Ascorbate peroxidase (APX):

The ascorbate peroxidase working (APX, EC: 1.11.1.11) will be determined from the decrease in absorbance of ascorbic at 290 nm (Asada and Chen, 1992). The enzyme activity will be assayed in 3 mL containing 50 mM phosphate buffer at pH 7, 0.5 mM ascorbic acid, and 0.5 mM H2O2. The reaction will be started by … Read more

Nitrate reductase (NR):

Nitrate reductase (NR; EC 1.6.6.1) activity will be performed by measuring the amount of NO2 formed in NR assay buffer (Wray and Filner, 1970). The assay medium for NR contained 0.5 ml potassium phosphate buffer (100 mM, pH 7.5), 0.1 ml KNO3 (100 mM), 0.1 ml NADH (0.15 mM) and 0.1 ml extract in a … Read more

Antioxidant enzymes activities:

Fresh leaves (0.5 g) will be finely ground under chilled conditions in 10 ml of phosphate buffer (50 mM with pH 7.8) for the extraction of antioxidant enzymes. Centrifugation of the mixture will be performed at 12000 × g for 20 min at 4ºC. The supernatant will be re-centrifuged at 15000 × g for 10 … Read more

Relative Membrane Permeability (%):

Fresh leaves (0.5 g) will be chopped and placed in 10 ml of distilled water. Then samples will be vortexed for 5s and the electrical conductivity (ECo) determined. The test tubes will be kept at 4°C overnight and the EC1 measured. Then the samples will be autoclaved for 1 h and EC2 measured after cooling … Read more

Relative water contents (RWC) (%):

The relative water content will be calculated following the method of Jones and Turner (1978). Fresh leaf samples will be weighed (Fw), soaked in deionized water in the dark for 24 h to re-hydrate and their turgid weight (TW) recorded. After that, the samples will be oven-dried at 80°C for 48 h to get their … Read more

Soil pH:

Soil samples were used to measure the pH of the soil. Each replicate was subjected to pH analysis to determine the average acidic or alkaline nature of the soil. The pH meter was used which was standardized with buffers provided by the manufacturers that were of 4.01, 7.00, 10.00 standards. 5g soil was added with … Read more

Soil texture:

The hydrometer method was used to determine soil texture. Particle size determination of samples was done through this method. 1% solution of sodium Hexa Meta phosphate was made and 30g soil was added with this solution and left it for 24 hours. Dilution was done with distilled water in a 1L cylinder, using a mechanical … Read more

Total soluble sugars:

Total soluble sugars were determined by Riazi et al., (1985). Fresh leaves (1 g) were grounded in 10 mL of 80% methanol. Centrifuged at 10,000rpm and filtered the extract and supernatant was used for the estimation of sugars. Plant extract 0.3mL was taken in test tubes and 3 ml of anthrone reagent was added to … Read more

Total flavonoids:

Flavonoids were determined by using the AlCl3 method. 1ml of the sample was taken in cuvette and 0.3ml of NaNO2 was added. Before adding the next chemical shaken well and then added 0.3ml of AlCl3. After mixing well, 2ml of NaOH was added. The optical density was taken at 510nm via a spectrophotometer.

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