A method for identifying wheat salt tolerance based on an AA3 flow analyzer

Abstract

This invention relates to the field of flow analyzer technology, and particularly to a method for identifying the salt tolerance of wheat based on an AA3 type flow analyzer. The method includes: Step S1, obtaining a number of wheat samples to be tested; Step S2, preparing sodium ion and potassium ion standard working solutions respectively; Step S3, injecting each standard working solution into a continuous flow analyzer and measuring the sodium and potassium ion standard working curves using a flame photometer; Step S4, constructing the detection parameters for the samples to be tested; Step S5, injecting the samples into a continuous flow analyzer and measuring the absorbance of each standard stock solution using a flame photometer; Step S6, analyzing the salt tolerance of wheat based on the measured sodium and potassium ion concentrations of the samples to be tested. This invention has advantages such as continuous testing of batches of samples, fast analysis speed, saving manpower and resources, high accuracy, and strong anti-interference ability.

Description

Technical Field

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

[0002] Soil salt stress significantly inhibits crop growth and development. Salt stress causes stunted plant development, inhibits root morphogenesis and organ growth and differentiation, leads to leaf curling and chlorosis, reduces thousand-grain weight, causes floret sterility, and ultimately results in a substantial reduction in crop yield. High concentrations of salt stress can cause osmotic stress and ion toxicity in plants, leading to secondary damage such as oxidative stress and nutrient depletion. Excessive soil salinity causes the soil water potential to fall below that of plant root cells, making it difficult for roots to absorb water and causing drought stress. Continuous salt stress reduces plant cell turgor pressure, affecting cell growth; plants must maintain cell elongation and growth through osmotic regulation. Salt stress shortens cell elongation time, affecting the development of the wheat main stem and reproductive structures, shortening flowering time, and thus accelerating plant maturation.

[0003] Multiple studies have shown that The ratio is one of the important indicators for evaluating the salt tolerance of plants. Under salt stress conditions, the ratio of salt content in plants... Concentration usually increases significantly, while The concentration is relatively reduced, resulting in The ratio increased. This change reflects the plant's response mechanism to salt stress, namely, by reducing... to reduce by ratio Toxic effects on cell function. For example, studies have shown that under salt stress, the aboveground parts of wheat... The significantly reduced ratio indicates that salt-tolerant varieties can more effectively eliminate [the pollutants]. and maintain The absorption and accumulation The ratio not only reflects the current salt tolerance status of wheat, but can also serve as a breeding target for screening and cultivating salt-tolerant varieties. For example, by measuring the salt tolerance of wheat seedlings under salt stress... The ratio can be used to quickly screen for materials with strong salt tolerance. Therefore, developing a simple, fast, accurate, and low-cost wheat [method / technology] is crucial. , There is an urgent need for a suitable analytical method. The AA3 flow analyzer, with its advantages of high automation, fast analysis speed, high accuracy, and low reagent consumption, has been widely used in water quality analysis, soil nutrient detection, and other fields. However, applying the AA3 flow analyzer to wheat... , No research or methods for salt content determination have been reported yet, which is of great significance for assessing the salt tolerance of wheat varieties and guiding wheat cultivation in saline-alkali land.

[0004] Chinese Patent Application Publication No. CN105612851A discloses a method for evaluating or screening salt-tolerant wheat based on potassium ion flow, comprising the following steps: dividing the wheat seeds to be tested into two groups, with the control group germinated under normal conditions and the experimental group germinated under salt stress conditions, and detecting the aleurone layer of the seeds during germination. The flow direction and velocity, and the control group Flow rate minus experimental group The flow rate is obtained as a numerical value M; if the numerical value M is less than or equal to 30 The wheat to be tested is a candidate salt-intolerant wheat; if the value M is greater than 30 The wheat to be tested was a candidate salt-tolerant wheat. This shows that this method only applies to wheat... Testing alone cannot fully reflect the salt tolerance of wheat, and the measurement process is complex and difficult to promote. Summary of the Invention

[0005] Therefore, this invention provides a method for identifying wheat salt tolerance based on an AA3 flow analyzer, to overcome the limitations of existing technologies for wheat salt tolerance identification. , The content determination process is complex and the accuracy of the determination cannot be precisely judged.

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

[0007] Step S1: Obtain a number of wheat samples that meet the consistency standard, and pre-process them to obtain the test sample;

[0008] Step S2: Prepare several sodium ion standard stock solutions and several potassium ion standard stock solutions with different concentrations.

