Wheat salt tolerance identification method based on AA3 type flow analyzer

Abstract

The invention relates to the technical field of flow analyzer detection, in particular to a wheat salt tolerance identification method based on an AA3 type flow analyzer, which comprises the following steps: S1, obtaining a plurality of wheat samples to be detected; s2, respectively preparing a sodium ion standard working solution and a potassium ion standard working solution; s3, respectively performing sample introduction on each standard working solution by using a continuous flow analyzer, and determining by using a flame photometer to obtain a sodium ion standard working curve and a potassium ion standard working curve; step S4, constructing detection parameters of the sample to be detected; s5, feeding a sample to be detected by using a continuous flow analyzer, and determining the absorbance of each standard stock solution by using a flame photometer; s6, analyzing the salt tolerance of the wheat according to the sodium ion concentration and the potassium ion concentration measured by the to-be-measured sample. The method has the advantages of being capable of continuously testing batch samples, high in analysis speed, capable of saving manpower and material resources, high in accuracy, high in anti-interference capability and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of flow analyzer detection, and in particular to a method for identifying salt tolerance of wheat based on an AA3 flow analyzer. Background Art

[0002] Soil salt stress significantly inhibits the growth and development of crops. Salt stress can cause plant development retardation, inhibit root morphology construction and organ growth and differentiation, cause leaf curling and chlorosis, reduce thousand-grain weight, cause floret sterility, etc., and ultimately lead to a significant reduction in crop yields. High-concentration salt stress can cause osmotic stress and ion toxicity in plants, and thereby cause secondary hazards such as oxidative stress and nutrient depletion. Excessive soil salinity can cause the soil water potential to be lower than the water potential of plant root cells, making it difficult for the roots to absorb water from the soil and causing drought stress. Continuous salt stress can reduce plant cell turgor pressure and affect cell growth. Plants must maintain cell elongation and growth through osmotic regulation. Salt stress shortens cell extension time, affects the development of wheat main stems and reproductive structures, shortens flowering time, and accelerates plant maturity.

[0003] Several studies have shown that The ratio is one of the important indicators for evaluating plant salt tolerance. Concentrations usually increase significantly, while The concentration is relatively reduced, resulting in This change reflects the plant's response mechanism to salt stress, which is to reduce Ratio to reduce Toxic effects on cell function. For example, studies have shown that under salt stress, the aboveground part of wheat The ratio was significantly reduced, indicating that salt-tolerant varieties can more effectively eliminate and maintain absorption and accumulation, The ratio not only reflects the current salt tolerance of wheat, but can also be used as a breeding target to screen and cultivate salt-tolerant varieties. Therefore, a wheat 、 There is an urgent need for a determination method. The AA3 flow analyzer has the advantages of high automation, fast analysis speed, high accuracy, and low reagent consumption. It has been widely used in water quality analysis, soil nutrient testing and other fields. However, the application of the AA3 flow analyzer to wheat 、 The research and methods of content determination have not been reported, which is of great significance for evaluating the salt tolerance of wheat varieties and guiding wheat cultivation in saline-alkali land.

[0004] Chinese patent application publication number: CN105612851A, discloses a method for evaluating salt tolerance of wheat or screening salt-tolerant wheat based on potassium ion flow, comprising the following steps: dividing the wheat seeds to be tested into two groups, the control group is germinated under normal conditions, and the test group is germinated under salt stress conditions, and detecting the salt tolerance of the aleurone layer of the seeds during the germination period. The flow direction and velocity of the control group The flow rate of the experimental group was subtracted The flow rate gets the value M; if the value M is less than or equal to 30 , the wheat to be tested is a candidate for salt-intolerant wheat; if the value M is greater than 30 , the wheat to be tested is a candidate salt-tolerant wheat. The test cannot fully reflect the salt tolerance of wheat, and the measurement steps are complicated and difficult to promote. Summary of the Invention

[0005] To this end, the present invention provides a method for identifying salt tolerance of wheat based on AA3 flow analyzer, which is used to overcome the problems in the prior art. 、 The content determination operation is complicated and the determination accuracy cannot be accurately judged.

