Method for detecting concentration of available phosphorus in rice tissue

Abstract

The invention discloses a method for detecting the concentration of available phosphorus in rice tissues, and belongs to the technical field of physiological and biochemical detection of plants. The method comprises the following steps: mixing and homogenizing a rice tissue sample, a grinding liquid and grinding beads, adding a weak acid extracting solution, and extracting under a low-temperature dark condition to obtain a crude extracting solution; adding hydrogel microspheres prepared by a specific method into the crude extract, swelling, centrifuging, and collecting concentrated and purified supernate; diluting the supernate, detecting the absorbance at the wavelength of 820 nm by adopting a molybdenum blue method, and calculating the available phosphorus concentration through a standard curve. Phosphate ions are passively concentrated through the selective water absorption characteristic of the hydrogel microspheres, macromolecular interferents such as pigments and proteins are removed through the size exclusion effect of the network structure of the hydrogel microspheres, and the detection sensitivity and the anti-interference capacity are remarkably improved. The method is simple, convenient and safe to operate, the detection limit reaches 0.015 mg / L, the adding standard recovery rate is 95.00%-105.00%, and the method is suitable for detecting rice samples of different tissue types, phosphorus content levels and indica and japonica rice varieties.

Description

Technical Field

[0001] This application relates to the field of plant nutrient analysis technology, and in particular to a method for detecting the effective phosphorus concentration in rice tissues. Background Technology

[0002] Phosphorus that rice roots can directly absorb and utilize is called available phosphorus, which usually exists in the form of inorganic phosphorus. Organic phosphorus needs to be converted into inorganic phosphorus through enzymatic hydrolysis before it can be absorbed. Therefore, detecting available phosphorus (inorganic phosphorus) in rice tissues is crucial for studying plant growth.

[0003] Currently, methods for determining the available phosphorus concentration in rice tissues include: molybdenum blue colorimetric method, malachite green colorimetric method, continuous flow analysis, ion chromatography, and nuclear magnetic resonance spectroscopy.

[0004] The molybdenum blue colorimetric method is a relatively traditional and widely used method, simple to operate and inexpensive, but it has certain accuracy issues. In using this method, sample pretreatment usually requires strong acid solutions such as trichloroacetic acid or sulfuric acid. Strong acid solutions can not only severely damage cell structures but also cause organic phosphorus to hydrolyze into inorganic phosphorus, resulting in overestimation of the measured values. Furthermore, residual strong acid in the extract can easily interfere with subsequent colorimetric reactions, and the strong acid solutions themselves are highly corrosive, leading to low operational safety and requiring high levels of protection for the experimental environment, equipment, and personnel, thus increasing experimental costs and operational complexity. While the malachite green colorimetric method has relatively high sensitivity, it has poor anti-interference capabilities and a narrow linear range for its standard curve, making it difficult to cover samples with high and low concentrations.

[0005] Furthermore, malachite green exhibits poor stability under acidic or strong light conditions, is prone to degradation, and the fuel itself possesses a degree of toxicity, resulting in low safety and limiting the application of this method. Continuous flow analysis offers a high degree of automation, suitable for the determination of large batches of samples; however, its detection time is long, the required equipment is expensive, and maintenance costs are high, making it unsuitable for widespread laboratory application. While ion chromatography and nuclear magnetic resonance spectroscopy offer high precision and resolution, they also suffer from expensive equipment, complex sample pretreatment, high operator skill requirements, and low detection efficiency, making them unsuitable for rapid, large-scale determination of available phosphorus in plant tissues.

[0006] Therefore, in view of the shortcomings of the above-mentioned technologies, there is an urgent need for a method for determining the available phosphorus concentration in rice tissues that is easy to operate, fast to detect, accurate and reliable, and suitable for large-scale application, so as to meet the actual needs of plant nutrition research and agricultural production management. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a method for detecting available phosphorus in rice tissues and its application. By optimizing the extraction solution and reaction conditions, the extraction efficiency is improved, and the detection sensitivity, precision, and accuracy are also enhanced.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: (1) The rice tissue sample was mixed with grinding liquid and grinding beads and then homogenized to obtain tissue homogenate; (2) Add the extraction solution to the tissue homogenate and extract for 10-30 minutes at 4°C in the dark to obtain crude extract; (3) Add hydrogel microspheres to the crude extract, mix and let stand to allow them to swell, then centrifuge and collect the supernatant; (4) The supernatant is diluted with a diluent to obtain the sample reaction solution; (5) The sample reaction solution and the colorimetric reaction solution are mixed and incubated. After the colorimetric reaction occurs, the absorbance is detected at 820 nm. The effective phosphorus concentration in the rice tissue is calculated based on the pre-established phosphorus concentration standard curve.