[0009] Step S3: Set the operating parameters of the flow analyzer, inject each standard stock solution into the continuous flow analyzer and measure the absorbance of each standard stock solution using a flame photometer, and obtain the sodium ion standard working curve and potassium ion standard working curve based on the absorbance.

[0010] Step S4: Construct the detection parameters of the sample to be tested based on the sodium ion standard working curve and the potassium ion standard working curve;

[0011] Step S5: Inject the sample to be tested using a continuous flow analyzer and measure 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 based on the concentration and absorbance of the sample to be tested before and after dilution.

[0013] Step S6: Analyze the salt tolerance of wheat based on the sodium ion concentration and potassium ion concentration measured on the sample to be tested.

[0014] Further, step S1 includes:

[0015] Step S11: Cut the collected wheat root or leaf samples into small segments and mix them to obtain sample segments.

[0016] Step S12: Weigh the sample segment and place it in a homogenizer, add ultrapure water, homogenize at a speed of 5000 r / min, centrifuge and filter to obtain wheat sample extract;

[0017] Step S13: Take the wheat sample extract and put it into a volumetric flask. Add 0.1 mol / L aluminum sulfate solution, dilute to volume with ultrapure water, and shake well to obtain the sample to be tested.

[0018] Further, step S2 includes:

[0019] Step S21: Prepare sodium standard stock solutions with a sodium chloride concentration of 1000 mg / L and potassium standard stock solutions with a potassium chloride concentration of 1000 mg / L.

[0020] Step S22: Take different volumes of sodium standard stock solution and potassium standard stock solution and make up to volume to obtain several sodium standard working solutions and potassium standard working solutions with different mass concentrations.

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

[0022] Further, step S3 includes:

[0023] Step S31: Set the operating parameters of the flow analyzer;

[0024] Step S32: Use the dilution of the corresponding standard working solution to calibrate the zero point of the corresponding standard working curve;

[0025] Step S33: Measure the absorbance of each sodium standard working solution and establish a sodium ion measurement curve based on the sodium ion content and corresponding absorbance in each sodium standard working solution.

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

[0027] Step S34: Calculate the mean point based on the average absorbance of each sodium standard working solution, and establish an ideal sodium ion curve based on the mean point and the zero point.

[0028] The mean point is calculated based on the average absorbance of each potassium standard working solution, and an ideal potassium ion curve is established based on the mean point and the zero point.

[0029] Step S35: Compare the sodium ion measurement curve with the corresponding ideal sodium ion curve to determine the sodium ion standard working curve; compare the potassium ion measurement curve with the corresponding ideal potassium ion curve to determine the potassium ion standard working curve.

[0030] Specifically, the absorbance of the ideal curve and the measured curve at the same concentration are compared. If the absorbance difference is within 5%, the standard working curve is selected from the curve segment corresponding to the ideal curve; if the absorbance difference exceeds 5%, the standard working curve is selected from the curve segment corresponding to the measured curve.

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

[0032] Step S51: Obtain the peak height of the absorbance of the sample to be tested, and record it as the first peak height;

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

[0034] When the height of the first peak 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. The dilution ratio is determined based on the height of the first peak and the corresponding standard working curve.

[0035] Step S53: Open the dilution valve, draw up the diluent according to the dilution ratio, draw up the test sample again and put it into the dilution container, mix the test sample and diluent evenly, and then retest.

[0036] Step S54: Re-measure the peak height of the diluted sample, wherein...

[0037] If the peak height of the retest 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 peak height of the retest still exceeds 80% of the maximum peak height, repeat step S52, redetermine the dilution ratio, and retest again.

[0039] Step S55: Based on the final dilution ratio and the measured absorbance, the actual concentration of the sample to be tested is calculated by reverse derivation.

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

[0041] Step S56: Obtain the actual concentration and corresponding absorbance of the diluted sample to be tested, and determine the first ratio;

[0042] Step S57: Obtain the absorbance of the stock solution determined by the first peak height of the sample before dilution, and determine the second ratio based on 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 deemed 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 a tested sample is determined to need dilution, the cleaning time 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 cleaning solution contains 3% nitric acid by volume.

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

[0049] The fuel gas is an air-propane mixture with a propane pressure of 0.08 MPa and an air pressure of 15 Psi.

[0050] Further, in step S4, the detection parameters of the sample to be tested are constructed, including:

[0051] Step S41: Open the AA3 flow analyzer software, set the concentration unit and decimal places, sampling time, rinsing time, set the standard curve type, select nonlinear fitting, and input the corresponding standard working solution concentration from high to low. 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 blank, run deionized water in the flow path for more than 10 minutes, and judge the baseline to be stable when the bubbles in the flow path are regular. Adjust the OD value to 000 by adjusting the flame photometer.

