Screening method of oilseed rape germplasm in saline-alkali soil based on rhizosphere comprehensive stress simulation

Abstract

The application belongs to the technical field of agricultural germplasm resource screening, and particularly relates to a screening method for oilseed rape germplasm in saline-alkali soil based on rhizosphere comprehensive stress simulation, which comprises the following steps: preparing rhizosphere comprehensive stress solution by using soil in the 0cm-20cm layer of the target saline-alkali soil; performing germination test on oilseed rape seeds by using the rhizosphere comprehensive stress solution, and screening out primary screening germplasm with a relative salt injury rate of less than or equal to 20%; performing potting test on the primary screening germplasm by using homologous soil, and screening out germplasm with a relative yield ratio of more than or equal to 80%, i.e. oilseed rape germplasm with salt-alkali tolerance and yield maintenance capability. The application realizes efficient and accurate screening of oilseed rape salt-alkali tolerant germplasm in coastal saline-alkali soil by unifying stress sources and setting quantitative screening thresholds in stages, and has good repeatability and application value.

Description

Technical Field

[0001] This invention belongs to the field of agricultural germplasm resource screening technology, specifically relating to a screening method for rapeseed germplasm in saline-alkali land based on rhizosphere integrated stress simulation. Background Technology

[0002] Saline-alkali land is an important reserve of arable land in my country, including various types such as coastal saline-alkali land, inland saline-alkali land, and desert saline-alkali land. Among them, coastal saline-alkali land, due to the influence of seawater infiltration, high groundwater level, and strong evaporation, has soil environment characterized by high salinity, complex ionic composition, high pH, ​​and low nutrient availability, which significantly limits crop growth and yield. Rapeseed, as a major oil crop, is considered one of the preferred crops for developing and utilizing such land due to its certain salt tolerance and its ability to improve soil during cultivation. However, its production and application highly depend on the accurate selection of salt-tolerant germplasm.

[0003] Existing screening techniques for rapeseed salt and alkali tolerance mostly use sodium chloride solution or artificially prepared saline-alkali solutions as stress sources under laboratory conditions to evaluate seed germination or seedling growth. These methods employ a single stress condition, making it difficult to reflect the comprehensive stress environment formed by the coexistence and interaction of multiple ions in the topsoil of coastal saline-alkali lands, resulting in insufficient consistency between screening results and field performance.

[0004] Existing technologies attempt to use soil extracts to replace artificial salt solutions, but the soil source for these extracts is deep soil, which is not limited to the cultivated layer environment where crop roots actually distribute and function. This still results in a mismatch between the stress environment and actual rhizosphere conditions. Furthermore, existing screening methods often lack clear and unified quantitative thresholds for evaluation indicators, leading to insufficient repeatability and comparability of screening standards. Therefore, how to construct a screening system that more closely resembles the actual rhizosphere stress environment of coastal saline-alkali land under experimental or semi-controlled conditions, and how to achieve consistency between the rapeseed germplasm obtained from laboratory screening and the final field performance through stable and quantifiable evaluation conditions, remains a pressing technical problem to be solved in this field. Summary of the Invention

[0005] This invention provides a method for screening rapeseed germplasm in saline-alkali land based on rhizosphere integrated stress simulation. By limiting the source of stress and evaluation conditions, it realistically simulates the integrated rhizosphere stress environment of crops and establishes stable quantitative screening criteria, thereby improving the accuracy and reproducibility of the screening results for salt-alkali tolerant rapeseed germplasm.

