Method for evaluating salt tolerance of wheat variety in seedling stage based on multiple physiological indexes

Abstract

The invention discloses a method for evaluating the salt tolerance of a wheat variety in a seedling stage based on various physiological indexes, which comprises the following steps: planting and treating: putting soaked wheat seeds into a culture dish for culturing, when leaves grow to 2-3cm, transferring into a culture box filled with a culture solution, and when the seedlings grow to a three-leaf stage, thinning; continuously culturing by using a culture solution containing NaCl from a trefoil stage; and measuring physiological indexes. The method solves the problem that the prior art cannot quickly and accurately evaluate the salt tolerance of wheat in the seedling stage, so that the yield in the mature stage is greatly reduced, and the wheat is seriously dead. By means of the method, the salt tolerance of the wheat variety in the seedling stage can be rapidly evaluated under the laboratory or greenhouse condition, and the salt tolerance SI < gt > of the wheat variety is obtained; 0.5 is a high-salt-tolerance variety, and 0.3 1t; the wheat variety is a medium salt-tolerant variety with SI < = 0.5, and the wheat variety is a low salt-tolerant variety with SI < = 0.3, so that the screening period of the wheat salt-tolerant variety is shortened, and the breeding efficiency is improved.

Description

Technical Field

[0001] The present invention relates to a method for evaluating the salt tolerance of wheat, and particularly to a method for evaluating the salt tolerance of wheat varieties at the seedling stage based on multiple physiological indexes. Background Art

[0002] Soil salinization is one of the important abiotic stress factors affecting agricultural production. During the wheat planting process, salt stress will have a serious impact on the growth, development and yield of wheat. With the continuous increase of the global salinized land area, it is particularly urgent to cultivate and screen salt-tolerant wheat varieties.

[0003] At present, the traditional method for screening salt-tolerant wheat varieties mainly relies on field planting observation in natural saline-alkali land. This method has a long cycle, is greatly affected by environmental factors and has low efficiency. Although there are also some attempts to preliminarily judge the salt tolerance of wheat based on a single physiological index (such as the content of leaf osmotic adjustment substances), a single index is difficult to comprehensively and accurately reflect the salt tolerance ability of wheat, resulting in insufficient reliability of the screening results. Moreover, in the traditional method for screening salt-tolerant wheat varieties, the evaluation is mostly based on the yield of wheat at maturity, and there is less research and evaluation on the seedling stage of wheat. However, the salt tolerance at the seedling stage of wheat is very important for the yield at maturity. If the wheat is not salt-tolerant at the seedling stage, it will lead to a great reduction in the yield at maturity, and in severe cases, the seedlings may die. Therefore, developing a method that can quickly and accurately evaluate the salt tolerance of wheat at the seedling stage is of great significance for the breeding of salt-tolerant wheat varieties and the production of wheat in saline-alkali land. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for evaluating the salt tolerance of wheat varieties at the seedling stage based on multiple physiological indexes, which solves the problem that the prior art cannot quickly and accurately evaluate that wheat is not salt-tolerant at the seedling stage, resulting in a great reduction in the yield at maturity and even death in severe cases. The salt tolerance SI of wheat varieties is classified and evaluated according to the comprehensive score: SI>0.5 is a highly salt-tolerant variety; 0.3<SI ≤ 0.5 is a moderately salt-tolerant variety; SI ≤ 0.3 is a low-salt-tolerant variety, which greatly shortens the screening cycle of salt-tolerant wheat varieties and improves the breeding efficiency.

[0005] To achieve the above purpose, the present invention provides a method for evaluating the salt tolerance of wheat varieties at the seedling stage based on multiple physiological indexes, and this method includes: S1. Planting and Treatment (1) Put the soaked wheat seeds into a culture dish for cultivation. When the leaves grow to 2-3 cm after germination, transfer them into a culture box filled with Hoagland nutrient solution. When the seedlings grow to the three-leaf stage, thin out the seedlings; (2) Starting from the three-leaf stage, adjust the Hoagland nutrient solution to Hoagland nutrient solution containing NaCl and continue to culture; (3) Performing lightless cultivation and light-treated cultivation daily; the lightless cultivation is performed at a temperature of 16 degrees; the light-treated cultivation is performed at a temperature of 22 degrees and a light intensity of 8000 lux; S2. Physiological index measurement After the cultivation in step S1 is completed, nine physiological indices are measured, namely: catalase activity, hydrogen peroxide content, malondialdehyde content, peroxidase activity, superoxide dismutase activity, soluble sugar content, soluble protein content, total chlorophyll content, and proline content; The data of the nine physiological indicators were collected and sorted, and variance analysis, correlation analysis and PCA analysis were performed to screen out significantly correlated indicators; the standardized indicator value and salt tolerance comprehensive score of each wheat variety were calculated according to the standardized formula and the salt tolerance comprehensive score formula; The comprehensive salt tolerance score > 0.5 is a high salt tolerance variety; 0.3 < the comprehensive salt tolerance score ≤ 0.5 is a medium salt tolerance variety; the comprehensive salt tolerance score ≤ 0.3 is a low salt tolerance variety.