[0053] Step S43: Set the gain adjustment, place the prepared highest concentration standard working solution in the sampling tray, perform manual sampling, and ensure that the liquid sampling time of the injection needle is consistent with the sampling time. Wait for the peak to appear and then adjust the gain to complete the setting of the detection parameters.

[0054] Further, in step S1, the sample is homogenized at 5000 r / min for 5 min, centrifuged at 5500 r / min for 12 min, and then filtered to obtain wheat sample extract.

[0055] Compared with the prior art, the beneficial effect of the present invention is that it provides a method for determining wheat using an AA3 flow analyzer. , The method, through , Recently, the salt tolerance of wheat has been assessed in order to address existing wheat [problems]. , The previous methods suffered from complex operation, cumbersome sample pretreatment, high analysis costs, and the need for professional technicians. This new method simplifies the complex manual operation into automated instrument detection, which can continuously test batches of samples. It is not only fast in analysis and saves manpower and resources, but also has high accuracy, strong anti-interference ability, low reagent consumption and low environmental pollution. The automated operation also reduces human error. The characteristics of this technology are that it is suitable for early generation populations with small size, does not harm the plants, and can lock in the breeding target in the early generation, thereby improving breeding efficiency.

[0056] Furthermore, by establishing an automated dilution system to replace manual dilution steps, this invention can reduce sample processing time by more than 70% in batch testing. The system can automatically adapt to samples of different concentrations: high-concentration samples are automatically diluted to the linear range, while low-concentration samples are prevented from being injected repeatedly, which greatly reduces labor costs and achieves full-process automation and continuous operation around the clock.

[0057] Furthermore, by dynamically correcting the standard working curve and calibrating the curve in real time, this invention reduces the problem of accuracy decay of traditional static curves, ensures long-term stability during the detection process, eliminates experimental rework caused by curve failure at the source, and significantly expands the detection range of the instrument. Attached Figure Description

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

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

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

[0061] Figure 4 This is a flowchart illustrating the method for determining sodium and potassium ions in wheat using an AA3 flow analyzer, as described in an embodiment of the present invention. Detailed Implementation

[0062] To make the objectives and advantages of the present invention clearer, the present invention will be further described below with reference to embodiments; it should be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the present invention.

[0063] Preferred embodiments of the present invention will now be described with reference to the accompanying drawings. Those skilled in the art should understand that these embodiments are merely illustrative of 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 this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

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

[0066] Step S1, Sample collection and processing: Obtain a number of wheat samples that meet the consistency standard, and pre-process them to obtain the test samples;

[0067] Cut the collected wheat root or leaf samples into small segments ≤1cm and mix them thoroughly. Weigh 10g of the sample segment and place it in a homogenizer, then add 50mL of ultrapure water and homogenize at 5000r / min for 5min. After centrifugation (5500r / min for 12min) and filtration, obtain the sample extract. Finally, take 5mL of the wheat sample extract and put it into a 25mL volumetric flask, add 1mL of 0.1mol / L aluminum sulfate solution, and dilute to the mark with ultrapure water. Shake well.

[0068] Step S2: Prepare the standard working solution;

[0069] Prepare sodium standard stock solution: Weigh 2.5421 g (accurate to 0.0001 g) of sodium chloride working standard reagent that has been dried to constant weight in an electric oven at 105℃, add 3% nitric acid (v / v) to dissolve and dilute to 1000 mL; this solution is sodium standard stock solution with a sodium chloride concentration of 1000 mg / L.

[0070] Prepare sodium standard working solutions: Take 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 up 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] Preparation of potassium standard stock solution: Weigh 0.1907 g of potassium chloride working standard reagent that has been dried in an electric oven at 110℃ for 2 h, add it to 1.0 mol / L ammonium acetate solution (pH 7.0) and make up to 100 mL. This solution is the potassium standard stock solution with a potassium chloride concentration of 1000 mg / L.

[0072] Preparation of potassium standard working solutions: Take 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 up to 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: Set the instrument operating parameters and plot the standard operating curve;

[0074] The cleaning solution used was 3% nitric acid by volume; the detector used 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 sodium ion standard working solution and potassium ion standard working solution were injected using the above-mentioned working parameters, and the absorbance of each standard working solution was measured by a flame photometer to obtain the absorbance data corresponding to each working solution.