[0006] The technical solution adopted in this invention is: This invention provides a method for screening rapeseed germplasm in saline-alkali land based on rhizosphere integrated stress simulation, the steps of which are as follows: S1. Collect soil from the topsoil layer of the target saline-alkali land from 0cm to 20cm. After drying and grinding, add water for extraction, filter, and adjust the salt content of the filtrate to 0.6g / L to 1.0g / L to obtain the rhizosphere stress solution. S2. Germination tests were conducted on rapeseed seeds using rhizosphere stress solution to obtain the germination rate of the rhizosphere stress solution; simultaneously, germination tests were conducted on rapeseed seeds cultured in deionized water to obtain the germination rate of the deionized water; based on the germination rates of the rhizosphere stress solution and the deionized water, the relative salt damage rate was determined, and rapeseed with a relative salt damage rate ≤20% was screened to obtain the initial screening germplasm. S3. Using the soil layer (0cm-20cm) of the target saline-alkali land in S1, a planting experiment was conducted on the pre-screened germplasm to obtain the yield per plant in the target saline-alkali land. Simultaneously, a planting experiment was conducted on slightly saline-alkali soil near the target saline-alkali land in S1 to obtain the yield per plant in slightly saline-alkali soil. The relative yield ratio was calculated based on the yield per plant in the target saline-alkali land and the yield per plant in slightly saline-alkali soil. Germplasm with a relative yield ratio ≥80% was selected, thus obtaining saline-alkali land rapeseed germplasm that combines salt and alkali tolerance with yield retention ability. Slightly saline-alkali soil refers to soil with a total salt content of <2g / kg.

[0007] Preferably, the formula for calculating the relative salt damage rate is: ; in, A Relative salt damage rate; B Germination rate of deionized water; C Germination rate of rhizosphere stress solution.

[0008] Preferably, the formula for calculating the relative yield ratio is: ; in, M : Relative output ratio; m 1 Target yield per plant in the topsoil layer of saline-alkali land; m 2 Yield per plant in slightly saline-alkali soil.

[0009] Preferably, the preparation conditions for the rhizosphere stress-reducing solution are as follows: Add water to the soil and extract by shaking at 25℃~28℃ and 160r / min~180r / min for 3min~5min. Filter the solution and concentrate or dilute the filtrate to make the salt content of the filtrate reach 0.6g / L~1.0g / L, which is the rhizosphere stress solution.

[0010] Preferably, the preparation conditions of the rhizosphere stress solution are as follows: Add water to the soil and extract by shaking at 25℃ and 180r / min for 3 minutes. Filter the solution and concentrate or dilute the filtrate to achieve a salt content of 0.8g / L, thus obtaining the rhizosphere stress solution.

[0011] Preferably, the concentration conditions are such that the temperature does not exceed 60°C.

[0012] Preferably, the conditions for the germination experiment are: 25℃, relative humidity 54%, photoperiod is 8h light / 16h darkness.

[0013] Preferably, the germination experiment lasts for 7 days.

[0014] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a method for screening rapeseed germplasm in saline-alkali land based on rhizosphere integrated stress simulation, the steps of which are as follows: S1. Collect soil from the topsoil layer of the target saline-alkali land from 0cm to 20cm. After drying and grinding, add water for extraction, filter, and adjust the salt content of the filtrate to 0.6g / L to 1.0g / L to obtain the rhizosphere stress solution. S2. Germination tests were conducted on rapeseed seeds using rhizosphere stress solution to obtain the germination rate of the rhizosphere stress solution; simultaneously, germination tests were conducted on rapeseed seeds cultured in deionized water to obtain the germination rate of the deionized water; based on the germination rates of the rhizosphere stress solution and the deionized water, the relative salt damage rate was determined, and rapeseed with a relative salt damage rate ≤20% was screened to obtain the initial screening germplasm. S3. Using the 0cm~20cm soil layer of the target saline-alkali land in S1, a planting experiment was conducted on the pre-screened germplasm to obtain the yield per plant in the target saline-alkali land. At the same time, a planting experiment was conducted on the slightly saline-alkali soil near the target saline-alkali land in S1 to obtain the yield per plant in the slightly saline-alkali soil. The relative yield ratio was calculated based on the yield per plant in the target saline-alkali land and the yield per plant in the slightly saline-alkali soil. Germplasm with a relative yield ratio ≥80% was selected, which is the saline-alkali land rapeseed germplasm that has both salt tolerance and yield retention ability. Slightly saline-alkali soil refers to soil with a total salt content <2g / kg.