[0006] To achieve the above object, the present invention provides a method for identifying salt tolerance of wheat based on an AA3 flow analyzer, comprising:

[0007] Step S1, obtaining a number of wheats that meet the consistency standards and pre-processing them to obtain a sample to be tested;

[0008] Step S2, preparing a plurality of sodium ion standard stock solutions with different concentrations and a plurality of potassium ion standard stock solutions with different concentrations;

[0009] Step S3, setting the working parameters of the flow analyzer, injecting each standard stock solution using the continuous flow analyzer and measuring the absorbance of each standard stock solution using a flame photometer, and obtaining a sodium ion standard working curve and a potassium ion standard working curve based on the absorbance;

[0010] Step S4, constructing the detection parameters of the sample to be tested according to the sodium ion standard working curve and the potassium ion standard working curve;

[0011] Step S5, injecting the sample to be tested using a continuous flow analyzer and measuring the absorbance of each standard stock solution using a flame photometer;

[0012] When the peak height of the absorbance of the sample to be tested exceeds 80% of the maximum concentration peak height in the corresponding standard working curve, it is determined that the sample needs to be diluted, and the corresponding standard working curve is corrected according to the concentration and absorbance of the sample to be tested measured before and after dilution;

[0013] Step S6, analyzing the salt tolerance of the wheat according to the sodium ion concentration and potassium ion concentration measured for the sample to be tested.

[0014] Furthermore, step S1 includes:

[0015] Step S11, cutting the collected wheat sample roots or leaves into small segments and then mixing them to obtain sample segments;

[0016] Step S12, weighing the sample segment, placing it into a homogenizer, adding ultrapure water, homogenizing at a speed of 5000 r / min, centrifuging, and filtering to obtain a wheat sample extract;

[0017] Step S13, aspirating the wheat sample extract, placing it into a volumetric flask, adding 0.1 mol / L aluminum sulfate solution, making up the volume with ultrapure water, and shaking well to obtain a sample to be tested.

[0018] Furthermore, step S2 includes:

[0019] Step S21, preparing a sodium standard stock solution with a sodium chloride concentration of 1000 mg / L and a potassium standard stock solution with a potassium chloride concentration of 1000 mg / L;

[0020] Step S22, aspirating different volumes of sodium standard stock solution and potassium standard stock solution and respectively fixing the volumes to obtain a plurality of sodium standard working solutions and potassium standard working solutions with different mass concentrations;

[0021] The concentration of the sodium standard working solution includes at least three different sodium chloride concentrations, and the concentration of the potassium standard working solution includes at least three different potassium chloride concentrations;

[0022] Furthermore, step S3 includes:

[0023] Step S31, setting the working parameters of the flow analyzer;

[0024] Step S32, using a dilution of the corresponding standard working solution to calibrate the zero point of the corresponding standard working curve;

[0025] Step S33, measuring the absorbance of each sodium standard working solution respectively, and establishing a sodium ion measurement curve based on the sodium ion content in each sodium standard working solution and the corresponding absorbance;

[0026] The absorbance of each potassium standard working solution is measured respectively, and a potassium ion measurement curve is established based on the potassium ion content in each potassium standard working solution and the corresponding absorbance;

[0027] Step S34, calculating a mean point according to the average values ​​of the absorbances of the respective sodium standard working solutions, and establishing a sodium ion ideal curve based on the mean point and the zero point;

[0028] The mean point is calculated according to the average value of the absorbance of each potassium standard working solution measured respectively, and the potassium ion ideal curve is established based on the mean point and the zero point;

[0029] Step S35, comparing the sodium ion measurement curve with the corresponding sodium ion ideal curve to determine a sodium ion standard working curve, and comparing the potassium ion measurement curve with the corresponding potassium ion ideal curve to determine a potassium ion standard working curve;

[0030] Among them, the absorbance of the ideal curve and the measurement curve at the same concentration are compared respectively. If the absorbance difference is within 5%, the standard working curve is determined to select the curve segment corresponding to the ideal curve; if the absorbance difference exceeds 5%, the standard working curve is determined to select the curve segment corresponding to the measurement curve.

[0031] Furthermore, in step S5, the process of determining whether the sample needs to be diluted includes:

[0032] Step S51, obtaining the peak height of the absorbance of the sample to be tested, recorded as the first peak height;

[0033] Step S52, comparing the first peak height with 80% of the maximum concentration peak height of the standard working curve;

[0034] When the first peak height is greater than 80% of the maximum concentration peak height of the standard working curve, it is determined that the sample to be tested needs to be diluted, and the dilution ratio is determined according to the first peak height and the corresponding standard working curve;

[0035] Step S53: Open the dilution valve, draw the diluent according to the dilution ratio, and re-draw the sample to be tested into the dilution container to mix the sample to be tested and the diluent evenly, and then retest;

[0036] Step S54, re-measure the peak height of the diluted sample to be tested, wherein,

[0037] If the re-measured peak height is less than 80% of the maximum concentration peak height of the standard working curve, drain the sample from the dilution container through the drain valve;

[0038] If the re-measured peak height still exceeds 80% of the maximum peak height, repeat step S52, redetermine the dilution ratio, and re-measure;

[0039] Step S55 , reversely deduce and calculate the actual concentration of the sample to be tested based on the final dilution ratio and the measured absorbance.

