evaluation method based on gas exchange parameters commonly used in this field, to test and evaluate the drought resistance of the same batch of potted Bambusa textilis plants, and compared and verified the evaluation results of the two methods.

[0156] To verify the universality and corrective ability of the bamboo drought resistance evaluation method of this invention, potted plants of the same batch of *Dracaena fragrans* were selected and divided into 6 experimental groups (denoted as group 1 to 6). Each group contained 9 plants as biological replicates for the determination of gas exchange parameters and anatomical structure indicators, as well as for the population statistics of the final rehydration survival rate.

[0157] The treatment process included a control group, a drought treatment group (natural drought for 30 days, soil moisture content ≤ 45%), and a rehydration treatment group (drought treatment followed by irrigation to restore soil moisture content to ≥ 80%, with a 24-hour waiting period). Gas exchange parameters were measured in the control and drought treatment groups to calculate... WUE D Threshold trigger determination is performed; anatomical structural indicators are extracted from sampled plants to calculate... DI and RI Based on the dynamic characteristics of anatomical structures, the structure-physiology coupling index was calculated according to the method in Example 1, and the drought resistance level of the plants was determined. Table 1 shows... WUE D Low-value distortion triggering and penalty correction results.

[0158] The control method employs a rapid evaluation pathway based on gas exchange parameters: the net photosynthetic rate of the control group is measured during the control phase. A C Compared with control transpiration rate E C Calculate the water use efficiency of untreated bamboo leaves. WUE C = A C / E C Net photosynthetic rate was measured under drought conditions. A With transpiration rate E Calculate the water use efficiency of bamboo leaves under drought treatment. WUE D = A D / E D .

[0159] To facilitate alignment with the five-level stress resistance rating of the survival value after rehydration, this experimental example includes the uncorrected control method. WUE D The grouped statistics are mapped to a five-level resilience rating: First, the grouping statistics are calculated. WUE D The mean value is then used to map the groups to extremely strong, strong, moderate, weak, and extremely weak based on the quantile thresholds (e.g., 20%, 40%, 60%, and 80%) of the mean value in all groups. The results are used as the drought resistance rating of the control method in Table 2.

[0160] To verify the effectiveness and consistency of the two rapid evaluation methods mentioned above, this experimental example refers to the criteria and time points for "statistical survival after drought-rehydration" in the seedling stage of the current national standard Technical Specification for Identification and Evaluation of Wheat Drought Resistance (GB / T 21127—2007), using the survival rate 120 hours after rehydration as the result criterion. This standard counts survival 120 hours after rehydration, with leaves turning bright green as the survival criterion. This experimental example only refers to its drought-rehydration treatment process, statistical time points, and survival criteria to construct an objective endpoint indicator for consistency verification, and does not impose mandatory limitations on bamboo species based on this standard.

[0161] In this experiment, the survival rate was calculated as "number of surviving plants / total number of plants × 100%" and used as a criterion for judging drought resistance results. This was used to verify the consistency between the control method and the bamboo drought resistance evaluation method in Example 1.

[0162] Comparison Results and Analysis:

[0163] The comparison results are shown in Tables 1 and 2. It occurred at the end of the drought period. E and / or Gs In samples entering the low-value range, the control method is prone to [failure]. WUE D As a ratio indicator, it can be abnormally amplified when the denominator decreases significantly, leading to the risk of misjudgment. For example, a sample with almost stopped gas exchange may be misjudged as highly drought-resistant.

[0164] Example 1: Bamboo Drought Resistance Evaluation Method Using Threshold Triggering and Penalty Correction to Inhibit WUE The abnormal amplification, and further introduction of structural dynamic features ( DI BC , DI FCAV , RI BC , RI FCAV Coupled, the resulting Coupling IndexThe grading results show higher consistency with the survival rate criterion 120 hours after rehydration, and stable grading can be output at the end of drought and 24 hours after rehydration, thereby improving screening efficiency and enhancing the robustness of the judgment.

[0165] It is evident that the grading results of the bamboo drought resistance evaluation method of the present invention have a higher consistency with the true survival value 120 hours after rehydration (see Table 2). This can avoid misjudging high drought resistance when gas exchange is almost stopped under drought stress, thereby achieving accurate and robust drought resistance level determination.