[0009] By adopting the above technical solution, hydrogel microspheres selectively adsorb water molecules through their hydrophilic three-dimensional network structure, thereby achieving passive concentration of phosphate ions. At the same time, they effectively remove large molecular interferences such as pigments and proteins from the extract by utilizing the size exclusion effect of their pore size, which significantly improves the anti-interference ability of the detection.

[0010] Optionally, the hydrogel microspheres are prepared by the following method: Acrylamide and N,N'-methylenebisacrylamide were dissolved in deionized water. After purging with nitrogen to remove oxygen, ammonium persulfate and N,N,N',N'-tetramethylethylenediamine were added. The mixture was stirred to form a hydrogel. The hydrogel was then broken up, sieved, and washed to obtain microspheres with a particle size of 50-100 μm. The mass ratio of acrylamide to N,N'-methylenebisacrylamide was (18-20):1.

[0011] Optionally, the mass ratio of the hydrogel microspheres to the rice tissue sample is (1-2).

[0012] By adopting the above technical solution, the hydrogel microspheres synthesized in a specific ratio have suitable water absorption and swelling properties and network pore size, which can effectively concentrate phosphate ions and fully remove macromolecular impurities; the optimized mass ratio ensures that the best concentration and purification effect is achieved within a limited processing volume.

[0013] Optionally, the extract can be any of the following aqueous solutions: acetic acid solution, citric acid solution, choline chloride-oxalic acid solution, MES buffer, or sodium acetate solution. When using acetic acid solution, its volume concentration is 0.5%-5% (v / v); When other solutions are used, their mass concentration is 0.5%-5% (w / v).

[0014] By adopting the above technical solutions, a variety of effective and mild extraction solutions are provided, and the concentration range is optimized to minimize the damage of the extraction solution to available phosphorus and the interference with subsequent colorimetric reactions. At the same time, the waste liquid generated is easy to treat and has little environmental pollution.

[0015] Optionally, when the extract is a sodium acetate solution, it also contains 0.005%-0.05% (w / v) of plant cell lysin.

[0016] Optionally, the plant cell lysin is selected from at least one of cellulase, pectinase, and hemicellulase.

[0017] By adopting the above technical solution, the selected enzymes can specifically hydrolyze the main components of rice tissue cell walls, including cellulose and pectin. Under the synergistic effect of multiple enzymes, the cell structure can be more thoroughly disintegrated, increasing the dissolution of available phosphorus. Moreover, these enzymes can maintain appropriate activity under low temperature conditions and will not interfere with subsequent detection.

[0018] Optionally, the colorimetric reaction step is based on the molybdenum blue method.

[0019] By adopting the above technical solution, the colorimetric and quantitative analysis of phosphorus is performed using the molybdenum blue method. The absorbance of the molybdenum blue complex is detected at 820 nm. This wavelength has high specificity and can effectively reduce the interference of impurities in the sample, ensuring the specificity of the colorimetric reaction and the reliability of the detection results.

[0020] Optionally, the standard curve exhibits a linear relationship within the phosphorus concentration range of 0-25 mg / L, with a linear regression equation of y=0.1504x+0.046 and a detection limit (LOD) of 0.015 mg / L; the spiked recovery rate of the detection method ranges from 95.00% to 105.00%.

[0021] By employing the above technical solution, the established standard curve exhibits good linearity within the phosphorus concentration range of 0-25 mg / L, covering the typical concentration range of available phosphorus dilutions from rice tissues, ensuring the accuracy of quantitative calculations. The extremely low detection limit indicates that the method has very high sensitivity and can accurately detect trace amounts of available phosphorus in rice tissues. The spiked recovery results demonstrate that the method has high accuracy, small systematic error, minimal loss of available phosphorus throughout the detection process, and the measured values ​​are close to the true values.