[0040] Furthermore, in step S5, the process of correcting the corresponding standard working curve includes:

[0041] Step S56, obtaining the actual concentration of the diluted sample and the corresponding absorbance to determine a first ratio;

[0042] Step S57, obtaining the absorbance of the stock solution determined by the first peak height before dilution of the test sample, and determining a second ratio according to the absorbance of the stock solution and the corresponding stock solution concentration on the standard working curve;

[0043] Step S58: Compare the first ratio and the second ratio to calculate the deviation, wherein:

[0044] If the deviation is within the preset error range, the detection accuracy of the undiluted sample is determined to be qualified, the undiluted concentration is set as the maximum concentration of the standard working solution, and the standard working curve is corrected.

[0045] Furthermore, in step S5, whenever the tested sample is determined to need to be diluted, the cleaning time of the tested sample after testing is extended by 30 seconds to reduce errors in subsequent measurements.

[0046] Furthermore, the operating parameters of the flow analyzer include:

[0047] The volume ratio of the cleaning solution is 3% nitric acid;

[0048] The absorbance detector used was a flame photometer, with an injection rate of 40 samples per hour, an injection time of 40 s, and an injection and cleaning time of 35 s;

[0049] The fuel gas is an air-propane mixture, the propane pressure is 0.08MPa, and the air pressure is 15Psi.

[0050] Furthermore, in step S4, constructing the detection parameters of the sample to be tested includes:

[0051] Step S41, open the AA3 flow analyzer software, set the concentration unit and decimal places, sampling time, flushing time, set the standard curve type, select nonlinear fitting, and enter the corresponding standard working solution concentration from high to low, wherein the standard working solution concentration is the ion concentration in the curve segment corresponding to the measurement curve in the standard working curve;

[0052] Step S42: Set a blank, run deionized water through the flow path for more than 10 minutes, and determine that the baseline is stable when bubbles running in the flow path are regular. Adjust the OD value to 000 by adjusting the flame photometer.

[0053] Step S43, set the adjustment gain, place the prepared highest concentration standard working solution on the sampling tray, perform manual sampling, and make the injection needle take the liquid time consistent with the sample aspiration time. Wait for the peak to appear and stabilize before adjusting the gain to complete the setting of the detection parameters.

[0054] Furthermore, in the step S1, the mixture is homogenized at a speed of 5000 r / min for 5 minutes, centrifuged at a speed of 5500 r / min for 12 minutes, and filtered after centrifugation to obtain a wheat sample extract.

[0055] Compared with the prior art, the present invention has the beneficial effect of providing a method for measuring wheat 、 method, through 、 Recently, the salt tolerance of wheat has been identified to solve the existing wheat 、 The determination method has problems such as complex operation, tedious sample pre-treatment, high analysis cost and the need for professional technicians to operate. This method simplifies the complex manual operation into automated detection of the instrument, which can continuously test batches of samples. It not only has fast analysis speed and saves manpower and material resources, but also has high accuracy and strong anti-interference ability, consumes less reagents and has little environmental pollution. Automated operation also reduces human errors. The characteristics of this technology are that it is suitable for small early-generation groups, does not harm plants, and can lock in breeding targets in the early generation, thereby improving breeding efficiency.

[0056] Furthermore, the present invention can reduce sample processing time by more than 70% in batch testing by establishing an automated dilution system to replace the manual dilution step; the system can automatically adapt to samples of different concentrations: high-concentration samples are automatically diluted to the linear range, and low-concentration samples avoid repeated injections, greatly reducing labor costs, realizing full process automation, and continuous operation throughout the day.

[0057] Furthermore, the present invention reduces the problem of accuracy attenuation of traditional static curves by dynamically correcting the standard working curve and calibrating the curve in real time, ensuring long-term stability during the detection process, eliminating experimental rework caused by curve failure from the root, and significantly broadening the detection range of the instrument. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is a diagram of the steps for determining the salt tolerance of wheat using an AA3 flow analyzer in an embodiment of the present invention.