[0015] This invention utilizes rhizosphere integrated stress solution prepared from topsoil in target coastal saline-alkali land. This accurately replicates the composition, proportion, and synergistic stress effects of complex salt and alkali ions, nutrients, and microorganisms in the rhizosphere microdomain, ensuring a high degree of consistency between the screening environment and the actual crop growth environment from the outset. Based on this, a tiered screening system is constructed using quantitative thresholds of "relative salt damage rate ≤20%" and "relative yield ratio ≥80%", providing a realistic and objective screening framework. This method fundamentally overcomes the predictive limitations of traditional laboratory screening due to "stress distortion," enabling early screening results to accurately reflect the stress tolerance and yield potential of varieties under real coastal saline-alkali land conditions. This significantly improves the consistency between the selected rapeseed germplasm and its final field performance. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the overall process of the method of the present invention. Detailed Implementation

[0017] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0018] The inventive concept of this invention is as follows: For screening rapeseed germplasm suitable for planting in saline-alkali land, a common approach in existing technologies, balancing efficiency and reliability, is a combined strategy of "primary screening in laboratory simulated solutions or salt ponds + secondary screening in pots / fields." For example, a large number of germplasms are first screened for germination or seedling tolerance using NaCl solution or a small salt pond, and then the superior lines selected in the primary screening are transferred to pots or small experimental fields for further evaluation of yield potential. This approach sounds ideal, aiming to balance the efficiency of primary screening with the reliability of secondary screening. However, in practical applications, the results are often less than ideal due to the following fundamental problems:

[0019] 1. Inconsistent and discontinuous stress environments at each screening stage lead to breakage of the prediction chain.

[0020] In existing technologies, initial screening typically uses artificially prepared, single-component "simulated environments," such as NaCl solutions or homogeneous saline soil; while secondary screening and validation are conducted in real saline-alkali soils or open fields with more complex compositions and structures. This fundamental difference in the nature of the stress sources means that the tolerance mechanisms evaluated in the initial screening (such as resistance to a single NaCl solution) are different. + or Cl -The tolerance of these samples cannot fully represent the overall performance of rapeseed varieties under real-world complex adversity. As a result, the predictive value of initial screening is significantly weakened, and many materials that perform well in the laboratory are eliminated in subsequent stages, leading to a waste of research and development resources.

[0021] 2. There is a lack of standardized and efficient secondary screening processes that are "output-oriented".

[0022] In existing combined programs, there is often a lack of an efficient intermediate screening step that directly focuses on yield potential as the core indicator between the initial screening and the final field validation. Pot experiments using general saline-alkali soil are not targeted enough; direct field trials are too inefficient. This results in a gap between "efficient but distorted" and "accurate but inefficient" screening processes, making it impossible to achieve precise convergence of traits.

[0023] Fundamentally, existing technical solutions fail to construct a standardized screening system that is "realistically consistent with the stress environment, with interconnected screening steps, and clearly oriented towards target traits."

[0024] Existing technologies, such as patent CN118592327A, although using soil leachate, draw leachate from deep soil layers, and its ionic environment differs from the actual environment of the cultivated layer where crops grow. Furthermore, its screening process involves multiple parallel steps, making it relatively complex. In contrast, the method described in this invention provides a dedicated screening method with a more realistic stress environment and a more efficient and direct screening process.

[0025] This invention overcomes the problems of poor predictability and low screening efficiency caused by "ion distortion" and "niche mismatch" of stress sources in existing screening methods. It provides a complete technical solution for efficient and accurate screening of rapeseed germplasm resources in coastal saline-alkali land by realistically simulating the rhizosphere complex ion stress environment and applying optimized and verified specific quantification thresholds.

[0026] The method for screening rapeseed germplasm in coastal saline-alkali land based on integrated rhizosphere stress simulation described in this invention is a screening system with "real ecological simulation" and "precise quantitative control" as its dual cores. The specific process is as follows: Figure 1 The reasons are as follows: 1. Construction of a real rhizosphere complex ion stress environment.