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

[0077] The zero point of the standard curve was corrected by nitric acid with a volume ratio of 3%. 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 based on the average absorbance of each sodium standard working solution, and an ideal sodium ion curve is established based on the mean point and the zero point.

[0079] Specifically, the process of plotting the potassium standard 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. 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 based on the average absorbance of each potassium standard working solution, and an ideal potassium ion curve is established based on the mean point and the zero point.

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

[0083] For example, given the obtained ideal sodium ion curve and sodium ion measurement curve, the two curves are divided into several segments based on concentration, depending on whether the absorbance difference between the two curves exceeds 5%. Within the same concentration range, the absorbance of the same concentration in two curve segments differs by either less than 5% or more than 5%. In this case, one curve segment is selected based on whether the absorbance difference exceeds 5%. This selection process is repeated for each curve segment to form the sodium ion standard working curve. The selection logic for the potassium ion standard working curve is the same and will not be elaborated upon here.

[0084] Step S4: Construct the detection parameters of the sample to be tested based on 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 on the analysis interface to enter the MethodsSetting interface, click Add to create new sodium and potassium methods. Click General to set the concentration unit and accuracy, sampling time, and rinsing time. Click OD / Curve / QC to set the standard curve type, using flame emission method. Preferably, select nonlinear fitting, and input 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 measured curve in the standard working curve, and the settings are complete.

[0086] Set the flow path to blank, run the deionized water for more than 10 minutes, and 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 highest concentration of standard working solution in the sampling tray, and perform manual sampling. The sampling time of the injection needle should be consistent with the set sampling time. Wait for the peak to appear and then adjust the gain to complete the setting of the detection parameters.

[0088] Step S5, Sample determination; the sample to be tested is injected using a continuous flow analyzer and the absorbance of each standard stock solution is measured using a flame photometer, wherein,

[0089] The peak height of the absorbance of the sample to be tested is obtained and recorded as the first peak height. 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] The dilution ratio is determined based on the height of the first peak and the corresponding standard working curve. To prevent the concentration after dilution from falling just at the limit of the detection range and affecting the accuracy of the detection, a redundancy coefficient is set (within the range of 1.1 to 1.4), with a preferred value of 1.2.

[0091]

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

[0093] When it is confirmed that the sample to be tested needs to be diluted, open the dilution valve, draw up the diluent according to the dilution ratio, draw up the sample to be tested again and put it into the dilution container, mix the sample to be tested with the diluent evenly, and then retest.

[0094] Specifically, the peak height of the diluted sample was re-measured, where...

[0095] If the peak height of the retest is less than 80% of the maximum concentration peak height of the standard working curve, the dilution ratio is deemed appropriate. The sample in the dilution container is then drained through the drain valve, and the cleaning time after the test sample is detected is extended by 30 seconds to reduce the error of subsequent measurements.

[0096] If the peak height of the retest 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. The dilution container is equipped with a level gauge; when it is predicted that the diluted sample will exceed the upper limit of the container's level (80% of the dilution container's capacity), half the sample volume is drained through the drain valve. Based on the final dilution ratio and the measured absorbance, the actual concentration of the sample is calculated in reverse. It is understandable that calculating the concentration of the sample before dilution using the dilution ratio and the concentration of the diluted sample after dilution is existing technology and will not be elaborated upon here.

[0097] In step S5, the corresponding standard working curve is corrected based on the concentration and absorbance of the sample before and after dilution.

[0098] Specifically, the corresponding standard operating curves are modified, including:

[0099] Obtain the actual concentration and corresponding absorbance of the diluted sample to be tested, and determine the first ratio;

[0100] The absorbance of the stock solution is determined by the first peak height of the sample before dilution, and the second ratio is determined based on the absorbance of the stock solution and the corresponding concentration of the stock solution 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 deviation is calculated by comparing the first ratio and the second ratio. If the deviation is within the preset error range (95% to 105%), the detection accuracy of the undiluted test sample is deemed 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 original solution concentration and original solution absorbance are added to the standard working curve data.

[0103] Step S6: Analyze the salt tolerance of wheat based on the sodium ion concentration and potassium ion concentration measured on the sample to be tested.