[0059] Figure 2 This is the standard working curve of wheat sodium ion measured in the embodiment of the present invention;

[0060] Figure 3 This is the wheat potassium ion standard working curve measured in the embodiment of the present invention;

[0061] Figure 4 This is a flow chart of a method for determining sodium and potassium ions in wheat using an AA3 flow analyzer according to an embodiment of the present invention. DETAILED DESCRIPTION

[0062] In order to make the objects and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with embodiments; it should be understood that the specific embodiments described herein are merely used to explain the present invention and are not intended to limit the present invention.

[0063] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only used to explain the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0064] Furthermore, it should be noted that, in the description of the present invention, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed connections, detachable connections, or integral connections; mechanical connections or electrical connections; direct connections or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0065] See also Figure 1 As shown, an embodiment of the present invention provides a method for determining salt tolerance of wheat using an AA3 flow analyzer, comprising:

[0066] Step S1, sample collection and processing: obtaining a number of wheats that meet the consistency standards and pre-processing them to obtain a sample to be tested;

[0067] Cut the collected wheat sample roots or leaves into small segments ≤1 cm and mix them evenly; weigh 10 g of the sample segment and place it in a homogenizer, then add 50 mL of ultrapure water and homogenize at a speed of 5000 r / min for 5 minutes. Centrifuge (speed of 5500 r / min, time for 12 minutes) and filter to obtain the sample extract; finally, draw 5 mL of the wheat sample extract and place it in a 25 mL volumetric flask, add 1 mL of 0.1 mol / L aluminum sulfate solution, dilute to the scale line with ultrapure water, and shake well.

[0068] Step S2, preparing a standard working solution;

[0069] Prepare the sodium standard stock solution: Weigh 2.5421 g (accurate to 0.0001 g) of the working standard reagent sodium chloride, which has been dried to constant weight in an electric oven at 105°C. Dissolve it in 3% by volume nitric acid and adjust the volume to 1000 mL. This solution is the sodium standard stock solution, and the sodium chloride concentration is 1000 mg / L.

[0070] Prepare sodium standard working solution: aspirate 0, 0.2, 0.4, 0.8, 1.2, and 1.6 mL of sodium standard stock solution in sequence, add 3% nitric acid to make the volume to 100 mL, and obtain standard solutions with sodium chloride concentrations of 0, 2, 4, 8, 12, and 16 mg / L, which are sodium ion standard working solutions with different concentrations.

[0071] Prepare potassium standard stock solution: weigh 0.1907 g of working standard reagent potassium chloride that has been dried in an electric oven at 110°C for 2 h, add it to 1.0 mol / L ammonium acetate solution (pH 7.0) and dilute to 100 mL. This solution is the potassium standard stock solution with a potassium chloride concentration of 1000 mg / L.

[0072] Prepare potassium standard working solution: aspirate 0, 0.2, 0.4, 0.8, 1.2, and 1.6 mL of potassium standard stock solution in sequence, add 1.0 mol / L ammonium acetate solution to make the volume 100 mL, and obtain standard solutions with potassium chloride concentrations of 0, 2, 4, 8, 12, and 16 mg / L, which are potassium ion standard working solutions with different concentrations.

[0073] Step S3, setting instrument operating parameters and drawing a standard working curve;

[0074] The cleaning liquid was 3% nitric acid by volume; the detector was an M410 flame photometer; the injection rate was 40 samples per hour, the injection time was 40 s; the injection cleaning time was 35 s, the fuel gas was an air-propane mixture, the propane pressure was 0.08 MPa, and the air pressure was 15 Psi.

[0075] In this embodiment, the above working parameters are used to inject each sodium ion standard working solution and the potassium ion standard working solution, and the absorbance of each standard working solution is measured by flame photometer to obtain the absorbance data corresponding to each working solution.

[0076] Specifically, the process of drawing the sodium standard working curve includes:

[0077] The zero point of the standard curve was calibrated with 3% by volume nitric acid, and the absorbance of each sodium standard working solution was measured. A sodium ion measurement curve was established based on the sodium ion content in each sodium standard working solution and the corresponding absorbance.

[0078] The mean point is calculated according to the average value of the absorbance of each sodium standard working solution measured respectively, and the sodium ion ideal curve is established based on the mean point and the zero point.

[0079] Specifically, the process of drawing a potassium standard working curve includes:

[0080] The zero point of the standard curve was calibrated with a 1.0 mol / L ammonium acetate solution. The absorbance of each potassium standard working solution was measured, and a potassium ion measurement curve was established based on the potassium ion content in each potassium standard working solution and the corresponding absorbance.