[0022] Optionally, the rice tissue sample includes rice tissues of different tissue types or different phosphorus content levels; the rice variety is indica rice and / or japonica rice.

[0023] The above-described technical solution demonstrates that this method has good universality and can be applied to different tissue types of rice, such as roots, stems, and leaves, as well as rice samples with different phosphorus nutritional statuses, including low-phosphorus, normal-phosphorus, and high-phosphorus varieties. The detection results are reliable. Furthermore, this method can overcome potential differences in structural tissues and chemical composition among different rice subspecies, exhibiting broad varietal adaptability and providing a universal method for phosphorus nutrition research on different rice varieties.

[0024] In summary, this application has the following beneficial effects: 1. The present invention uses hydrogel microspheres prepared with a specific ratio to effectively remove macromolecular impurities such as pigments and proteins from crude extract through size exclusion effect, and at the same time achieves passive concentration of phosphate ions through selective water absorption, which significantly improves the detection signal-to-noise ratio.

[0025] 2. The detection method of the present invention exhibits good linearity in the range of 0-25 mg / L, with a coefficient of determination R0. 2 It is approximately 0.9999.

[0026] 3. The detection method of the present invention is accurate, with a spiked recovery rate between 95.00% and 105.00%.

[0027] 4. This invention is applicable to rice tissues of different varieties and rice tissue samples with different phosphorus contents, and can detect a wide range while maintaining detection sensitivity and accuracy. Attached Figure Description

[0028] Figure 1 This is the standard curve graph of this application; Figure 2 This is the result of the determination of available phosphorus concentration in rice tissues in Example 11 of this application; Figure 3 This is the result of measuring the effective phosphorus concentration in rice tissues in Example 12 of this application. Detailed Implementation

[0029] Instruments and reagents: 1.5 mL centrifuge tubes, 2 mL centrifuge tubes, 15 mL centrifuge tubes, 96-well microplates, biochemical incubator (Shanghai Yiheng), microplate reader (Thermo Fisher Scientific Varioskan LUX), tissue homogenizer (Shanghai Jingxin), centrifuge (Eppendorf).

[0030] Acetic acid and sulfuric acid were of analytical grade and purchased from Sinopharm Reagents; all other reagents involved were commercially available products.

[0031] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this application, the technical solution of the present invention will now be described in detail with reference to the following specific embodiments and accompanying drawings. However, this should not be construed as limiting the scope of implementation of the present invention.

[0032] Preparation example of hydrogel microspheres Materials: Acrylamide, N,N'-methylenebisacrylamide, ammonium persulfate, N,N,N',N'-tetramethylethylenediamine (TEMED).

[0033] Preparation method: Add 50 mL of deionized water to a 100 mL beaker, and dissolve 4.5 g of acrylamide and 0.25 g of N,N'-methylenebisacrylamide sequentially. Purge with nitrogen for 10 min to remove oxygen interference. Then add 0.05 g of ammonium persulfate and 50 μL of TEMED, and react at room temperature for 30 min with rapid stirring (500 rpm) to form a hydrogel. After crushing the synthesized hydrogel using a pulverizer, collect microspheres with a diameter of 50-100 μm through a sieve. Finally, wash three times with deionized water and store at 4℃ for later use.

[0034] Sample preparation: Rice seeds were treated in a 0.1% NaOH solution for half an hour to break dormancy; then soaked in clean water and placed in a 37℃ incubator for germination. Once the seeds showed signs of germination, they were transferred to a mesh tray with tweezers. The mesh tray was placed in clean water for 2-3 days. Afterward, the clean water was replaced with normal nutrient solution, and the pH was adjusted to 5.5. The control group samples were cultured continuously in the normal nutrient solution for 28 days. The phosphorus content in the normal nutrient solution was 200 μM, which is considered high-phosphorus culture. Low-phosphorus culture involved culturing in the normal nutrient solution for 14 days, then transferring to a low-phosphorus solution for another 28 days. The phosphorus content in the low-phosphorus nutrient solution was 10 μM. During both high-phosphorus and low-phosphorus cultures, the nutrient solution concentration was changed every three days. Samples were taken from the second leaf (old leaf), the fifth leaf (new leaf), and root tissue for analysis.