[0027] This invention creatively and explicitly specifies that the rhizosphere integrated stress solution must be prepared from the topsoil (0cm~20cm) of the target coastal saline-alkali land as the sole stress source for germination screening. The scientific basis for this technical selection lies in the fact that the topsoil is the core micro-domain for seed germination, root establishment, and water and nutrient absorption, and its soil solution ionic composition is the direct and sole environmental signal for crops to perceive saline-alkali stress. Using topsoil to prepare the stress solution can maximally preserve and reproduce the complete ionic spectrum, dynamic ion ratios, and resulting synergistic stress effects of the field rhizosphere micro-domain in the laboratory, such as osmotic stress, ion toxicity, nutrient imbalance, and low microbial activity. This fundamentally unifies the stress faced in early screening with the final field environment for crop growth, laying a real ecological foundation for solving the problem of prediction accuracy.

[0028] 2. Determination and application of specificity quantitative screening thresholds.

[0029] This invention does not simply follow general screening procedures or thresholds. Instead, it identifies the optimal combination of quantitative criteria for the specific screening scenario of "coastal saline-alkali land-rapeseed" through extensive verification experiments, targeting the real stress environment described above: a relative salt damage rate of ≤20% for initial screening during germination, and a relative yield ratio of ≥80% for secondary screening during the entire growth period of potted plants. This threshold combination is non-obvious: "≤20%" is an extremely stringent initial screening threshold for salt tolerance, aiming to efficiently screen salt-tolerant germplasm from a vast amount of resources; "≥80%" is a key standard to ensure that these salt-tolerant materials can maintain near-normal yield potential under adversity. The organic combination of these two criteria forms a progressive and precise screening logic of "initial screening emphasizing stress tolerance, secondary screening emphasizing yield."

[0030] The method described in this invention is as follows: Step 1: Realistic simulation and rigorous initial screening.

[0031] Soil samples (0cm-20cm) from the topsoil layer of the target coastal saline-alkali land were collected, air-dried, and ground. Deionized water was added at a predetermined soil-to-water ratio (1:5-7), and the mixture was extracted by shaking for a predetermined time (3-5 minutes). The extract was then filtered through filter paper, and the filtrate was concentrated or diluted at a temperature not exceeding 60℃ to obtain the rhizosphere stress solution used for germination experiments. Deionized water was used as a "control solution." Rapeseed seeds to be screened were placed in petri dishes lined with filter paper. The experimental group received the rhizosphere stress solution, while the control group received an equal volume of the control solution. A standard germination experiment was conducted in a controlled-light incubator. A relative salt tolerance rate of ≤20% was used as the core evaluation index. Germplasm with a salt tolerance rate below or equal to this threshold was identified as "preliminary high-tolerance germplasm" and proceeded to the next screening stage.

[0032] Step 2: Production-oriented and precise rescreening.

[0033] The "highly salt-tolerant germplasm" obtained from the initial screening were subjected to pot experiments. Two treatments were set up: A. Saline-alkali soil treatment group: using coastal saline-alkali soil from the same source as in the first step; B. Slightly saline-alkali soil control group: using soil from the surrounding area of ​​the treatment group, but with a significantly lower salt content (preferably <2 g / kg total salt content). All management measures were kept consistent. After the crop matured, all plants were harvested and the grain yield was measured, and the average grain yield per plant was calculated. Using a relative yield ratio of ≥80% as the core evaluation index, germplasm with a yield higher than or equal to this threshold was identified as "high-yielding and salt-tolerant germplasm after secondary screening".

[0034] Step 3: Verify production potential.

[0035] The obtained "high-yielding and salt-tolerant germplasm after secondary screening" was planted in the target coastal saline-alkali environment. The core purpose of this step was to verify the effectiveness of the screening system in the first two steps. By comparing the yield of the "high-yielding and salt-tolerant germplasm after secondary screening" with local common control varieties, it was demonstrated that the materials screened by the method of this invention have a significant yield advantage in real field conditions. Preferably, further multi-point or multi-year comparative trials can be conducted with the locally cultivated salt-tolerant varieties to comprehensively evaluate its yield stability and adaptability.

[0036] The production potential verification test in the third step proves that the present invention can obtain rapeseed germplasm suitable for saline-alkali land through realistic simulation and strict initial screening, plus yield-oriented and precise re-screening.

[0037] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0038] 1. Basic information involved in the experiment of this invention.

[0039] Experimental site: Saline-alkali land in Binhai New Area, Tianjin.