[0081] The mean point is calculated according to the average value of the absorbance of each potassium standard working solution measured respectively, and the potassium ion ideal curve is established based on the mean point and the zero point.

[0082] Specifically, the absorbance of the ideal curve and the measurement curve at the same concentration are compared. If the absorbance difference is within 5%, the standard working curve is determined to select the curve segment corresponding to the ideal curve; if the absorbance difference exceeds 5%, the standard working curve is determined to select the curve segment corresponding to the measurement curve.

[0083] For example, for the obtained sodium ion ideal curve and sodium ion measurement curve, the two curves are divided into several curve segments divided by concentration based on whether the absorbance difference between the two curves exceeds the range of 5%. If the absorbance corresponding to the same concentration in two curve segments within the same concentration range is within 5% or exceeds 5%, one of the curve segments is selected based on whether the absorbance difference exceeds 5%. The selection of each curve segment is completed in sequence to form the sodium ion standard working curve. The selection logic of the potassium ion standard working curve is the same as above and will not be repeated here.

[0084] Step S4, constructing the detection parameters of the sample to be tested according to the sodium ion standard working curve and the potassium ion standard working curve;

[0085] Specifically, open the software of the AA3 flow analyzer, click the icon in the analysis interface to enter the Methods Setting interface, click Add, and create a new sodium and potassium method. Click General to set the concentration unit and accuracy, sampling time, and rinse time. Click OD / Curve / QC to set the standard curve type, using the flame emission method. Preferably, select nonlinear fitting and enter the standard working solution concentration from high to low; wherein the standard working solution concentration is the ion concentration in the curve segment corresponding to the measurement curve in the standard working curve, and the setting is completed.

[0086] Set up a blank and run deionized water through the entire flow path for more than 10 minutes. When the baseline is stable, adjust the OD value in the software to 000 by rotating the "blank" knob in the flame photometer.

[0087] Adjust the gain, place the standard working solution with the highest concentration on the sampling tray, and perform manual sampling. The injection needle should take the liquid at the same time as the set sampling time. Wait for the peak to appear and stabilize before adjusting the gain to complete the setting of the detection parameters.

[0088] Step S5, sample determination: injecting the sample to be tested using a continuous flow analyzer and measuring the absorbance of each standard stock solution using a flame photometer, wherein,

[0089] Obtaining the peak height of the absorbance of the sample to be tested, recorded as the first peak height, and when the first peak height is greater than 80% of the maximum concentration peak height of the standard working curve, it is determined that the sample to be tested needs to be diluted;

[0090] Determine the dilution ratio based on the first peak height and the corresponding standard working curve; in order to prevent the concentration after dilution from being exactly at the limit of the detection range and affecting the detection accuracy, set a redundancy factor (between 1.1 and 1.4), preferably 1.2;

[0091]

[0092] Where K is the dilution ratio, To detect the peak height of the sample, 80% of the peak height of the maximum concentration of the standard working curve, is the redundancy coefficient.

[0093] When it is confirmed that the sample to be tested needs to be diluted, open the dilution valve, draw the diluent according to the dilution ratio, and re-draw the sample to be tested into the dilution container to mix the sample to be tested and the diluent evenly, and then retest;

[0094] Specifically, the peak height of the diluted sample to be tested is re-measured, wherein,

[0095] If the re-measured peak height is less than 80% of the maximum concentration peak height of the standard working curve, the dilution ratio is determined to be appropriate. The sample in the dilution container is drained through the drain valve, and the cleaning time of the sample to be tested is extended by 30 seconds after testing to reduce errors in subsequent measurements.

[0096] If the re-measured peak height still exceeds 80% of the maximum concentration peak height of the standard working curve, the dilution ratio is re-determined and the test is repeated. A liquid level gauge is installed in the dilution container. When the sample is predicted to exceed the upper limit of the container liquid level (80% of the dilution container capacity) after dilution, half of the sample volume is discharged through the drain valve. The actual concentration of the test sample is calculated by reverse deduction based on the final dilution ratio and the measured absorbance. It is understood that the calculation of the pre-dilution concentration of the test sample using the dilution ratio and the measured concentration of the diluted sample after dilution is a prior art technique and will not be further elaborated here.