[0035] Reagent preparation: (1) 1% (v / v) acetic acid solution: Measure 10 mL of analytical grade acetic acid, add it to 80 mL of deionized water, mix well and make up to 1000 mL; (2) 1% (w / v) citric acid solution: Weigh 1 g of analytical grade citric acid, add it to 80 mL of deionized water, mix well and make up to 100 mL; (3) 1% (w / v) choline chloride-oxalic acid (1:2) solution: Weigh 1 g of choline chloride and 1.81 g of oxalic acid dihydrate, add to 80 mL of deionized water, mix well and make up to 100 mL; (4) 1% (w / v) MES solution: Weigh 1 g of analytical grade citric acid, add it to 80 mL of deionized water, mix well and make up to 100 mL; (5) 1% sodium acetate solution containing 0.01% cellulase: Dissolve 1 g of sodium acetate in about 80 mL of water, make up to 100 mL, and mix well; weigh 0.01 g of cellulase, add it to the 1% sodium acetate solution, and stir gently until well mixed; (6) 1% sodium acetate solution containing 0.01% pectinase: Dissolve 1 g of sodium acetate in about 80 mL of water, make up to 100 mL, and mix well; weigh 0.01 g of pectinase, add it to the 1% sodium acetate solution, and stir gently until well mixed; (7) 1% sodium acetate solution containing 0.01% cellulase and 0.01% pectinase: Dissolve 1 g of sodium acetate in about 80 mL of water, make up to 100 mL, and mix well; weigh 0.01 g of cellulase and 0.01 g of pectinase respectively, add them to the 1% sodium acetate solution, and stir gently until well mixed; (8) 1% sodium acetate solution containing 0.01% hemicellulase: Dissolve 1 g of sodium acetate in about 80 mL of water, bring the volume to 100 mL and mix well; weigh 0.01 g of hemicellulase, add it to the 1% sodium acetate solution, and stir gently until well mixed; (9) 1 N H2SO4 solution: Measure 27.2 mL of analytical grade sulfuric acid, slowly add it to 800 mL of deionized water, mix well and then make up to 1000 mL; (10) 0.42% (w / v) ammonium molybdate [(NH4)6Mo7O 24 •4H2O] solution: Weigh 0.42 g of ammonium molybdate, dissolve it in 80 mL of 1N H2SO4 solution, mix well and then make up to 100 mL; (11) 4.10% (w / v) ascorbic acid solution: Weigh 1.0 g of ascorbic acid, add about 8 mL of deionized water to dissolve, mix well and make up to 10 mL. This solution should be prepared fresh or stored at -20 ℃; (12) 1000 mg / L KH2PO4 standard stock solution: Weigh 4.39 g KH2PO4, add about 800 mL of deionized water to dissolve, mix well and then make up to 1000 mL. (13) Standard phosphorus solution: Using a 50 mL volumetric flask, add 40 mL of 1% acetic acid solution, then dilute by multiples according to Table 1 and add a certain amount of KH2PO4 standard stock solution. Mix well and slowly dilute to 50 mL to prepare a series of standard curve solutions of 0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 15, 20, 25 mg / L.

[0036] Table 1. Preparation of Standard Curve Solutions

[0037] 1. Mix ammonium molybdate solution and ascorbic acid solution in a volume ratio of 6:1 beforehand to prepare a colorimetric reaction solution. Pipette 210 μL into a centrifuge tube and add 90 μL of the standard curve series solution to the centrifuge tube, with reagent ① serving as a blank control. After mixing thoroughly, incubate at 37°C for 30 min to promote the formation of the blue molybdate complex.