[0040] The soil used in this invention was collected from saline-alkali farmland south of Zhujiang Road in the Lingang Industrial Zone of Binhai New Area, Tianjin. The basic physicochemical properties of the topsoil (0cm~20cm) are: pH 9.33, total salt content of soil is 3.39g / kg, and soil organic matter content is 11.27g / kg, which belongs to a moderate saline-alkali obstacle area.

[0041] 2. Relevant terms in this invention.

[0042] (1) Relative salt damage rate.

[0043] .

[0044] In the formula, A Relative salt damage rate;B Germination rate of the control group; C Germination rate of rhizosphere stress solution.

[0045] (2) Relative output ratio.

[0046] .

[0047] In the formula, M : Relative output ratio; m 1 Average yield per plant in the coastal saline-alkali soil treatment group at the target location; m 2 Average yield per plant in the mildly saline-alkali soil control group.

[0048] (3) Superior plants: In field trials, the actual yield reaches or exceeds the preset absolute yield threshold. The absolute yield threshold is measured by the average yield of rapeseed in my country, i.e., 140 kg / mu.

[0049] (4) The rate of superior plants.

[0050] .

[0051] In the formula, N : Rate of superior plants; n 1 Number of superior plants; n 2 The total number of germplasm samples selected for field verification after secondary screening.

[0052] (5) Initial screening pass rate.

[0053] .

[0054] In the formula, D : Rate of superior plants; d : Number of germplasms that passed the initial screening; 10: Total number of germplasms.

[0055] 3. Materials and Environment.

[0056] Ten rapeseed germplasm accessions were tested, labeled Y01-Y10. Basic information on the rapeseed germplasm used in this invention is shown in Table 1.

[0057] Table 1 Basic information on rapeseed germplasm Example 1 The screening method for rapeseed germplasm in saline-alkali land based on integrated rhizosphere stress simulation is as follows: S1. Prepare rhizosphere stress solution.

[0058] Soil samples were collected from the topsoil layer (0cm-20cm) of coastal saline-alkali land. After air drying and grinding, deionized water was added, and the mixture was extracted by shaking at 25℃ and 180r / min for 3 minutes. The filtrate was filtered through quantitative filter paper, and the salt content of the filtrate was adjusted to 0.8g / L, which is the rhizosphere stress solution.

[0059] S2. Initial screening: Rapeseeds were cultured using rhizosphere integrated stress solution and germination tests were conducted to screen out rapeseeds with a relative salt damage rate of ≤20% and obtain the initial screening germplasm.

[0060] Germination test: 30 plump seeds were randomly selected from each germplasm sample and placed in 10cm diameter petri dishes lined with filter paper. The prepared rhizosphere stress solution was then added, with the amount added enough to saturate the filter paper and leave a small amount of solution in the petri dish. After covering, the petri dishes were placed in a controlled-light incubator (8h light / 16h darkness) at 25℃ and 54% relative humidity for germination testing. A certain volume of rhizosphere stress solution was added to the petri dishes every two days to maintain the water potential required for seed germination. Germination rate was recorded after seven days, with three replicates per group.

[0061] In this step, the relative salt damage rate was used as the core evaluation index, with a relative salt damage rate of ≤20% set as the first threshold. An equal amount of deionized water was used to replace the rhizosphere stress solution as a control group. The number of germinations and germination rate of various germplasm resources were counted after treatment with the rhizosphere stress solution or deionized water, and the relative salt damage rate was calculated. The results are shown in Table 2.

[0062] Table 2 Relative Salt Injury Rate As shown in Table 2, there are two rapeseed germplasms that meet the requirement of relative salt damage rate ≤20%, namely Y01 and Y05, with an initial screening pass rate of 20.0%.

[0063] S3. Secondary screening: Using the soil from the 0cm to 20cm topsoil layer of the coastal saline-alkali land in S1, pot experiments were conducted on the germplasm that were initially screened. The relative yield ratio was calculated, and germplasm with a relative yield ratio ≥80% was selected, which is the rapeseed germplasm that has both salt and alkali tolerance and yield retention ability.