[0097] In step S5, the corresponding standard working curve is corrected according to the concentration and absorbance of the sample to be tested measured before and after dilution;

[0098] Specifically, the corresponding standard working curve is corrected, including:

[0099] Obtaining the actual concentration of the diluted sample and the corresponding absorbance to determine a first ratio;

[0100] Obtaining the absorbance of the stock solution determined by the first peak height before dilution of the test sample, and determining a second ratio according to the absorbance of the stock solution and the corresponding stock solution concentration on the standard working curve;

[0101] Specifically, the first ratio is the ratio of the actual concentration to the corresponding absorbance, and the second ratio is the ratio of the stock solution concentration to the stock solution absorbance;

[0102] The first ratio and the second ratio are compared to calculate the deviation. If the deviation is within a preset error range (95% to 105%), the detection accuracy of the undiluted sample is determined to be qualified, the undiluted concentration is set as the maximum concentration of the standard working solution, the standard working curve is corrected, and the stock solution concentration and the stock solution absorbance are added to the standard working curve data.

[0103] Step S6, analyzing the salt tolerance of the wheat according to the sodium ion concentration and potassium ion concentration measured for the sample to be tested.

[0104] According to the above step S5, the sodium ion concentration and potassium ion concentration of the sample to be tested are obtained or calculated, and the data processing software processes the data and generates a report. Concentration and Concentration analysis of wheat salt tolerance provides reliable data support for wheat salt tolerance research and production practice.

[0105] It can be understood that the present invention ultimately obtains or calculates the sodium ion concentration and potassium ion concentration of the sample to be tested, and the data can be used to provide accurate data support for the salt tolerance study of wheat. As for how to analyze the salt tolerance of wheat based on the sodium ion concentration and potassium ion concentration of wheat, the present invention does not limit it, and it is all within the scope of protection of the present invention.

[0106] Example 1:

[0107] 1. Sample collection and processing: 10 varieties of wheat materials used in production, each with more than 50 samples, were obtained and subjected to salt stress treatment in 300 mmol / L NaCl solution. After incubation for 10 days, young leaves and rhizomes of wheat were collected as samples. The collected samples were cut into small segments ≤10 mm and mixed. 10 g of the sample segment was weighed and placed in a homogenizer. 50 mL of ultrapure water was added and homogenized at 5000 r / min for 5 min. The plant sample extract was obtained by centrifugation (speed: 5500 r / min, time: 12 min) and filtration. Finally, 5 mL of the plant sample extract was aspirated and placed in a 25 mL volumetric flask. 1 mL of 0.1 mol / L aluminum sulfate solution was added and the volume was made up to the mark with ultrapure water. The extract was then shaken to obtain the final product.

[0108] 2. Standard curve drawing: Please refer to Figure 2 and Figure 3 As shown, accurately weigh a certain amount of sodium chloride and potassium chloride to prepare the standard working solution, measure the absorbance and draw a standard curve (the specific steps are as in the above implementation steps). In the figure, y is the peak height, x is the ion concentration, is the goodness of fit.

[0109] 3. Sample determination: Please refer to Figure 4 As shown, the prepared 、 The standard working solution and sample are sucked into the peristaltic pump through the sampler and flow through the entire system. At the same time, the peristaltic pump continuously delivers the reagents required for each analytical method and draws in air to divide the fluid into segments. Under the same conditions (including time, flow rate, temperature, cleaning ratio, etc.), each segment is fully mixed and reacted in the mixing circle to generate colored compounds. The colorimetry is performed by the detector, and finally the data is processed and a report is generated.

[0110] Example 2:

[0111] The difference between this embodiment and Example 1 is that this embodiment only changes the selected samples in Example 1, and uses normally grown young leaves and rhizomes of wheat that have not been subjected to salt stress as samples. The rest is the same as Example 1.

[0112] Comparative Example 1:

[0113] In this comparative example, the samples selected in Example 1 were tested using conventional dry ashing technology.

[0114] Comparative Example 2:

[0115] In this comparative example, the samples selected in Example 2 were tested using conventional dry ashing technology.

[0116] Part of wheat tested by the present invention 、 The test results are shown in Table 1:

[0117] Table 1 Results of wheat rhizome salt tolerance determined by the improved method

[0118]

[0119] Table 2 Results of salt tolerance test of young wheat leaves determined by the improved method

[0120]

[0121] As can be seen from Tables 1 and 2 above, the sodium-potassium ion ratio of Jiemai 19 wheat changes the least under salt stress, indicating that this variety can more effectively eliminate sodium ions and maintain the absorption and accumulation of potassium ions. It can be used as a breeding target to screen and cultivate salt-tolerant varieties. The wheat salt tolerance identification method based on the AA3 flow analyzer of the present invention can accurately and quantitatively identify the salt tolerance of wheat samples. 、 The content can be accurately measured, and it has the advantages of high degree of automation, fast analysis speed, high precision and low reagent consumption.