[0038] 2. Transfer 200 μL of the incubated reaction solution to an ELISA plate for measurement. Set the detection wavelength to 820 nm. The results are then obtained as follows: Figure 1 The standard curve shown indicates a good linear relationship between effective phosphorus concentration and absorbance value within the phosphorus concentration range of 0-25 mg / L, with a correlation coefficient (R0). 2 The value reached 0.9999, and the regression fitting equation was: y = 0.1504x + 0.046, where y is the measured absorbance value and x is the phosphorus concentration (mg / L) of the standard solution.

[0039] In the relevant embodiments of this invention, a standard curve was established using a 1% acetic acid solution as a reference. Experimental verification showed that the linear range of this standard curve (0-25 mg / L) could cover the theoretical concentration range of all solutions in this application. Comparative experiments confirmed that different solution systems exhibited a highly consistent linear relationship between concentration and response signal under the same detection conditions, and the matrix effect had negligible influence on the quantitative results. Therefore, the quantitative data in all embodiments of this invention are calculated based on this reference standard curve and do not affect the reproducibility and effectiveness evaluation of the technical solution.

[0040] Precision testing: Phosphorus standard solutions with mass concentrations of 1 mg / L, 10 mg / L, and 20 mg / L were selected as samples representing low, medium, and high concentration levels, respectively. These samples were mixed with the colorimetric reaction solution, and their absorbance at 820 nm was measured. Each concentration was measured 10 times to obtain relevant data. The standard deviation and relative standard deviation were calculated. The measurement results are shown in Table 2.

[0041] Table 2 Precision Measurement Results

[0042] The results showed that the standard deviations for the corresponding concentrations were 0.02 mg / L, 0.14 mg / L, and 0.23 mg / L, with RSD values ​​ranging from 1.18% to 2.06%. Furthermore, based on the absorbance of the blank sample and the slope of the standard curve, the limit of detection (LOD) of this method was calculated to be 0.015 mg / L, which is superior to the typical LOD (0.05–0.1 mg / L) of the traditional molybdenum blue method.

[0043] The formula for calculating the limit of detection is:

[0044] Among them: SD Blank Concentration deviation of absorbance value of blank sample S: Slope of the corresponding standard curve Comparative Example Available phosphorus in the sample was extracted using the H₂SO₄-H₂O₂ digestion method. The method was as follows: Fresh sample was ground with liquid nitrogen, weighed, and then 1 mL of concentrated sulfuric acid (98%) and 500 μL of 30% H₂O₂ were added. The mixture was placed in a digestion furnace at 180℃ for 2 hours until the solution became clear. After cooling, the volume was adjusted to 10 mL, which was used as the test solution. A certain amount of the test solution was mixed with the colorimetric reaction solution and incubated. After the determination, a standard curve was used for calculation. Example 1

[0045] (1) Weigh 50 mg of rice tissue sample (new leaves of 28-day-old rice); (2) Add 500 μL of 1% acetic acid solution and ordinary zirconia grinding beads to the rice tissue sample and grind it using a tissue homogenizer. Set the grinding frequency to 60 Hz and the grinding time to 1.5 min. Grind three times to obtain a homogenized sample tissue. (3) Add 1 mL of 1% acetic acid extract to the sample tissue homogenate, mix well, and place in the dark under low temperature conditions for 20 min. Shake gently once during the process. (4) Add 50 mg of the hydrogel microspheres obtained in the preparation example to the crude extract after standing, gently shake and mix, and let stand for 10 min at 4°C in the dark to allow the hydrogel microspheres to fully swell. During this period, it is necessary to shake appropriately once to ensure that the hydrogel microspheres can absorb all the liquid except for impurities visible to the naked eye. (5) Centrifuge the hydrogel microspheres at 10000 g for 5 minutes, collect the supernatant into a new centrifuge tube, add 4 mL of 1% acetic acid solution to dilute and mix well to obtain the sample reaction solution. Example 2

[0046] The difference between this embodiment and embodiment 1 is that a 1% citric acid solution is used instead of an acetic acid solution in steps (3) and (5). Example 3

[0047] The difference between this embodiment and embodiment 1 is that a 1% choline chloride-oxalic acid solution is used in steps (3) and (5). Example 4

[0048] The difference between this embodiment and embodiment 1 is that 1% MES solution is used in both steps (3) and (5). Example 5