[0166] Table 1 WUE D Low-value distortion triggering and penalty correction results

[0167]

[0168] Table 2 Comparison of drought resistance evaluation results and survival verification

[0169]

[0170] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for evaluating the drought resistance of bamboo based on the coupling of leaf anatomical structure dynamics and photosynthetic physiology, characterized in that, include: Bamboo was subjected to drought treatment and rehydration treatment in sequence to obtain bamboo leaves treated with drought and bamboo leaves treated with rehydration. Using untreated bamboo leaves, drought-treated bamboo leaves, and rehydrated bamboo leaves as subjects, photosynthetic gas exchange parameters were collected to obtain net photosynthetic rate, transpiration rate, and stomatal conductance, and the water use efficiency of drought-treated bamboo leaves was calculated. Microscopic images of cross-sections of bamboo leaves were acquired for each group. Two adjacent bundles of vascular bundles were identified in the images, and the area between the two bundles of bundles was defined as the inter-bundle region based on the inner boundary of the two bundles of vascular bundles. The area of ​​the inter-bundle region was calculated. Cavities of vesicular cells and spindle cells within the inter-bundle region were identified, and the areas of vesicular cells and spindle cells were calculated. The areas of vesicular cells and spindle cells were normalized using the area of ​​the inter-bundle region as an internal benchmark to obtain the normalized proportions of vesicular cells and spindle cells. The dynamic characteristics of the anatomical structure were calculated using the normalized proportion of vesicular cells and the normalized proportion of cavitary cells; the dynamic characteristics of the anatomical structure included: the drought deformation index of vesicular cells, the drought deformation index of cavitary cells, the rehydration index of vesicular cells, and the rehydration index of cavitary cells. The dynamic features of the anatomical structure are weighted and summed with the water use efficiency of bamboo leaves under drought treatment to obtain the structure-physiology coupling index; wherein, a threshold triggering rule is set to identify the water use efficiency distortion risk interval, and when the water use efficiency is entered into the water use efficiency distortion risk interval, the water use efficiency is penalized and / or reduced in weight. The penalty correction is to multiply the measured water use efficiency of bamboo leaves under drought treatment by a penalty factor less than 1. The weighting process involves making the weighting coefficient of the water use efficiency of bamboo leaves under drought treatment smaller than the sum of the weighting coefficients of the dynamic features of the anatomical structure. The drought resistance level of plants is determined based on the structure-physiology coupling index.

2. The method for evaluating bamboo drought resistance based on the coupling of leaf anatomical structure dynamics and photosynthetic physiology according to claim 1, characterized in that, The threshold triggering rules are as follows: The area is considered to be in the risk zone of water use efficiency distortion when any of the following conditions are met: (i) E ≤ E th (ii) Gs ≤ Gs th (iii) E and / or Gs The proportion is lower than the preset threshold of the average value of its corresponding untreated bamboo leaf group; in, E For transpiration rate, E th The threshold for transpiration rate. Gs For porosity, Gs th This is the porosity conductance threshold.

3. The method for evaluating bamboo drought resistance based on the coupling of leaf anatomical structure dynamics and photosynthetic physiology according to claim 2, characterized in that, The threshold is determined using an absolute threshold or a relative threshold; When using an absolute threshold E th Take 0.20~0.35 mmol·m -2 ·s -1 , Gs th Take 0.005~0.02 mol·m -2 ·s -1 Furthermore, the unit of the absolute threshold is consistent with the unit of the measured value; When a relative threshold is used, the preset ratio is 10% to 30%.

4. The method for evaluating bamboo drought resistance based on the coupling of leaf anatomical structure dynamics and photosynthetic physiology according to claim 1, characterized in that, The penalty factor is between 0.01 and 0.20; And / or, the weighting factor for the water use efficiency of bamboo leaves under drought treatment is 0.3~0.45; And / or, the weighted coefficients of the dynamic features of the anatomical structure are summed to 0.55~0.

7.

