The foxtail millet variety Changnong 47 was selected for this study. This variety, which has been successfully commercialized, exhibits excellent quality and palatability, and is well-suited for planting in the middle and late maturing areas of south-central Shanxi Province, Shaanxi, Liaoning, and Gansu.The experiment was initiated on May 18, 2023, with a single row measuring 5 meters in length, a row spacing of 0.4 meters, and a density of 30,000 plants per square meter. According to the policy of promoting fertilizer and pesticide reduction and efficiency improvement, Two nitrogen gradients, no nitrogen N0 (0 kg/hm2) and normal nitrogen N1 (75 kg/hm2), were used in the main area of the split plot experiment design. During the cereal pulling period, nitrogen was applied as a furrow fertilizer in the form of urea (45% N), while molybdenum was provided as ammonium molybdate (NH4)6Mo7O24 56% Mo) in the secondary zone, with five gradients of Mo0 (0%). Foliar sprays of the following treatments were applied at the nodulation stage: Mo0 (0%), Mo1 (0.1%), Mo2 (0.2%), Mo3 (0.3%), and Mo4 (0.4%). Three replications were set up for each treatment. The harvest was carried out on October 6, 2023, using a direct cutting method for the ears, which were then dried and threshed.
The test site was situated within the experimental land of the Cereal Research Institute of Shanxi Agricultural University, Luzhou District, Changzhi City, Shanxi Province. Its geographical coordinates are longitude 113°8′27″E, latitude 36°12′57″N (
Overview of the geographic location of the test site.
Overview of the soil elements in the experiment site.
| elemental | content | elemental | content |
|---|---|---|---|
| Total potassium (g/kg) | 19.73 | Slow-acting potassium (mg/kg) | 641 |
| Iron (mg/kg) | 43705.3 | Copper (mg/kg) | 121 |
| Total Nitrogen (g/kg) | 1.36 | Total Phosphorus (%) | 0.107 |
| Quick Potassium (mg/kg) | 175 | Organic Carbon (g/kg) | 17.5 |
| Manganese (mg/kg) | 824 | Zinc (mg/kg) | 187 |
Five cereal plants exhibiting uniform growth were selected from the middle four rows of each treatment at the grain-pulling stage seven days after fertilizer spraying, at the filling stage, and at the completion stage. The following variables were measured: plant height, spike weight, spike length, and spike thickness. The latter three variables were measured at the filling stage and at the completion stage after spiking (
Once the ripening process was complete, samples were taken from five plants of uniform growth for each treatment. The stems, sheaths, leaves, and spikes were separated, weighed individually, and then subjected to a 105°C heat treatment for 30 minutes and subsequent drying at 80°C for 72 hours. This process was employed to determine the dry matter content of each part (
SPAD measurements were conducted using the SPAD-502 Plus (Konica Minolta Sensing, Inc., Japan) during the grain filling period. Photosynthesis indexes were determined by the CIRAS-2 Photosynthesis Rate Meter (PPSYSTEMS) during the filling period to ascertain the net photosynthesis rate (Pn), transpiration rate (E), and intercellular CO2 concentration (Ci) of the flag leaf of the grain. The net photosynthesis rate (Pn), transpiration rate (E), and intercellular CO2 concentration (Ci) were measured during the grain filling period.
Flag leaves of millet with uniform growth were sampled in the field during grout filling period, and placed in liquid nitrogen and brought back to the freezer at -80°C.
The activity of superoxide dismutase (SOD) was determined by nitro blue tetraolium (NBT) photochemical reduction method (
Reagent:
0.05mol/L phosphate buffer (PH7.8): 21.25mL of liquid A + 228.25mL of liquid B, with a constant volume of 1L;
130mmol/L methionine (Met) solution: 1.9399g Met was filled with phosphoric acid buffer to 100ml;
750μmol/L nitrogen blue tetrazole solution: 0.06133g NBT was weighed and stored to 100mL with phosphoric acid buffer, away from light;
100μmol/L EDTA-Na2 solution: Weigh 0.03721g EDTA-Na2 and volume it with phosphoric acid buffer to 1000 ml;
20μmol/L riboflavin solution: Weigh 0.0753g riboflavin and fill it with distilled water to 1000mL, and store it away from light.
Enzyme solution preparation:remove 0.5g of leaves in a pre-cooled mortar, add 1mL of pre-cooled phosphoric acid buffer ice bath to grind into pulp, add buffer to make the final volume of 5mL. Take 1.5-2 mL and centrifuge at 1000r/min for 20min, the supernatant is the crude extract of SOD.
Color reaction: Take 5mL test tubes (requiring good transparency), 2 tubes are measuring tubes, and the other 2 tubes are monitoring, add each solution and mix.Put 1 tube monitoring in the dark, and the other tubes react for 15-20min under 4000lx sunlight (requiring the same light condition of each tube, high temperature, shorter time, and longer at low times).
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The activity of peroxidase (POD) was determined by guaiacol method (
Reagent: 50 mL phosphate buffer and 28μL guaiacol were heated and mixed on a magnetic stirrer until the guaiacol was completely dissolved. After the solution was cooled, 30%H2O2 19 μL was added, mixed evenly and stored in a refrigerator at 4°C.