[0006] Preferably, in step S1 (1), the germination time is 2 to 3 days, and the components of the Hoagland culture medium are: KNO3, NH4NO3, KH2PO4, MgSO4, FeNaEDTA, KI, H3BO3, MnSO4, ZnSO4, Na2MoO4, CuSO4, CoCl2 and Ca(NO3)2; The concentrations of KNO3, NH4NO3, KH2PO4, MgSO4, FeNaEDTA, KI, H3BO3, MnSO4, ZnSO4, Na2MoO4, CuSO4, CoCl2 and Ca(NO3)2 are 506 mg / L, 80 mg / L, 136 mg / L, 241 mg / L, 36.7 mg / L, 0.83 mg / L, 6.2 mg / L, 22.3 mg / L, 8.6 mg / L, 0.25 mg / L, 0.025 mg / L, 0.025 mg / L and 945 mg / L, respectively.

[0007] Preferably, in step S1 (2), the concentration of NaCl is 150 mmol / L.

[0008] Preferably, in step S1 (3), the humidity of the non-light culture and the light culture is 71.2%; the time of the non-light culture is 8 hours; and the time of the light culture is 16 hours.

[0009] Preferably, in step S2, the step of the catalase activity is: a. Reagent preparation: Mix Na2HPO4 and NaH2PO4 and dilute to volume with distilled water; b. Reaction solution preparation: Take PBS, add 30% H2O2 and shake well; c. Sample determination: Take the reaction solution and add enzyme solution to measure OD 240 ; d. Calculation of enzyme activity: A decrease of 0.01 OD value per minute is defined as 1 unit of enzyme activity (U); CAT=[ΔA240.Vt] / (W.Vs.0.01)(u / g min) ΔA240=(A240f-A240i) / (tf-ti) Where A240f is the end value, A240i is the initial value, tf is the end time, and ti is the initial reaction time; W is the fresh weight of the sample, in g; t is the reaction time, in min; Vt is the total volume of the extracted enzyme solution, in 9 mL; Vs is the volume of the enzyme solution used during the assay, in 0.2 mL; The determination method of the hydrogen peroxide content is: a. The leaf samples were ground evenly in liquid nitrogen to obtain frozen samples, and HClO4 was added to the frozen samples three times; b. Centrifuge at 6000 g at 4°C. Add KOH dropwise to the supernatant until the pH reaches 6-7. c. Add activated carbon to adsorb the pigment, vortex, and centrifuge at 12,000 g at 4 degrees Celsius; remove the supernatant and pass it through a membrane for H2O2 determination; d. Horseradish peroxidase, H2O2 sample and reaction buffer were added, and the absorption peak was measured at 412nm; The determination method of the malondialdehyde content is: a. Add trichloroacetic acid to the sample, grind the resulting homogenate and centrifuge at 3000 r / min; b. Add total bile acids to the supernatant, mix, boil at 100°C, cool, and centrifuge. c. Measure the absorbance of the supernatant at 450 nm, 532 nm, and 600 nm, and calculate the MDA concentration according to the formula. Then, calculate the MDA content in the fresh tissue. The peroxidase activity was determined as follows: a. Take the plant tissue into a mortar, add phosphate buffer and grind into a homogenate, centrifuge at 3000 rps, transfer the supernatant into a volumetric flask, rinse the precipitate with phosphate buffer, and the supernatant was added to a volumetric flask and brought to volume with phosphate buffer; b. Adjust the setting to zero at 470 nm, then add enzyme solution to the assay tube, start the timer, mix thoroughly, perform colorimetric analysis, and read the absorbance.

[0010] Preferably, in step S2, the soluble sugar content is determined by: a. Draw a standard curve; b. Weigh the chopped mixed fresh sample, add quartz sand and grind to a homogenous slurry, adjust the volume together with the residue, filter at room temperature, discard the residue, and obtain a sample extract; c. Add the sample extract to the anthrone reagent, shake well, bring to a boil, cool, and compare color at 620 nm on a spectrophotometer. Use a blank as the zero point, record the absorbance, and find the corresponding sugar in micrograms on the standard curve. The method for determining the soluble protein content is: a. Take G-250 as dimethylcyanine brilliant blue dissolved in 90% ethanol, add 85% phosphoric acid, dilute to volume, and filter to obtain a G-250 solution; b. Take the enzyme solution and G-250 solution and place them at 595 nm for colorimetry; c. Calculate the result based on: Soluble protein (mg / Gfw) = (C x V / Va) / W; The determination method of the total chlorophyll content is: a. Take 0.2 g of leaves, chop them, add 20 mL of a mixture of anhydrous ethanol and acetone (volume ratio: 2:1), and soak in a dark place at room temperature. Shake the mixture until it turns completely white. b. Adjust the transmittance to 100%, and then measure the absorbance of the material at 663 and 645 nm respectively; c. Calculate according to the formula Ca=12.7A 663 -2.69A 645 , Cb=22.9A 645 -4.68A 663 ,Ca+b=8.02A 663 +20.21A 645 ; The proline content is determined as follows: a weighed fresh leaves, added 3% sulfosalicylic acid solution, ground into a homogenate, extracted in a boiling water bath, cooled and filtered; b. Take the filtrate, add glacial acetic acid and acidic ninhydrin solution, heat at 100 ° C, and cool rapidly after the reaction is completed; c. Add toluene and extract by oscillation. After standing for stratification, measure the absorbance of the upper toluene phase at 520 nm. Calculate the proline content in the sample based on the proline standard curve.