5. The method for evaluating bamboo drought resistance based on the coupling of leaf anatomical structure dynamics and photosynthetic physiology according to any one of claims 1 to 4, characterized in that, The formula for calculating water use efficiency is as follows: ； in, WUE D Water use efficiency of bamboo leaves under drought conditions; A Net photosynthetic rate; E This refers to the transpiration rate; The calculation formula for the normalization process is as follows: ； ； in, R BC The normalized proportion of vesicular cells; A BC The area of ​​the vesicular cells; A IBS The area of ​​the inter-beam region; R FCAV The normalized proportion of spindle cell cavities; A FCAV The area of ​​the cavity of the spindle cell; The formula for calculating the drought deformation index of vesicular cells is as follows: ； in, The drought deformation index of vesicular cells; The proportion of vesicular cells in bamboo leaves after drought treatment is normalized. The proportion of vesicular cells in untreated bamboo leaves after normalization. The lower limit of the denominator; It is a very small positive number, and its value ranges from 10. -8 ~10 -3 ; The formula for calculating the cavity drought deformation index of spindle cells is as follows: ； in, The drought deformation index of spindle cell cavities; The proportion of spindle-shaped cells in bamboo leaves after drought treatment; The proportion of spindle-shaped cells in untreated bamboo leaves after normalization. The lower limit of the denominator; It is a very small positive number, and its value ranges from 10. -8 ~10 -3 ; The formula for calculating the vesicular cell rehydration index is as follows: ； in, The rehydration index of vesicular cells; The proportion of vesicular cells in bamboo leaves after rehydration treatment and normalization. The proportion of vesicular cells in bamboo leaves after drought treatment is normalized. The proportion of vesicular cells in untreated bamboo leaves after normalization. The lower limit of the denominator; It is a very small positive number, and its value ranges from 10. -8 ~10 -3 ; The formula for calculating the rehydration index of spindle cell cavities is as follows: ； in, The rehydration index of spindle cell cavities; The proportion of spindle-shaped cells in bamboo leaves after rehydration treatment; The proportion of spindle-shaped cells in bamboo leaves after drought treatment; The proportion of spindle-shaped cells in untreated bamboo leaves after normalization. The lower limit of the denominator; It is a very small positive number, and its value ranges from 10. -8 ~10 -3 ; The formula for calculating the structure-physiology coupling index is as follows: ； in, Coupling Index The structure-physiology coupling index; Anatomy Dynamic i For the first i Dynamic characteristics of the anatomical structure Anatomy Dynamic 1 =1- DI BC , Anatomy Dynamic 2 =1- DI FCAV , Anatomy Dynamic 3 = RI BC , Anatomy Dynamic 4 = RI FCAV ; For the first i Weighting coefficients for the dynamic characteristics of the anatomical structure; WUE D The measured values ​​of water use efficiency of bamboo leaves under drought treatment; f penalty As a penalty factor, when entering the water use efficiency distortion risk zone, f penalty Take a value less than 1, when the water use efficiency distortion risk range has not been entered. f penalty Take 1; The weighting coefficient for water use efficiency of bamboo leaves under drought treatment.

6. The method for evaluating bamboo drought resistance based on the coupling of leaf anatomical structure dynamics and photosynthetic physiology according to any one of claims 1 to 4, characterized in that, The method for determining the drought resistance level of plants based on the structure-physiology coupling index is as follows: The structure-physiology coupling index is ≥ 0.80, indicating extremely strong drought resistance; A structure-physiology coupling index of 0.60 ≤ < 0.80 indicates strong drought resistance; A structure-physiology coupling index of 0.45 ≤ < 0.60 indicates moderate drought resistance. A structure-physiology coupling index of 0.30 ≤ < 0.45 indicates weak drought resistance; The structure-physiology coupling index is < 0.30, indicating extremely weak drought resistance.

7. The method for evaluating bamboo drought resistance based on the coupling of leaf anatomical structure dynamics and photosynthetic physiology according to any one of claims 1 to 4, characterized in that, Also includes: After determining the drought resistance level of the plants, data on differences in the expression of photosynthesis-related gene families and / or differences in the content of photosynthesis-related metabolites are used to compare the results with the evaluation results or generate matching indexes for consistency verification or confidence enhancement.

8. An apparatus for implementing the bamboo drought resistance evaluation method based on the coupling of leaf anatomical structure dynamics and photosynthetic physiology as described in any one of claims 1 to 7, characterized in that, The device includes: Standardized sampling module; used to collect bamboo leaves and determine the sampling area; Anatomical sampling and microscopic imaging module; used to acquire cross-sectional microscopic images of bamboo leaves; Image analysis and anatomical feature extraction module; used to extract the cavity area of ​​vesicular and spindle cells in the interfascicular region and calculate and output dynamic features of anatomical structures; Photosynthetic gas exchange parameter acquisition module; used to determine water use efficiency. Low-value distortion range identification and correction module; used to determine whether water use efficiency has entered the distortion risk range, and to perform penalty correction and / or weight reduction processing on water use efficiency. The structure-physiology coupling evaluation module is used to weight and sum the dynamic characteristics of anatomical structures with water use efficiency, and output the structure-physiology coupling index. And a drought resistance level determination module; used to determine and output the drought resistance level of plants based on the structure-physiology coupling index.

9. The apparatus according to claim 8, characterized in that, The device further includes: Consistency verification module; used to include data on differences in expression of photosynthesis-related gene families and / or differences in the content of photosynthesis-related metabolites, and output consistency comparison results or matching indexes of the evaluation results.

10. The method for evaluating bamboo drought resistance based on the coupling of leaf anatomical structure dynamics and photosynthetic physiology as described in any one of claims 1 to 7, or the device described in claim 8 or 9, is used in the screening of drought-resistant bamboo germplasm resources or in the breeding of drought-resistant bamboo.

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