[0064] New Y01 and Y05 seedlings were planted in pots in coastal saline-alkali soil from the same source as the rhizosphere integrated stress solution, with 10 seeds per pot. Thinning was performed when the rapeseed reached the 2-leaf stage, and final thinning was done at the 4-leaf stage, ultimately retaining 3 uniformly growing rapeseed plants per pot. Each treatment was replicated three times. The potted plants were placed in a greenhouse without temperature or humidity control, but regular watering was maintained to ensure adequate soil moisture. Yield was measured after rapeseed maturity, and the relative yield ratio was calculated. A slightly saline-alkali soil near the same source, with a total salt content <2 g / kg, served as a control.

[0065] In this step, the relative yield ratio is used as the core evaluation indicator, and a relative yield ratio of ≥80% is set as the second threshold. The results of the relative yield ratios of Y01 and Y05 are shown in Table 3.

[0066] Table 3 Relative Yield Ratio As shown in Table 3, after secondary screening in pot experiments on coastal saline-alkali soil, Y01 had the highest relative yield ratio, at 81.3%. Thus, this invention obtains a high-yielding, salt-tolerant germplasm.

[0067] After the above process is completed, Y01 will be compared with local common commercial germplasm (Qingza 11, nationally approved oil variety 2012015) in a field test on saline-alkali land in the soil mining area. The management methods will be kept the same, and the yield will be calculated after maturity.

[0068] The results showed that the average yield of the high-yield and salt-tolerant germplasm Y01, which was screened twice, was 155 kg / mu, an increase of 11% compared with the average yield of rapeseed in my country of 140 kg / mu. Meanwhile, the growth of ordinary commercial germplasm in the local area was severely inhibited, indicating that the selected salt-tolerant germplasm has application value.

[0069] In summary, the method described in Example 1 successfully and efficiently screened one superior germplasm with outstanding high-yield and salt-tolerant potential from 10 materials.

[0070] To demonstrate the inventiveness and synergy of the "rhizosphere stress solution in coastal saline-alkali land at depths of 0cm-20cm" + "20% / 80% threshold" combination in Example 1, the present invention established the following Comparative Examples 1 to 3. Comparative Examples 1 to 3 used 10 rapeseed germplasm accessions from the same source and batch as screening targets, and independently conducted a complete screening process from initial screening to secondary screening to field validation. Finally, all germplasm accessions that passed the secondary screening in each treatment group were subjected to a randomized block yield comparison experiment in the same moderately saline-alkali coastal land field, with no duplication of the same germplasm accessions from different groups.

[0071] It should be noted that in this invention, the cultivated layer refers to the soil at a depth of 0cm to 20cm, and the deep layer refers to the soil at a depth of 100cm to 120cm. Since the groundwater level in this area is 120cm, deeper soil can be selected in other areas for comparison with patent CN118592327A.

[0072] Comparative Example 1 The screening method for rapeseed germplasm in saline-alkali land based on integrated rhizosphere stress simulation is as follows: During the initial screening, deep soil from the same plot as in Example 1 was used to prepare the rhizosphere stress solution. The soil, screening threshold, and all subsequent management for the potted plants were exactly the same as in Example 1. Deep soil refers to soil at a depth of 100cm to 120cm.

[0073] Table 4 shows the performance of various qualities in Comparative Example 1. Note: "-" in Table 4 indicates that this item is not available.

[0074] Comparative Example 2 The screening method for rapeseed germplasm in saline-alkali land based on integrated rhizosphere stress simulation is as follows: The first threshold is set to ≤40%, the second threshold is set to ≥70%, and the remaining subsequent management is exactly the same as in Example 1.

[0075] Table 5 shows the performance of various qualities in Comparative Example 2. Note: "-" in Table 5 indicates that this item is not present.

[0076] Comparative Example 3 The screening method for rapeseed germplasm in saline-alkali land based on integrated rhizosphere stress simulation is as follows: During the initial screening, deep soil from the same plot as in Example 1 was selected to prepare the rhizosphere integrated stress solution. The first threshold was set to ≤40%, and the second threshold was set to ≥70%. All subsequent management was exactly the same as in Example 1. Deep soil refers to soil at a depth of 100cm to 120cm.