[0011] Preferably, in step S2, the analysis of variance and correlation analysis are performed using GraphPad Prism statistical analysis software.

[0012] Preferably, in step S2, the normalization formula is: X ′ = ( X - X min) / ( X max - X min), where X is the original data, Xmin is the minimum value of all data of this index, Xmax is the maximum value.

[0013] Preferably, in step S2, the comprehensive score formula for salt tolerance is: SI = ∑ i = 1 nWiXi ′, where Wi is the weight of each index, Xi ′ is the standardized index value.

[0014] Preferably, the significantly correlated indicators are that the hydrogen peroxide content, peroxidase activity, soluble protein content, relative leaf water content, proline content are positively correlated with wheat salt tolerance; the catalase activity, malondialdehyde content, superoxide dismutase activity and soluble sugar content are negatively correlated with wheat salt tolerance.

[0015] A method for evaluating the salt tolerance of wheat varieties at the seedling stage based on multiple physiological indicators according to the present invention solves the problems in the prior art that it cannot quickly and accurately evaluate the salt intolerance of wheat seedlings at the seedling stage, resulting in a great reduction in the yield at the mature stage and serious death, and has the following advantages: 1. By comprehensively analyzing multiple physiological indicators, the present invention constructs a comprehensive and accurate evaluation system for wheat salt tolerance at the seedling stage. Compared with the traditional single-index evaluation method, it can more reliably and comprehensively reflect the salt tolerance ability of wheat.

[0016] 2. Using this method, the salt tolerance of wheat varieties at the seedling stage can be quickly evaluated under laboratory or greenhouse conditions (grading and evaluating the salt tolerance SI of wheat varieties according to the comprehensive score: SI > 0.5 is a highly salt-tolerant variety; 0.3 < SI ≤ 0.5 is a moderately salt-tolerant variety; SI ≤ 0.3 is a low-salt-tolerant variety), greatly shortening the screening period of wheat salt-tolerant varieties and improving the breeding efficiency.

[0017] 3. It provides a scientific basis for wheat variety selection in saline-alkali land, accelerates the breeding process of salt-tolerant varieties, helps to reasonably plan the wheat planting layout in saline-alkali land, and improves the yield and quality of wheat in saline-alkali land. Brief Description of the Drawings

[0018] Figure 1 This is a comparison chart of proline content in different wheat varieties under control and salt treatment.

[0019] Figure 2 PCA analysis of different wheat varieties under control and salt treatment.

[0020] Figure 3 This is a correlation analysis chart of the hydrogen peroxide (H2O2) content in different wheat varieties under control and salt treatment. DETAILED DESCRIPTION

[0021] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0022] The experimental materials prepared in the following examples are as follows: 1. Prepare seeds of 30 wheat varieties (these varieties come from different geographical regions and breeding pedigrees and have a certain degree of genetic diversity. The seeds should be plump and free of pests and diseases). Sterilize the seeds with 75% alcohol for 5 minutes, then rinse with deionized water, and soak the seeds in a constant temperature incubator at 25°C for 24 hours to ensure similar seed vigor and germination rate.

[0023] 2. Prepare the hydroponic culture apparatus, clean and disinfect it, and then fill it with Hoagland nutrient solution. The control group used a standard nutrient solution, while the salt-treated group used Hoagland nutrient solution supplemented with 150 mmol / L NaCl. The composition and content of Hoagland nutrient solution are as follows: KNO₃ (506 mg / L), NH₄NO₃ (80 mg / L), KH₂PO₄ (136 mg / L), MgSO₄ (241 mg / L), FeNaEDTA (36.7 mg / L), KI (0.83 mg / L), H₃BO₃ (6.2 mg / L), MnSO₄ (22.3 mg / L), ZnSO₄ (8.6 mg / L), Na₂MoO₄ (0.25 mg / L), CuSO₄ (0.025 mg / L), CoCl₂ (0.025 mg / L), and Ca(NO₃)₂ (945 mg / L); purchased from Coolaibo, item number NS1010.