Enzyme solution preparation: Take a small number of leaves into a mortar, add 2 mL phosphate buffer (0.2 mol/L,pH 7.0) and grind into a homogenate. After all the homogenates were transferred into the 5 mL centrifuge tube, the mortar was cleaned twice with 4 mL phosphate buffer and transferred into the centrifuge tube. When centrifuged at 4°C for 20 min at 4000 r/min, the supernatant was the crude enzyme liquid.
Reaction determination: Take 3mL of reaction mixture for enzyme activity determination, add 20μL crude enzyme solution, and use the reaction mixture with 20μL phosphoric acid buffer as control. The absorbance was measured with 756 ultraviolet spectrophotometer at 470nm wavelength, and the absorbance value was recorded every 1 min, a total of 5 times. The unit μ of peroxidase activity is defined as a 0.01 change in A470 per minute.
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malondialdehyde (MDA) content was determined by thiobarbituric acid (TBA) colorimetry (
Reagents: 5% trichloroacetic acid (TAC), 0.67% thiobarbituric acid (TBA).
Preparation of enzyme solution: Take a small amount of plant tissue (leaves), mark the mass as m, add 5% TAC 5 ml, and centrifuge the homogenate obtained after grinding at 3000 r/min for 10 min.
Reaction: Use pipette gun to slowly absorb 2 ml of supernatant, not too fast, so as not to inhale the plant body, add 0.67% TBA 2 ml, mix and boil in 100°C water bath environment for 15 min, cool and then centrifuge once (10 min). The absorbance values of the supernatant at 450 nm, 532 nm and 600 nm were determined, respectively, with 2 ml distilled water plus 2 ml0.67%TBA as the control.
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Catalase was determined by ultraviolet spectrophotometry (
Reagent:
Buffer solution: weigh 65.52 g Na2HPO4·12H2O, 2.66 g NaH2PO4·2H2O, 10 g polyethylpyrrolidone, dissolve in about 900 mL water, use a pH meter to check the pH, adjust to 7.80, fixed volume to 1 L; Absorb 50 mL concentrated sulfuric acid and slowly add it to about 400 mL water. After cooling, the volume is fixed to 500 mL.
Hydrogen peroxide solution:absorbed 6 mL of 30% H2O2 solution, the volume was fixed to 1 L, and the concentration was calibrated before clinical use. The oxalic acid solution was dissolved in 3.1518 g of oxalic acid (H2C2O4·H2O) and transferred to a 500 mL volumetric bottle with a concentration of 0.1 mol·L-1.
Reagent calibration: Calibration of potassium permanganate solution: absorb 0.1M 5 mL of oxalic acid, add 10% 5 mL of sulfuric acid, and then slowly titrate with potassium permanganate solution until purple is maintained for half a minute. Start to react slowly, each drop, must wait for its fading before dropping.
Calibration of hydrogen peroxide solution: calibrated with calibrated potassium permanganate, absorb 2.5 mL of hydrogen peroxide solution to be calibrated, add 2.5 mL of 10% sulfuric acid, and titrate with known concentration of potassium permanganate solution until the pink color remains for 30 s as the end point; Because the molar absorption coefficient of hydrogen peroxide at 240 nm wavelength is 43.6 mol·L-1·cm-1, the concentration of the solution can be calculated by measuring the absorbance of the solution at 240 nm. Absorb 2.5mL hydrogen peroxide solution to be calibrated into a 25 mL colorimetric tube, add 2.5mL 10% sulfuric acid, set the volume to 25 mL, shake well, measure the absorbance at 240 nm, and set the volume of 2.5mL 10% sulfuric acid to 25 mL as blank.
Enzyme solution preparation: The treated leaf sample was cut and mixed well, weighed with a 1% electronic balance, 2-3 g was ground, 10 mL buffer solution and a small amount of quartz sand were added, and ground for 3 min at room temperature of 25°C to form a homogenization, then transferred to a 50 mL colorimetric tube, washed and ground with a small amount of buffer solution several times, and placed in the colorimetric tube each time. Finally, the buffer solution is set to 50 mL scale. After shaking, filter or centrifuge at 4000 r·min-1, the clear liquid is the enzyme extract and stored in the refrigerator at 4°C for use.
Reaction: Standard curve: Take 6 50 mL colorimetric tubes, add 2 mL buffer solution and 2.5 mL sulfuric acid each, then add 0, 1, 2, 3, 4 and 5 mL hydrogen peroxide solution respectively, shake well at a constant volume and measure absorbance at 240 nm. The regression equation y=ax+b was calculated with absorbance as the independent variable x and mass of H2O2 as the dependent variable y.
Determination: Two 50 mL colorimetric tubes were taken, and 1-2 mL of enzyme extract, 2.5 mL of sulfuric acid and 2.5 mL of hydrogen peroxide were added in turn. The sample tube was successively added with 1.0-2.0 mL of enzyme extract and 2.5 mL of hydrogen peroxide, then timed immediately, kept at 25°C for 10-20 min, and immediately added with 2.5 mL of sulfuric acid to terminate the reaction. Then the charge and sample tubes were filled to 50 mL and shaken well, and the absorbance As and Ac of the charge and sample tubes were determined at 240 nm. The amount of hydrogen peroxide calculated by the regression equation of the standard curve using the difference △A between the charge and the absorbance of the sample tube is converted to the enzyme activity.
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Significance difference analysis was performed in IBM SPSS Statistics 27, graph rendering in Origin 2021, correlation analysis and graph rendering in RStudio.