[0077] Table 6 shows the performance of various qualities in Comparative Example 3. Note: "-" in Table 6 indicates that this item is not present.

[0078] Given the severe salt stress in the experimental field, conventional control varieties could not grow normally. To objectively evaluate the practical application value of the selected germplasm, this invention defines "superior plant" as: germplasm whose actual yield in field trials reaches or exceeds a preset absolute yield threshold. The absolute yield threshold is measured by the average yield of rapeseed in my country, i.e., 140 kg / mu. The superior plant rate of Example 1 and Comparative Examples 1 to 3 was calculated based on this. This indicator directly reflects the accuracy and efficiency of different screening methods.

[0079] Experimental results and analysis: Key data are shown in Table 7.

[0080] Table 7. Comparison and Analysis of Screening Efficiency and Accuracy among Different Treatment Groups As shown in Table 7: 1) Example 1 demonstrates extremely high screening accuracy: Example 1 achieved a 100% superior plant rate with the lowest initial screening pass rate (20%) and the fewest number of germplasms selected in the second screening (1). This means that every germplasm selected by the method described in Example 1 of this invention and sent to the field for verification has been proven to be a "superior plant" with high yield potential, greatly saving the expensive and time-consuming resources of subsequent field trials.

[0081] 2) In Comparative Example 1, after screening 10 rapeseed germplasm samples with a rhizosphere stress solution at a depth of 100cm-120cm and a relative salt damage rate of ≤20%, 3 salt-tolerant germplasm samples were initially screened. Further screening with a relative yield ratio of ≥80% yielded 2 more salt-tolerant germplasm samples. Field testing of these 2 more salt-tolerant germplasm samples revealed that only 1 plant met the criteria for a superior plant, with a superior plant rate (50%) significantly lower than that of Example 1 (100%). This indicates that without addressing the authenticity of the stress source (using deep soil), simply increasing the screening threshold cannot achieve high-precision screening.

[0082] The screening conditions for Comparative Example 2 were: rhizosphere stress solution in the 0cm~20cm topsoil layer + ≤40% relative salt damage rate + ≥70% relative yield ratio, with a superior plant rate of 33%. Even with a more realistic stress source, Comparative Example 2, using conventionally lenient thresholds in this field, still introduced a large number of "false positive" materials, leading to a significant decrease in screening accuracy.

[0083] In summary, Example 1 combines the rhizosphere stress solution in the 0cm~20cm topsoil layer with a strict threshold of 20% / 80%, which specifically solves the technical problem of "how to balance the authenticity of stress and the accuracy of screening".

[0084] 3) The superior plant rate of Example 1 (100%) was much higher than that of Comparative Example 1 (50%) and Comparative Example 2 (33%). This is by no means a simple sum of the two characteristic effects, but rather a synergistic gain effect of "1+1>2".

[0085] The stress fluid from the topsoil provides a realistic stress environment, and the "20% / 80% strict threshold" sets a precise "evaluation standard" to match it. Both are indispensable; their combined effect maximizes the accuracy of the screening. This strongly demonstrates the inherent inventiveness of the technical solution of this invention.

[0086] In summary, the "High-efficiency screening method for rapeseed germplasm in coastal saline-alkali land based on integrated rhizosphere stress simulation" provided by this invention effectively overcomes the industry bottleneck of poor predictability of existing screening technologies through creative combination of technical features, and has significant theoretical value and broad prospects for industrial application.

[0087] This invention fundamentally improves the accuracy of ecological prediction in screening: by forcibly using topsoil extract, it achieves for the first time a true replication of the field rhizosphere compound ion stress environment on a laboratory scale, enabling early tolerance screening to be based on an ecology highly consistent with that in the field, thus solving the problem of prediction failure caused by "ion distortion" in traditional methods from the source.

[0088] It provides a validated and precise quantitative screening standard: the clearly defined thresholds of "≤20%" and "≥80%" provide a repeatable and operable precise quantitative standard for the screening of rapeseed germplasm in coastal saline-alkali land, overcoming the subjectivity and arbitrariness of experience-based judgment, and greatly improving the standardization level and reproducibility of the screening work.