[0122] Part of wheat determined by dry ashing 、 The test results are shown in Tables 3 and 4:

[0123] Table 3 Results of wheat root and stem salt tolerance test using traditional techniques

[0124]

[0125] Table 4 Results of salt tolerance test of young wheat leaves using traditional techniques

[0126]

[0127] As can be seen from Tables 1 to 4 above, the sodium-potassium ion ratios measured using the present invention are highly consistent with those obtained using conventional detection methods, demonstrating that the method's detection accuracy is highly comparable to that of conventional methods. Furthermore, the working curve can be promptly corrected when high-concentration samples are detected during the detection process, reducing manual intervention. This method significantly reduces reagent consumption and labor costs, significantly increases detection speed, and is simple to operate, making it easy to promote and implement.

[0128] Thus far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art may make equivalent changes or substitutions to the relevant technical features, and the technical solutions after such changes or substitutions will fall within the scope of protection of the present invention.

Claims

1. A method for identifying salt tolerance of wheat based on AA3 flow analyzer, characterized in that: include: Step S1, obtaining a number of wheats that meet the consistency standards and pre-processing them to obtain a sample to be tested; Step S2, preparing a plurality of sodium ion standard working solutions with different concentrations and a plurality of potassium ion standard working solutions with different concentrations; Step S3, setting the working parameters of the flow analyzer, injecting each standard working solution using the continuous flow analyzer and measuring the absorbance of each standard working solution using a flame photometer, and obtaining a sodium ion standard working curve and a potassium ion standard working curve based on the absorbance; Step S4, constructing the detection parameters of the sample to be tested according to the sodium ion standard working curve and the potassium ion standard working curve; Step S5, injecting the sample to be tested using a continuous flow analyzer and measuring the absorbance of each standard stock solution using a flame photometer; When the peak height of the absorbance of the sample to be tested exceeds 80% of the maximum concentration peak height in the corresponding standard working curve, it is determined that the sample needs to be diluted, and the corresponding standard working curve is corrected according to the concentration and absorbance of the sample to be tested measured before and after dilution; Step S6, analyzing the salt tolerance of the wheat according to the sodium ion concentration and potassium ion concentration measured for the sample to be tested.

2. The method for identifying salt tolerance of wheat based on AA3 type flow analyzer according to claim 1, wherein The step S1 comprises: Step S11, cutting the collected wheat sample roots or leaves into small segments and then mixing them to obtain sample segments; Step S12, weighing the sample segment, placing it into a homogenizer, adding ultrapure water, homogenizing at a speed of 5000 r / min, centrifuging, and filtering to obtain a wheat sample extract; Step S13, aspirating the wheat sample extract, placing it into a volumetric flask, adding 0.1 mol / L aluminum sulfate solution, making up the volume with ultrapure water, and shaking well to obtain a sample to be tested.

3. The method for identifying salt tolerance of wheat based on AA3 type flow analyzer according to claim 2, wherein: The step S2 includes: Step S21, preparing a sodium standard stock solution with a sodium chloride concentration of 1000 mg / L and a potassium standard stock solution with a potassium chloride concentration of 1000 mg / L; Step S22, aspirating different volumes of sodium standard stock solution and potassium standard stock solution and respectively fixing the volumes to obtain a plurality of sodium standard working solutions and potassium standard working solutions with different mass concentrations; The concentration of the sodium standard working solution includes at least three different sodium chloride concentrations, and the concentration of the potassium standard working solution includes at least three different potassium chloride concentrations.

4. The method for identifying salt tolerance of wheat based on AA3 type flow analyzer according to claim 3, wherein: The step S3 comprises: Step S31, setting the working parameters of the flow analyzer; Step S32, using a diluent of the corresponding standard working solution to calibrate the zero point of the corresponding standard working curve; Step S33, measuring the absorbance of each sodium standard working solution respectively, and establishing a sodium ion measurement curve based on the sodium ion content in each sodium standard working solution and the corresponding absorbance; The absorbance of each potassium standard working solution is measured respectively, and a potassium ion measurement curve is established based on the potassium ion content in each potassium standard working solution and the corresponding absorbance; Step S34, calculating a mean point according to the average values ​​of the absorbances of the respective sodium standard working solutions, and establishing a sodium ion ideal curve based on the mean point and the zero point; The mean point is calculated according to the average value of the absorbance of each potassium standard working solution measured respectively, and the potassium ion ideal curve is established based on the mean point and the zero point; Step S35, comparing the sodium ion measurement curve with the corresponding sodium ion ideal curve to determine a sodium ion standard working curve, and comparing the potassium ion measurement curve with the corresponding potassium ion ideal curve to determine a potassium ion standard working curve; Among them, the absorbance of the ideal curve and the measurement curve at the same concentration are compared respectively. If the absorbance difference is within 5%, the standard working curve is determined to select the curve segment corresponding to the ideal curve; if the absorbance difference exceeds 5%, the standard working curve is determined to select the curve segment corresponding to the measurement curve.