[0024] 3. Prepare the required solution The activity of antioxidant enzymes (SOD, POD and CAT) was determined using existing technology (Li Hesheng, ed.: Principles and Techniques of Plant Physiological and Biochemical Experiments. Higher Education Press, 2000: 267-268).

[0025] The solutions required for the configuration in this embodiment are specifically as follows: (1) Preparation of enzyme solution: Weigh 0.5 g of wheat leaves and put them into a mortar. Add 5 ml of phosphate buffer with a pH of 7.8, grind them in an ice bath, pour the homogenate into a centrifuge tube, centrifuge for 20 minutes at 10,000 × g, pour the supernatant (enzyme solution) into a test tube, and store it at 0-40°C until use.

[0026] (2) Preparation of phosphate buffer: Solution A: 0.2M KH2PO4 solution, analytically pure KH2PO4 27.216 g, diluted to 1000 ml with distilled water; Solution B: 0.2M K2HPO4 solution, analytically pure K2HPO4•3H2O4 5.644 g, diluted to 1000 ml with distilled water.

[0027] or, Solution A: 0.2M NaH2PO4 solution, analytically pure NaH2PO4•2H2O31.21 g, diluted to 1000 ml with distilled water; Solution B: 0.2M Na2HPO4 solution, analytically pure Na2HPO4•12H2O71.64 g, diluted to 1000 ml with distilled water.

[0028] (3) Determination of SOD: Preparation of SOD (superoxide dismutase) reaction solution: Preparation of mother liquor: 0.5M phosphate buffer (pH=7.8): 21.25ml of solution A + 228.25ml of solution B to make up to 1000ml; 130 mM Met (methionine): Take 1.9399 g of Met and dilute to 100 ml with phosphate buffer; 750 μM NBT: Take 0.06133 g NBT and dilute to 100 ml with phosphate buffer (protect from light); 100 μM EDTA-Na2: Take 0.0372 g EDTA-Na2 and dilute to 1000 ml with phosphate buffer; 20μM FD (riboflavin): dilute 0.00753g FD to 1000ml with phosphate buffer (prepare immediately before use).

[0029] SOD reaction solution: The solution was prepared according to a volume ratio of phosphate buffer: Met: NBT: EDTA-Na2: riboflavin (FD): H2O of 15:3:3:3:3:2.5.

[0030] Determination of SOD: Take a test tube of the same model, draw out 20 microliters of enzyme solution, add 3 milliliters of SOD reaction solution, and illuminate at 4000 Lux (usually use an illumination incubator with a ring fluorescent lamp) for 30 minutes (try to make the illumination conditions consistent). At the same time, take four test tubes, three as controls and one as blank (no enzyme solution, buffer solution instead); place the blank in a dark place, and place the control (CK) and enzyme solution in the same place under 4000 Lux conditions for 30 minutes, store in a dark place, adjust to zero with the blank, and perform colorimetry at 560 nm.

[0031] Result calculation: SOD total activity (absorbance / g·FW) = (A CK —A E )×V / (W×0.5×A CK Unit: 50% NBT photoreduction; SOD specific activity (enzyme unit / mg protein) = SOD total activity / soluble protein concentration (4) Determination of POD (peroxidase): Preparation of 0.1M pH 6.0 phosphate buffer: 219.25 solution A + 30.75 solution B, dilute to 500 ml; Preparation of POD reaction solution: 50 ml of 0.1 M pH 6.0 phosphate buffer was placed in a beaker, 28 μl of guaiacol was added, and the mixture was heated and stirred with a magnetic stirrer to completely dissolve it. After cooling, 19 μl of 30% H2O2 was added and mixed, and the mixture was stored in a refrigerator.

[0032] POD determination: 20 μl enzyme solution + 3 ml reaction solution were placed in a cuvette and the reading was taken at 470 nm every 1 minute for three times. The absorbance change per minute (ΔA470 / min·mg pr or ΔA470 / mgFW) was used to represent the enzyme activity.

[0033] Result calculation: POD activity (ΔA470 / min·gFW) = ΔA470×V / Va / W = ΔA470×5 / 0.02 / 0.5 =ΔA470×500 (5) CAT (catalase) assay: Preparation of CAT reaction solution: 0.1MH2O2 solution: Add 0.568 ml of 30% H2O2 to 100 ml.

[0034] 0.1MPH7.0 phosphate buffer: 97.5 ml of solution A + 152.5 ml of solution B, adjust the volume to 500 ml.

[0035] Mix 25 ml of 0.1 M H2O + 20 ml of 0.1 M pH 7.0 phosphate buffer (i.e., at a ratio of 1:4) to obtain the CAT reaction solution.

[0036] CAT determination: 0.1 ml (or 50 μl) enzyme solution + 2.5 ml reaction solution, colorimetric at 240 nm, read once every 1 minute, for a total of 3 times.

[0037] Result calculation: CAT activity (Δ240 / min·gFW) = Δ240×V / Va / W = Δ240× / 0.05 / 0.5 = Δ240×200.