[0049] The difference between this embodiment and embodiment 1 is that magnetic zirconia grinding beads are used for grinding in step (2). Example 6

[0050] The difference between this embodiment and embodiment 1 is that in step (3), 1 mL of a 1% sodium acetate solution containing 0.01% cellulase is added. Example 7

[0051] The difference between this embodiment and embodiment 1 is that in step (3), 1 mL of a 1% sodium acetate solution containing 0.01% pectinase is added. Example 8

[0052] The difference between this embodiment and embodiment 1 is that in step (3), 1 mL of a 1% sodium acetate solution containing 0.01% cellulase and 0.01% pectinase is added. Example 9

[0053] The difference between this embodiment and embodiment 1 is that in step (3), 1 mL of a 1% sodium acetate solution containing 0.01% hemicellulase is added. Example 10

[0054] Using the samples prepared in the comparative examples and Examples 1-9, 90 μL of the sample reaction solution was mixed with the colorimetric reaction solution, following the same procedure as the standard curve determination. After the determination, the effective phosphorus concentration ρ in the sample reaction solution was calculated using the standard curve.

[0055] Substitute the calculated effective phosphorus concentration ρ into the following calculation formula to obtain the effective phosphorus concentration of the sample:

[0056] In the formula: ρ — The effective phosphorus concentration calculated in the sample reaction solution, in mg / L; V — Total volume of the sample after dilution, in mL. In this method, it is calculated as 5.5 mL. m — Fresh weight of the sample, in grams; ω — effective phosphorus concentration in the sample, in mg / g; The measurement results are shown in Table 3.

[0057] Table 3. Test results of each group of samples

[0058] As can be seen from Table 3, in the comparative example, the conversion of organic phosphorus to inorganic phosphorus is caused by the action of sulfuric acid. In addition, the competition between sulfate and molybdate in the colorimetric reaction system will interfere with the detection results, resulting in a higher measured value of effective phosphorus concentration.

[0059] All examples used safe and environmentally friendly weak acid solutions to promote rice tissue lysis. The weak acid system prevents the hydrolysis of available phosphorus, and low-temperature, light-protected operation prevents the conversion of available phosphorus forms. Although extraction efficiencies varied, the RSD of all groups was lower than that of the comparative example, indicating that the weak acid system did not significantly interfere with the detection method, was safer, had less environmental pollution, posed no corrosive risk, and the generated waste liquid was easy to treat. Furthermore, compared to the comparative example, the reaction system of the examples was milder, the measured data showed less variation, good data consistency, and excellent experimental reproducibility.

[0060] Example 1, serving as the foundation of this method, showcases the unique advantages of combining hydrogel microspheres with a weak acid (acetic acid). The mild nature of the acetic acid solution prevents the hydrolysis and speciation of available phosphorus, ensuring the accuracy of the detection target from the source. Furthermore, the hydrogel microspheres, through selective adsorption of water molecules, achieve passive concentration of the target analyte, compensating for potential signal deficiencies due to the weak extraction power of the weak acid. Simultaneously, their size exclusion effect effectively removes large molecular impurities such as pigments and proteins from the extract. This synergistic effect of ensuring the authenticity of the weak acid combined with the enhanced efficiency of the hydrogel is the fundamental reason why Example 1 achieves a reliable extraction yield (1.29 mg / g) while maintaining an extremely low RSD (0.47%).

[0061] Examples 2, 3, and 4 further validated the universal compatibility advantages of hydrogel microspheres with different weak acid / buffer systems. Whether using citric acid, choline chloride-oxalic acid, or MES buffer, the hydrogel microspheres effectively performed their concentration and purification functions, ensuring that these systems, with their varying extraction capabilities, could ultimately produce highly reproducible detection results (RSD 1.44%-1.90%). This demonstrates that the introduction of hydrogel microspheres significantly enhances the inclusiveness of weak acid extracts, providing a flexible and reliable technical option for addressing different sample characteristics.