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

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

[0106] Example 1:

[0107] 1. Sample Collection and Processing: More than 50 samples of each of 10 approved wheat varieties were collected for production. These samples were subjected to salt stress treatment in a 300 mmol / L NaCl solution for 10 days. Young leaves and rhizomes were collected as samples. The collected samples were cut into small segments ≤10 mm and mixed thoroughly. 10 g of sample segments were weighed and placed in a homogenizer, along with 50 mL of ultrapure water. The homogenizer was homogenized at 5000 rpm for 5 min, centrifuged (5500 rpm for 12 min), and filtered to obtain the plant sample extract. Finally, 5 mL of the plant sample extract was transferred to a 25 mL volumetric flask, 1 mL of 0.1 mol / L aluminum sulfate solution was added, and the solution was diluted to the mark with ultrapure water and shaken well.

[0108] 2. Standard curve plotting: Please refer to [link / reference]. Figure 2 and Figure 3 As shown, a certain amount of sodium chloride and potassium chloride were accurately weighed to prepare the standard working solution. The absorbance was measured and a standard curve was plotted (the specific steps are as described in the implementation steps above). In the figure, y is the peak height and x is the ion concentration. The goodness of fit is denoted as .

[0109] 3. Sample Measurement: Please refer to [link / reference needed]. Figure 4 As shown, the prepared , The standard working solution and sample are drawn 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, washing ratio, etc.), each segment is fully mixed in the mixing ring and reacts to generate a colored compound. The compound is then measured by colorimetry using a detector. 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 samples used in Example 1, using normally grown wheat young leaves and rhizomes that have not been subjected to salt stress as samples; the rest is the same as in Example 1.

[0112] Comparative Example 1:

[0113] This comparative example uses the traditional dry ashing technique to test the sample selected in Example 1.

[0114] Comparative Example 2:

[0115] This comparative example uses the traditional dry ashing technique to test the samples selected in Example 2.

[0116] The wheat samples measured by this invention , The test results are shown in Table 1:

[0117] Table 1. Results of salt tolerance test of wheat roots and stems using the improved method

[0118]

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

[0120]

[0121] As shown in Tables 1 and 2 above, the sodium-potassium ion ratio of Jiemai 19 wheat showed the smallest change under salt stress, indicating that this variety can more effectively expel sodium ions and maintain potassium ion absorption and accumulation. It can be used as a breeding target for screening and cultivating salt-tolerant varieties. The wheat salt tolerance identification method based on the AA3 flow analyzer of this invention can accurately quantify the salt tolerance of wheat samples. , It allows for precise measurement of content and has advantages such as high automation, fast analysis speed, high accuracy, and low reagent consumption.

[0122] Partial wheat determined by dry ashing method , The test results are shown in Tables 3 and 4:

[0123] Table 3. Results of salt tolerance test of wheat roots and stems 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 ratio measured using this invention is highly consistent with the results of traditional detection methods, indicating that its detection accuracy is very close to that of traditional methods. Furthermore, the working curve can be promptly corrected when high-concentration samples are encountered during the detection process, reducing manual intervention. This method can significantly reduce reagent consumption and labor costs, greatly improve detection speed, and is simple to operate and easy to promote and implement.

[0128] The technical solution of the present invention has been described above with reference to 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 can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the scope of protection of the present invention.