[0038] (6) Determination of MDA (malondialdehyde): Preparation of MDA reaction solution: 0.6 g TBA (thiobarbituric acid) was first dissolved in a small amount of 1 M NaOH and then diluted to 100 ml with 10% TCA (trichloroacetic acid).

[0039] Determination of MDA: 1 ml enzyme solution + 2 ml 0.6% TBA, seal and boil in water bath for 15 minutes, cool quickly and then centrifuge, take the supernatant, and compare colorimetry at three wavelengths of 600, 532, and 450 nm.

[0040] Calculation of results: MDA (μmol / gfw) = (6.45 × (D 532 -D 600 )-0.56D 450 ) × 0.015 / W or (6.45 × (D 532 -D 600 )-0.56D 450 ) × 0.03 / W (7) Determination of soluble protein Preparation of reaction solution: Dissolve 0.1 g of G-250 (G-250 is dimethocyanine brilliant blue) in 50 ml of 90% ethanol, add 100 ml of 85% phosphoric acid, dilute to 1000 ml, and filter.

[0041] Determination: 20 μl enzyme solution + 3 ml G-250, incubate for 2 minutes, colorimetry at 595 nm, and a blank (20 μl buffer + 3 ml G-250) is also prepared.

[0042] Result calculation: Soluble protein (mg / Gfw) = (C×V / Va) / W (8) Determination of proline content Reagent preparation: 0.6 g sulfosalicylic acid, dilute to 200 ml: Acidic ninhydrin (freshly prepared): 3.75 g ninhydrin + 90 ml glacial acetic acid + 36 ml distilled water Determination: Add 5 ml of sulfosalicylic acid to 0.3 g of leaves, cover, boil in water for 10 minutes, filter, aspirate 2 ml of the filtrate (simultaneously serve as a blank, aspirate 2 ml of distilled water), 2 ml of glacial acetic acid and 3 ml of acidic ninhydrin, boil in water for 40 minutes, cool, add 5 ml of toluene, shake thoroughly, let it stand to separate, take the upper toluene solution and place it in a cuvette, and compare the color at 520 nm.

[0043] Result calculation: Proline content (μg / g fresh weight) = C×V / Va / W.

[0044] (9) APX (ASA-POD) - Ascorbate peroxidase measurement: Take 0.5g of the material, put it in a mortar, add 5ml of the extract (same as above) pre-cooled at 4℃ and a small amount of quartz sand, grind it at 12000g·min -1 Centrifuge for 20 minutes. The supernatant is the crude APX extract and store at 4°C until needed. (For long-term storage, the extract should contain 0.186g EDTA, 0.4718g ASA, and be diluted to 500ml with pH 7.0 buffer. This is to prevent APX inactivation. However, for fresh materials, the difference between the two extraction methods will not be significant.)

[0045] APX reaction solution: 11.2 L 30% H2O2 + 0.04718 g AsA (ascorbic acid - Vc), phosphate buffer (pH 7.0) to 500 ml. (This reaction solution must be prepared immediately before use)

[0046] 0.1ml enzyme extract + 2.9ml APX reaction solution, 290nm colorimetric (0-40s).

[0047] Example 1 A method for evaluating salt tolerance of wheat varieties at the seedling stage based on multiple physiological indicators, the method comprising: 1. Planting and processing (1) Place the soaked wheat seeds in a culture dish, 50 seeds per dish. After germination for 2 to 3 days, when the leaves grow to 2 to 3 cm, transfer them to a culture box containing Hoagland culture medium. When the seedlings grow to the three-leaf stage, thin them out to 30 seedlings of uniform growth per pot.

[0048] (2) Starting from the three-leaf stage, the salt-treated group was cultured in Hoagland medium containing 150 mmol / L NaCl for one week; the control group was cultured in Hoagland medium for another week. Three replicates were set for both the control group and the salt-treated group, with 30 wheat plants in each replicate.

[0049] (3) Place both the control and salt-treated groups in a light incubator and culture them at the set temperature, light intensity, and photoperiod. Change the nutrient solution regularly during the culture period (16 hours of light and 8 hours of no light per day; specific parameters are: temperature 16.7 degrees, humidity 71.2%, photoperiod 8 hours, light intensity 0 lux (no light); temperature 22 degrees, humidity 71.2%, photoperiod 16 hours, light intensity 8000 lux, culture solution is changed once a week), and keep the nutrient solution well ventilated. Both the salt-treated and control groups were cultured for 7 days.

[0050] 2. Physiological index measurement After the above steps are completed, the following 9 physiological indicators are measured: (1) Catalase (CAT) activity: It was determined by ultraviolet absorption method, with the decomposition of 1 μmol of hydrogen peroxide per minute as one unit of enzyme activity.