[0062] Example 5 demonstrates that the combination of hydrogel microspheres and a weak acid system remains stable and reliable even with more efficient physical fragmentation (magnetic zirconia grinding beads) releasing more available phosphorus. This combination effectively treats high-concentration phosphorus extracts, avoids co-concentration of impurities, ensures the specificity of the detection signal, and achieves both high extraction yield (1.52 mg / g) and high precision (RSD 1.67%).

[0063] Examples 6, 7, and 9 demonstrate the enhanced effect of introducing enzymatic hydrolysis into a weak acid-hydrogel-based system. The use of enzymes improves phosphorus release efficiency, while the hydrogel microspheres ensure the cleanliness of the enzymatic hydrolysis system. It effectively eliminates soluble macromolecular byproducts generated during enzymatic hydrolysis, which often constitute complex interferences in traditional detection methods. Therefore, the combination of hydrogel microspheres and the weak acid-enzyme system achieves a seamless integration of bio-enhancement and physical purification, ensuring accurate and stable detection.

[0064] The composite enzyme system used in Example 8 represents the peak efficiency of this approach, and its success also relies on the synergy between the hydrogel microspheres and the weakly acidic environment. After the composite enzyme completely breaks down the cell wall, the mild weakly acidic environment protects the released available phosphorus from being destroyed or transformed, while the hydrogel microspheres immediately and efficiently capture and purify it, ultimately achieving a perfect balance between extraction efficiency and data stability.

[0065] Spike recovery of different extracts After grinding, 100 μL, 500 μL, and 1 mL of 1000 mg / L standard phosphorus solution were added to the subsamples prepared in Examples 1-9 for spiked detection. The results of the spiked detection are shown in Table 4.

[0066] Table 4 Spike Detection of Samples in Each Group

[0067] As shown in Table 4, this method is more accurate than the method using sulfuric acid for extracting available phosphorus from rice tissue, especially when the phosphorus content of the sample tissue is low. In Examples 1-9, the recoveries of spiked samples ranged from 95.00% to 105.00%, while the recoveries of comparative samples ranged from 60.00% to 132.8%. The comparative samples showed greater fluctuations, and the recoveries of the method in the examples were closer to the theoretical value (100%), indicating that this method is more accurate, especially in samples with low phosphorus content.

[0068] When the phosphorus content of the sample is relatively low, the recovery rate of the method in the example can still remain stable at 95.00%-102.00%, while the recovery rate of the comparative example in the low content group has shown a significant deviation, indicating that the method in the example is very sensitive and reliable for the detection of low phosphorus samples.

[0069] Furthermore, the method described in the examples is well adaptable to different concentrations of added standard phosphorus solutions, and changes in the amount of spiking do not significantly affect the detection results.

[0070] Examples 1-9 used samples with different phosphorus contents, but the recovery rates of all groups were relatively stable, indicating that the method is applicable to samples with different background phosphorus contents, and the sample matrix did not significantly interfere with the detection.

[0071] In summary, the methods in the examples showed stability under different spiking amounts and different sample matrices, while the comparative examples were more susceptible to interference, indicating that the methods involved in this application can effectively reduce the interference from sample matrices and operational errors. Example 11

[0072] Determination and analysis of available phosphorus concentration in different tissues of rice The difference between this embodiment and Example 1 is that the samples selected were taken under high-phosphorus and low-phosphorus conditions, and the available phosphorus concentration in various tissues of rice (new leaves, old leaves, and roots) was determined using this method. The results are as follows: Figure 2 As shown, under high phosphorus conditions, the effective phosphorus concentrations measured in roots, new leaves, and old leaves were 0.96 mg Pi / g, 1.22 mg Pi / g, and 2.37 mg Pi / g fresh weight, respectively. Under low phosphorus conditions, the effective phosphorus concentrations measured in roots, new leaves, and old leaves were 0.11 mg Pi / g, 0.25 mg Pi / g, and 0.41 mg Pi / g, respectively. The effective phosphorus concentrations in various tissues of rice under both high and low phosphorus conditions were within a reasonable range, indicating that the method involved in this application can be applied to samples with significant differences in phosphorus content in different tissues. It has a wide detection range while maintaining sensitivity, accuracy, and data reliability. Example 12