5. The method for identifying salt tolerance of wheat based on AA3 type flow analyzer according to claim 4, characterized in that: In step S5, the process of determining whether the sample needs to be diluted includes: Step S51, obtaining the peak height of the absorbance of the sample to be tested, recorded as the first peak height; Step S52, comparing the first peak height with 80% of the maximum concentration peak height of the standard working curve; When the first peak height is greater than 80% of the maximum concentration peak height of the standard working curve, it is determined that the sample to be tested needs to be diluted, and the dilution ratio is determined according to the first peak height and the corresponding standard working curve; Step S53: Open the dilution valve, draw the diluent according to the dilution ratio, and re-draw the sample to be tested into the dilution container to mix the sample to be tested and the diluent evenly, and then retest; Step S54, re-measure the peak height of the diluted sample to be tested, wherein, If the re-measured peak height is less than 80% of the maximum concentration peak height of the standard working curve, drain the sample from the dilution container through the drain valve; If the re-measured peak height still exceeds 80% of the maximum peak height, repeat step S52, redetermine the dilution ratio, and re-measure; Step S55 , reversely deduce and calculate the actual concentration of the sample to be tested based on the final dilution ratio and the measured absorbance.

6. The method for identifying salt tolerance of wheat based on AA3 type flow analyzer according to claim 5, characterized in that: In step S5, the process of correcting the corresponding standard working curve includes: Step S56, obtaining the actual concentration of the diluted sample and the corresponding absorbance to determine a first ratio; Step S57, obtaining the absorbance of the stock solution determined by the first peak height before dilution of the test sample, and determining a second ratio according to the absorbance of the stock solution and the corresponding stock solution concentration on the standard working curve; Step S58: Compare the first ratio and the second ratio to calculate the deviation, wherein: If the deviation is within the preset error range, the detection accuracy of the undiluted sample is determined to be qualified, the undiluted concentration is set as the maximum concentration of the standard working solution, and the standard working curve is corrected.

7. The method for identifying salt tolerance of wheat based on AA3 type flow analyzer according to claim 6, characterized in that: In step S5, whenever the tested sample is determined to need dilution, the cleaning time of the tested sample after testing is extended by 30 seconds.

8. The method for identifying salt tolerance of wheat based on AA3 type flow analyzer according to claim 1 or 4, characterized in that: The operating parameters of the flow analyzer include: The volume ratio of the cleaning solution is 3% nitric acid; The absorbance detector used was a flame photometer, with an injection rate of 40 samples per hour, an injection time of 40 s, and an injection and cleaning time of 35 s; The fuel gas is an air-propane mixture, the propane pressure is 0.08MPa, and the air pressure is 15Psi.

9. The method for identifying salt tolerance of wheat based on AA3 type flow analyzer according to claim 7, characterized in that: In step S4, constructing the detection parameters of the sample to be tested includes: Step S41, open the AA3 flow analyzer software, set the concentration unit and decimal places, sampling time, flushing time, set the standard curve type, select nonlinear fitting, and enter the corresponding standard working solution concentration from high to low, wherein the standard working solution concentration is the ion concentration in the curve segment corresponding to the measurement curve in the standard working curve; Step S42: Set a blank, run deionized water through the flow path for more than 10 minutes, and determine that the baseline is stable when bubbles running in the flow path are regular. Adjust the OD value to 000 by adjusting the flame photometer. Step S43, set the adjustment gain, place the prepared highest concentration standard working solution on the sampling tray, perform manual sampling, and make the injection needle take the liquid time consistent with the sample aspiration time. Wait for the peak to appear and stabilize before adjusting the gain to complete the setting of the detection parameters.

10. The method for identifying salt tolerance of wheat based on AA3 type flow analyzer according to claim 1, characterized in that: In the step S1, the mixture is homogenized at a speed of 5000 r / min for 5 minutes, centrifuged at a speed of 5500 r / min for 12 minutes, and filtered after centrifugation to obtain a wheat sample extract.

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