[0089] This system achieves a balance between screening efficiency and precision under extreme conditions: rigorous primary screening based on real stress environments efficiently eliminates the vast majority of unsuitable materials; while rigorous secondary screening based on yield potential accurately identifies high-yielding individuals from salt-tolerant populations. The entire system is particularly suitable for high-salt stress environments, maximizing the screening of germplasm resources with practical value with minimal post-validation costs.

[0090] Synergy among technical features: The core technical features of this invention—"tillage layer extract" and "20% / 80% strict threshold"—form an interdependent and synergistic organic whole. Comparative experimental data fully demonstrate that any single technical adjustment deviating from this specific combination (such as simply changing the soil source or simply adjusting the threshold) significantly reduces the final screening accuracy (measured by "superior plant rate"). This indicates that this technical combination is not something that those skilled in the art could easily conceive of or necessarily obtain through limited experiments, but rather a creative solution proposed to solve the deep-seated technical problem of "achieving precise quantitative screening in a realistic simulation environment," resulting in a synergistic benefit.

[0091] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0092] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for screening rapeseed germplasm in saline-alkali land based on integrated rhizosphere stress simulation, characterized in that, The steps are as follows: S1. Collect soil from the topsoil layer of the target saline-alkali land from 0cm to 20cm. After drying and grinding, add water for extraction, filter, and adjust the salt content of the filtrate to 0.6g / L to 1.0g / L to obtain the rhizosphere stress solution. S2. Germination tests were conducted on rapeseed seeds using rhizosphere stress solution to obtain the germination rate of the rhizosphere stress solution; simultaneously, germination tests were conducted on rapeseed seeds cultured in deionized water to obtain the germination rate of the deionized water; based on the germination rates of the rhizosphere stress solution and the deionized water, the relative salt damage rate was determined, and rapeseed with a relative salt damage rate ≤20% was screened to obtain the initial screening germplasm. S3. Using the soil layer (0cm-20cm) of the target saline-alkali land in S1, a planting experiment was conducted on the pre-screened germplasm to obtain the yield per plant in the target saline-alkali land. Simultaneously, a planting experiment was conducted on slightly saline-alkali soil near the target saline-alkali land in S1 to obtain the yield per plant in slightly saline-alkali soil. The relative yield ratio was calculated based on the yield per plant in the target saline-alkali land and the yield per plant in slightly saline-alkali soil. Germplasm with a relative yield ratio ≥80% was selected, thus obtaining saline-alkali land rapeseed germplasm that combines salt and alkali tolerance with yield retention ability. Slightly saline-alkali soil refers to soil with a total salt content of <2g / kg.

2. The screening method according to claim 1, characterized in that, The formula for calculating the relative salt damage rate is: ； in, A Relative salt damage rate; B Germination rate of deionized water; C Germination rate of rhizosphere stress solution.

3. The screening method according to claim 1, characterized in that, The formula for calculating the relative output ratio is: ； in, M : Relative output ratio; m 1 Target yield per plant in the topsoil layer of saline-alkali land; m 2 Yield per plant in slightly saline-alkali soil.

4. The screening method according to claim 1, characterized in that, The preparation conditions for the rhizosphere stress solution are as follows: Add water to the soil and extract by shaking at 25℃~28℃ and 160r / min~180r / min for 3min~5min. Filter the solution and concentrate or dilute the filtrate to make the salt content of the filtrate reach 0.6g / L~1.0g / L, which is the rhizosphere stress solution.

5. The screening method according to claim 4, characterized in that, The preparation conditions for the rhizosphere stress solution are as follows: Add water to the soil, shake and extract for 3 minutes at 25℃ and 180r / min, filter, concentrate or dilute the filtrate to make the salt content of the filtrate reach 0.8g / L, and the rhizosphere stress solution is obtained.

6. The screening method according to claim 5, characterized in that, Concentration is achieved at a temperature not exceeding 60℃.

7. The screening method according to claim 1, characterized in that, The conditions for the germination experiment are: 25℃, relative humidity 54%, photoperiod is 8h light / 16h darkness.

8. The screening method according to claim 1, characterized in that, The germination experiment lasted for 7 days.

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