[0073] Analysis of available phosphorus concentration in different rice varieties The difference between this embodiment and Example 11 is that the available phosphorus concentration in each tissue of two rice varieties—Nipponbare (a representative japonica rice variety) and Kasalath (a representative indica rice variety)—grown for 14 days under different phosphorus concentrations (200 μM Pi for high phosphorus and 10 μM Pi for low phosphorus) were measured. The results are as follows: Figure 3As shown, in the root tissues, new leaves, and old leaves of both varieties, the phosphorus content measured in tissues cultured with high phosphorus levels was significantly higher than that measured in tissues cultured with low phosphorus levels. This indicates that the method described in this application can not only be applied to both indica and japonica rice varieties, but also, to a certain extent, reflect the phosphorus uptake capacity of different rice genotypes. Using this method, the phosphorus allocation characteristics of different rice varieties can be precisely quantified, providing technical support for subsequent genetic breeding and variety screening.

[0074] The embodiments described herein are preferred embodiments and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made to the structure, shape, and principle of this application should be included within the scope of protection of this application.

Claims

1. A method for detecting tissue effective phosphorus concentration in rice, characterized by, The method comprises the following steps: (1) homogenizing the rice tissue sample with grinding liquid and grinding beads to obtain tissue homogenate; (2) adding extraction liquid to the tissue homogenate and extracting at 4°C in the dark for 10-30 minutes to obtain crude extract; (3) adding hydrogel microspheres to the crude extract, mixing, standing to swell, then centrifuging, and collecting the supernatant; (4) diluting the supernatant with diluent to obtain sample reaction solution; (5) mixing the sample reaction solution with color developing reaction solution, incubating, detecting absorbance at 820 nm after color development, and calculating the effective phosphorus concentration in the rice tissue based on a pre-established phosphorus concentration standard curve.

2. The method for detecting tissue effective phosphorus concentration of rice according to claim 1, wherein, The hydrogel microspheres are prepared by the following method: Dissolve acrylamide and N,N'-methylenebisacrylamide in deionized water, introduce nitrogen to remove oxygen, then add ammonium persulfate and N,N,N',N'-tetramethyl ethylenediamine, stir to form hydrogel, then crush, sieve and wash the hydrogel to obtain microspheres with a particle size of 50-100 μm; the mass ratio of the acrylamide to the N,N'-methylenebisacrylamide is (18-20):

1.

3. The method for detecting tissue effective phosphorus concentration of rice according to claim 1, wherein, The mass ratio of the hydrogel microspheres to the rice tissue sample is (1-2):

1.

4. The method for detecting tissue effective phosphorus concentration of rice according to claim 2, wherein, The grinding liquid, extraction liquid and diluent are any of the following aqueous solution systems: acetic acid solution, citric acid solution, choline chloride-oxalic acid solution, MES buffer, sodium acetate solution; When acetic acid solution is selected, its volume concentration is 0.5 %-5 %(v / v); When the remaining solutions are selected, their mass concentration is 0.5 %-5 %(w / v).

5. The method for detecting tissue effective phosphorus concentration of rice according to claim 4, wherein, When the extraction liquid is sodium acetate solution, it also contains 0.005 %-0.05 %(w / v) of plant cell lysing enzyme.

6. The method for detecting tissue available phosphorus concentration of rice according to claim 5, wherein, The plant cell lysing enzyme is at least one selected from cellulase, pectinase and hemicellulase.

7. The method for detecting tissue effective phosphorus concentration of rice according to claim 1, wherein, The color development step is based on the principle of molybdenum blue method.

8. The method for detecting tissue effective phosphorus concentration of rice according to claim 1, wherein, The phosphorus concentration standard curve is linear within the range of 0-25 mg / L of phosphorus concentration, the linear regression equation is y=0.1504x+0.046, and the detection limit LOD=0.015 mg / L.

9. The method for detecting tissue effective phosphorus concentration of rice according to claim 1, wherein, The recovery rate of the detection method is 95.00%-105.00%.

10. The method for detecting the effective phosphorus concentration in rice tissues according to any one of claims 1 to 9, characterized in that, The rice tissue sample includes rice tissues of different tissue types or different phosphorus content levels, and the rice varieties are indica rice and / or japonica rice.

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