Claims

1. A method for identifying wheat salt tolerance based on an AA3 flow analyzer, characterized in that, include: Step S1: Obtain a number of wheat samples that meet the consistency standard, and pre-process them to obtain the test sample; Step S2: Prepare several sodium ion standard working solutions and several potassium ion standard working solutions of different concentrations respectively. Step S3: Set the operating parameters of the flow analyzer, inject each standard working solution into the continuous flow analyzer, and measure the absorbance of each standard working solution using a flame photometer. Based on the absorbance, derive the sodium ion standard working curve and the potassium ion standard working curve; Step S3 includes: Step S31: Set the operating parameters of the flow analyzer; Step S32: Use the diluent of the corresponding standard working solution to calibrate the zero point of the corresponding standard working curve; Step S33: Measure the absorbance of each sodium standard working solution and establish a sodium ion measurement curve based on the sodium ion content and corresponding absorbance in each sodium standard working 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 and the corresponding absorbance in each potassium standard working solution. Step S34: Calculate the mean point based on the average absorbance of each sodium standard working solution, and establish an ideal sodium ion curve based on the mean point and the zero point. The mean point is calculated based on the average absorbance of each potassium standard working solution, and an ideal potassium ion curve is established based on the mean point and the zero point. Step S35: Compare the sodium ion measurement curve with the corresponding ideal sodium ion curve to determine the sodium ion standard working curve; compare the potassium ion measurement curve with the corresponding ideal potassium ion curve to determine the potassium ion standard working curve. Specifically, the absorbance of the ideal curve and the measured curve at the same concentration are compared. If the absorbance difference is within 5%, the standard working curve is selected from the curve segment corresponding to the ideal curve; if the absorbance difference exceeds 5%, the standard working curve is selected from the curve segment corresponding to the measured curve. Step S4: Construct the detection parameters of the sample to be tested based on the sodium ion standard working curve and the potassium ion standard working curve; Step S5: Inject the sample to be tested using a continuous flow analyzer and measure 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 based on the concentration and absorbance of the sample to be tested before and after dilution. In step S5, the process of determining whether the sample needs to be diluted includes: Step S51: Obtain the peak height of the absorbance of the sample to be tested, and record it as the first peak height; Step S52: Compare the height of the first peak with 80% of the maximum concentration peak height of the standard working curve; When the height of the first peak 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. The dilution ratio is determined based on the height of the first peak and the corresponding standard working curve. Step S53: Open the dilution valve, draw up the diluent according to the dilution ratio, draw up the test sample again and put it into the dilution container, mix the test sample and diluent evenly, and then retest. Step S54: Re-measure the peak height of the diluted sample, wherein... If the peak height of the retest 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 peak height of the retest still exceeds 80% of the maximum peak height, repeat step S52, redetermine the dilution ratio, and retest again. Step S55: Based on the final dilution ratio and the measured absorbance, the actual concentration of the sample to be tested is calculated in reverse. In step S5, the process of correcting the corresponding standard working curve includes: Step S56: Obtain the actual concentration and corresponding absorbance of the diluted sample to be tested, and determine the first ratio; Step S57: Obtain the absorbance of the stock solution determined by the first peak height of the sample before dilution, and determine the second ratio based on 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. If the deviation meets the preset error range, the detection accuracy of the undiluted test 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. Step S6: Analyze the salt tolerance of wheat based on the sodium ion concentration and potassium ion concentration measured on the sample to be tested.

2. The method for identifying wheat salt tolerance based on an AA3 flow analyzer according to claim 1, characterized in that, Step S1 includes: Step S11: Cut the collected wheat root or leaf samples into small segments and mix them to obtain sample segments. Step S12: Weigh the sample segment and place it in a homogenizer, add ultrapure water, homogenize at a speed of 5000 r / min, centrifuge and filter to obtain wheat sample extract; Step S13: Take the wheat sample extract and put it into a volumetric flask. Add 0.1 mol / L aluminum sulfate solution, dilute to volume with ultrapure water, and shake well to obtain the sample to be tested.

3. The method for identifying wheat salt tolerance based on an AA3 flow analyzer according to claim 2, characterized in that, Step S2 includes: Step S21: Prepare sodium standard stock solutions with a sodium chloride concentration of 1000 mg / L and potassium standard stock solutions with a potassium chloride concentration of 1000 mg / L. Step S22: Take different volumes of sodium standard stock solution and potassium standard stock solution and make up to volume to obtain several sodium standard working solutions and potassium standard working solutions with different mass concentrations. The sodium standard working solution contains at least three different sodium chloride concentrations, and the potassium standard working solution contains at least three different potassium chloride concentrations.

4. The method for identifying wheat salt tolerance based on an AA3 flow analyzer according to claim 3, characterized in that, In step S5, whenever a tested sample is determined to need dilution, the cleaning time after testing is extended by 30 seconds.

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

6. The method for identifying wheat salt tolerance based on an AA3 flow analyzer according to claim 5, characterized in that, In step S4, the detection parameters of the sample to be tested are constructed, including: Step S41: Open the AA3 flow analyzer software, set the concentration unit and decimal places, sampling time, rinsing time, set the standard curve type, select nonlinear fitting, and input the corresponding standard working solution concentration from high to low. 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 blank, run deionized water in the flow path for more than 10 minutes, and judge the baseline to be stable when the bubbles in the flow path are regular. Adjust the OD value to 000 by adjusting the flame photometer. Step S43: Set the gain adjustment, place the prepared highest concentration standard working solution in the sampling tray, perform manual sampling, and ensure that the liquid sampling time of the injection needle is consistent with the sampling time. Wait for the peak to appear and then adjust the gain to complete the setting of the detection parameters.

7. The method for identifying wheat salt tolerance based on an AA3 flow analyzer according to claim 1, characterized in that, In step S1, the sample is homogenized at 5000 r / min for 5 min, centrifuged at 5500 r / min for 12 min, and then filtered to obtain wheat sample extract.

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