[0051] (2) Hydrogen peroxide (H2O2) content: The content is determined by horseradish peroxidase (HRP) coupled fluorescence method, and the fluorescence value is used to determine the content. The specific method is as follows: a. Leaf samples (0.3 g, control and salt-treated groups) were ground in liquid nitrogen to obtain frozen samples. 1.0 M HClO4 (3 mL) was added to the frozen samples in three portions (1 mL each, while grinding).

[0052] b. Transfer to a 10 mL centrifuge tube and centrifuge at 6000 g for 5 min at 4°C. Transfer 2.5 mL of the supernatant (strictly quantitative) to a 10 mL centrifuge tube and add 4 M KOH dropwise to the supernatant until the pH reaches 6-7.

[0053] c. Add 0.05 g of activated carbon to absorb the pigment (excessive addition may result in the adsorption of H₂O₂). Vortex for 20 seconds, then centrifuge at 12,000 g for 5 minutes at 4°C. Pass 2 mL of the supernatant through a membrane (optional). Measure H₂O₂.

[0054] d. Add horseradish peroxidase, H2O2 sample, and reaction buffer (994 mL) and measure the absorbance peak at 412 nm.

[0055] (3) Malondialdehyde (MDA) content: The content was determined by the thiobarbituric acid method and calculated by colorimetry.

[0056] (4) Peroxidase (POD) activity: The activity was determined by the guaiacol method, with a change of 0.01 in absorbance per minute as one unit of enzyme activity.

[0057] (5) Superoxide dismutase (SOD) activity: The activity was determined by the nitroblue tetrazolium photoreduction method, with 50% inhibition of nitroblue tetrazolium photoreduction being considered one unit of enzyme activity.

[0058] (6) Soluble sugar content: The anthrone colorimetric method is used to determine the content. The content is obtained based on the absorbance value on the standard curve. The specific method is as follows: a. Draw a standard curve.

[0059] b. Extraction of soluble sugars from samples: Weigh 0.5 g to 1.0 g of the chopped and mixed fresh sample, add a small amount of quartz sand and grind into a homogenous slurry. Add the remaining residue and dilute to 100 mL. Filter at room temperature and discard the residue.

[0060] c. Determination: Add 5 mL of anthrone reagent to 1 mL of sample extract (distilled water control). Shake well and boil in a boiling water bath for 10 minutes. Remove and cool. Compare the color at 620 nm on a spectrophotometer. Adjust the zero point to the blank. Record the absorbance. Find the corresponding sugar in micrograms on the standard curve.

[0061] (7) Soluble protein content: Determined by the Coomassie Brilliant Blue method (Bradford method), the content was obtained based on the absorbance value on the standard curve.

[0062] (8) Total chlorophyll content: The total chlorophyll content is the sum of the chlorophyll a content and the chlorophyll b content. The ethanol-acetone mixed solution extraction method is used, the absorbance value is measured by spectrophotometer, and the chlorophyll a content and chlorophyll b content are calculated according to the formula. The specific method is as follows:

[0063] a. Sampling: Use a brush to remove dust from the leaf surface. Take 0.2 g of the leaf, cut it into small pieces, and place it in a stoppered test tube. Add 20 mL of a mixture of anhydrous ethanol and acetone in a volume ratio of 2:1 (or directly use 80% acetone). Soak the sample in a dark place at room temperature (25°C). Shake the sample several times until the sample turns completely white.

[0064] b. Read the absorbance: Use the mixed solution as a blank control, adjust the transmittance to 100%, and then measure the absorbance of the material at 663 and 645 nm.

[0065] c. Calculate according to the formula Ca=12.7A 663 -2.69A 645 , Cb=22.9A 645 -4.68A 663 ,Ca+b=8.02A 663 +20.21A 645 .

[0066] (9) Proline content: The proline content in the sample was calculated by the acid ninhydrin method and a standard curve was drawn.

[0067] 3. Data analysis and evaluation system construction Data on nine physiological indicators of all wheat varieties under control and salt treatment were collected and entered into Excel tables. Variance analysis and correlation analysis were performed using GraphPad Prism statistical analysis software to screen out significantly correlated indicators. The standardized indicator values ​​and salt tolerance comprehensive score formulas were used to calculate the standardized indicator values ​​and salt tolerance comprehensive score for each wheat variety. The results are as follows: (1) Statistical analysis was performed on the physiological index data of each wheat variety in the control group and the salt treatment group. Analysis of variance (ANOVA) was used to determine the significant differences in each index between the salt treatment and the control group (P < 0.05 was considered significant).

[0068] (2) Through correlation analysis, physiological indicators significantly correlated with wheat salt tolerance were screened out. The analysis found that hydrogen peroxide (H2O2) content, peroxidase (POD) activity, soluble protein content, leaf relative water content, and proline content were significantly positively correlated with wheat salt tolerance; while catalase (CAT) activity, malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and soluble sugar content were significantly negatively correlated with wheat salt tolerance.

[0069] (3) Standardize the selected significant correlation indicators to eliminate the influence of different indicator dimensions. The standardization formula is: X ′=( X -min) / (max-min), where X is the original data, min is the minimum value of all data of this indicator. max is the maximum value.

[0070] (4) Determine the weight of each indicator based on its correlation with salt tolerance. Principal component analysis determined that the weights of proline content and peroxidase (POD) activity were both 0.122; the weight of SOD activity was 0.117; the weights of catalase (CAT) activity and malondialdehyde (MDA) content were both 0.113; the weights of soluble protein content and chlorophyll content were 0.105; the weight of hydrogen peroxide (H2O2) content was 0.102; and the weight of soluble sugar content was 0.100.

[0071] (5) Calculate the comprehensive salt tolerance score (SI) of each wheat variety using the following formula: ,in W i is the weight of each indicator,X ′ i is the standardized index value, i is the number of wheat varieties, taking integers from 1 to 30, and n is the total number of wheat varieties, that is, 30 kinds. The salt tolerance of wheat varieties is classified and evaluated according to the comprehensive score: SI > 0.5 is a highly salt-tolerant variety; 0.3 < SI ≤ 0.5 is a moderately salt-tolerant variety; SI ≤ 0.3 is a low salt-tolerant variety.

[0072] As shown in Figure 1, the comparison chart of proline content of different wheat varieties under control and salt treatment. Figure 1 It can be seen that under salt stress treatment, the proline content of each variety has increased by more than ten times, which is the index with the largest change difference among the 9 indexes.

[0073] Such as Figure 2 shown, the PCA analysis chart of different wheat varieties under control and salt treatment, where salt in the figure is salt and control is control. Figure 2 It can be seen that the PCA principal component analysis of 9 seedling stage indexes of 30 wheat varieties can significantly separate the control group and the salt treatment group.

[0074] Such as Figure 3 shown, the correlation analysis chart of hydrogen peroxide (H2O2) content of different wheat varieties under control and salt treatment. Figure 3 It can be seen that there is a significant positive correlation between the hydrogen peroxide content of 30 wheat varieties in the control group and the salt treatment group.

[0075] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be considered as a limitation of the present invention. After those skilled in the art have read the above content, various modifications and substitutions of the present invention will be obvious. Therefore, the protection scope of the present invention should be defined by the appended claims.

Claims

1. A method for evaluating salt tolerance of wheat varieties at the seedling stage based on multiple physiological indicators, characterized in that: The method includes: S1. Planting and processing (1) Place the soaked wheat seeds in a culture dish and culture them. When the leaves grow to 2-3 cm, transfer them to a culture box filled with Hoagland culture medium. When the seedlings grow to the three-leaf stage, thin them out. (2) Starting from the three-leaf stage, the Hoagland culture medium was adjusted to Hoagland culture medium containing NaCl and continued to be cultured; (3) Perform cultivation without light and with light every day; The light-free culture is carried out at a temperature of 16 degrees; The light culture is carried out at a temperature of 22 degrees and a light intensity of 8000 lux; S2. Physiological index measurement After the cultivation in step S1 is completed, nine physiological indices are measured, namely: catalase activity, hydrogen peroxide content, malondialdehyde content, peroxidase activity, superoxide dismutase activity, soluble sugar content, soluble protein content, total chlorophyll content, and proline content; The data of the nine physiological indicators were collected and sorted, and variance analysis, correlation analysis and PCA analysis were performed to screen out significantly correlated indicators; the standardized indicator value and salt tolerance comprehensive score of each wheat variety were calculated according to the standardized formula and the salt tolerance comprehensive score formula; The salt tolerance comprehensive score> 0.5 is a high salt tolerance variety; 0.3 < The comprehensive salt tolerance score ≤ 0.5 is a medium salt tolerance variety; the comprehensive salt tolerance score ≤ 0.3 is a low salt tolerance variety.

2. The method according to claim 1, characterized in that In step S1 (1), the germination time is 2 to 3 days, and the components of the Hoagland culture medium are: KNO3, NH4NO3, KH2PO4, MgSO4, FeNaEDTA, KI, H3BO3, MnSO4, ZnSO4, Na2MoO4, CuSO4, CoCl2 and Ca(NO3)2.

3. The method according to claim 1, characterized in that In step S1 (2), the concentration of NaCl is 150 mmol / L.

4. The method according to claim 1, wherein In step S1 (3), the humidity of the non-light culture and the light culture is 71.2%; the time of the non-light culture is 8 hours; and the time of the light culture is 16 hours.

5. The method according to claim 1, characterized in that In step S2, the steps of the catalase activity are: a. Reagent preparation: Mix Na2HPO4 and NaH2PO4 and dilute to volume with distilled water; b. Reaction solution preparation: Take PBS, add 30% H2O2 and shake well; c. Sample determination: Take the reaction solution and add enzyme solution to measure OD 240 ; d. Calculation of enzyme activity: A decrease of 0.01 OD value per minute is defined as 1 unit of enzyme activity (U); CAT=[ΔA240.Vt] / (W.Vs.0.01)(u / g min) ΔA240=(A240f-A240i) / (tf-ti) Where A240f is the end value, A240i is the initial value, tf is the end time, and ti is the initial reaction time; W is the fresh weight of the sample, in g; t is the reaction time, in min; Vt is the total volume of the extracted enzyme solution, in 9 mL; Vs is the volume of the enzyme solution used during the assay, in 0.2 mL; The determination method of the hydrogen peroxide content is: a. The leaf samples were ground evenly in liquid nitrogen to obtain frozen samples, and HClO4 was added to the frozen samples three times; b. Centrifuge at 6000 g at 4°C. Add KOH dropwise to the supernatant until the pH reaches 6-7. c. Add activated carbon to adsorb the pigment, vortex, and centrifuge at 12000g at 4 degrees Celsius; take the supernatant and pass it through a membrane, and measure H2O2; d. Horseradish peroxidase, H2O2 sample and reaction buffer were added, and the absorption peak was measured at 412nm; The determination method of the malondialdehyde content is: a. Add trichloroacetic acid to the sample, grind the resulting homogenate and centrifuge at 3000 r / min; b. Add total bile acids to the supernatant, mix, boil at 100°C, cool, and centrifuge. c. Measure the absorbance of the supernatant at 450 nm, 532 nm, and 600 nm, and calculate the MDA concentration according to the formula. Then, calculate the MDA content in the fresh tissue. The peroxidase activity was determined as follows: a. Take the plant tissue into a mortar, add phosphate buffer and grind into a homogenate, centrifuge at 3000 rps, transfer the supernatant into a volumetric flask, rinse the precipitate with phosphate buffer, and the supernatant was added to a volumetric flask and brought to volume with phosphate buffer; b. Adjust the setting to zero at 470 nm, then add enzyme solution to the assay tube, start the timer, mix thoroughly, perform colorimetric analysis, and read the absorbance.

6. The method according to claim 1, wherein In step S2, the soluble sugar content is determined by: a. Draw a standard curve; b. Weigh the chopped mixed fresh sample, add quartz sand and grind to a homogenous slurry, adjust the volume together with the residue, filter at room temperature, discard the residue, and obtain a sample extract; c. Add the sample extract to the anthrone reagent, shake well, bring to a boil, cool, and compare color at 620 nm on a spectrophotometer. Use a blank as the zero point, record the absorbance, and find the corresponding sugar in micrograms on the standard curve. The method for determining the soluble protein content is: a. Take G-250 as dimethylcyanine brilliant blue dissolved in 90% ethanol, add 85% phosphoric acid, dilute to volume, and filter to obtain a G-250 solution; b. Take the enzyme solution and G-250 solution and place them at 595 nm for colorimetry; c. Calculate the result based on: Soluble protein (mg / Gfw) = (C x V / Va) / W; The determination method of the total chlorophyll content is: a. Take 0.2 g of leaves, chop them, add 20 mL of a mixture of anhydrous ethanol and acetone (volume ratio: 2:1), and soak in a dark place at room temperature. Shake the mixture until it turns completely white. b. Adjust the transmittance to 100%, and then measure the absorbance of the material at 663 and 645 nm respectively; c. Calculate according to the formula Ca=12.7A 663 -2.69A 645 , Cb=22.9A 645 -4.68A 663 ,Ca+b=8.02A 663 +20.21A 645 ; The proline content is determined as follows: a weighed fresh leaves, added 3% sulfosalicylic acid solution, ground into a homogenate, extracted in a boiling water bath, cooled and filtered; b. Take the filtrate, add glacial acetic acid and acidic ninhydrin solution, heat at 100 ° C, and cool rapidly after the reaction is completed; c. Add toluene and extract by oscillation. After standing for stratification, measure the absorbance of the upper toluene phase at 520 nm. Calculate the proline content in the sample based on the proline standard curve.

7. The method according to claim 1, characterized in that In step S2, the variance analysis and correlation analysis were performed using GraphPad Prism statistical analysis software.

8. The method according to claim 1, characterized in that In step S2, the normalization formula is: X ′=( X - X min) / ( X max- X min), where X is the original data, Xmin is the minimum value of all data of this indicator. Xmax is the maximum value.

9. The method according to claim 1, characterized in that In step S2, the salt tolerance comprehensive score formula is: SI =∑ i =1 nWiXi ' ,in Wi is the weight of each indicator, Xi ′ is the normalized index value.

10. The method according to claim 1, characterized in that The significant correlation indicators are hydrogen peroxide content, peroxidase activity, soluble protein content, leaf relative water content, and proline content, which are positively correlated with wheat salt tolerance; catalase activity, malondialdehyde content, superoxide dismutase activity, and soluble sugar content, which are negatively correlated with wheat salt tolerance.

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