Wheat grains (0.2 g) were dry grinded and extracted in 2 ml (50 mM) potassium phosphate buffer (pH 7.4) or in buffer specified in method. Samples were centrifuged at 14,462 × g for 10 min at 4°C. The supernatant was separated and used for the determination of different enzymatic and non-enzymatic activities. All the data was taken in triplicate.

For the estimation of APX activity, seeds were homogenized in a medium composed of 50 mM potassium phosphate buffer (pH 7.0). APX activity was measured using the method of (Dixit et al., 2001). Assay buffer was prepared by mixing 200 mM potassium phosphate buffer (pH 7.0), 10 mM ascorbic acid and 0.5 M EDTA. For measurement of APX, the activity assay solution contained assay buffer made up of 10 mM ascorbic acid, 0.5 M EDTA and 200 mM potassium phosphate buffer, H₂O₂ (1 ml), and supernatant 50 μl. The oxidation rate of ascorbic acid was estimated by following the decrease in absorbance at 290 nm after every 30 s (Chen and Asada, 1989).

For the estimation of catalase activity, seeds were homogenized in a medium composed of 50 mM potassium phosphate buffer (pH 7.0) and 1 mM dithiothreitol (DTT). Catalase (CAT) was estimated using the method described by Beers and Sizer (1952). For measurement of CAT activity, the assay solution contained 50 mM phosphate buffer (pH 7.0), 59 mM H₂O₂, and 0.1 ml enzyme extract. The decrease in absorbance of the reaction solution at 240 nm was recorded after every 20 s. An absorbance change of 0.01 min⁻¹ was defined as 1 U of CAT activity. Enzyme activity was expressed on seed weight basis.

For the estimation of POD, seeds were homogenized in a medium composed of 50 mM potassium phosphate buffer (pH 7.0), 0.1 M EDTA, and 1 mM DTT. Activity of peroxidase (POD) was measured using the method of Chance and Maehly (1955) with some modification. For measurement of POD activity, the assay solution contained distilled water (545 μl), 200 mM phosphate buffer (pH 7.0), 200 mM guaiacol, 400 mMH₂O₂, and 15 μl enzyme extract. The reaction was initiated after adding the enzyme extract. The increase in absorbance of the reaction solution at 470 nm was recorded after every 20 s. One unit of POD activity was defined as an absorbance change of 0.01 min⁻¹. Enzyme activity was expressed on seed weight basis.

For the estimation of SOD activity, seeds were homogenized in a medium composed of 50 mM potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, and 1 mM dithiothreitol (DTT) as described by Dixit et al. (2001). The activity of SOD was assayed by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) following the method of (Giannopolitis and Ries, 1977). One unit of SOD activity was defined as the amount of enzyme that caused 50% inhibition of photochemical reduction of NBT.

A micro colorimetric method as described by Ainsworth and Gillespie (2007) was applied with some modifications for total phenolics content, in which Folin-Ciocalteau (F-C) reagent was used. For analysis, 0.5 g seed samples were homogenized in 500 μl ice cold 95% methanol using an ice cold mortar and pestle. The samples were then incubated at room temperature for 48 h in the dark. The samples were then subjected to centrifugation at 14,462 × g for 5 min at room temperature. The supernatant was removed and used for TPC measurement. A 100 μl of supernatant was mixed with 100 μl of 10% (v/v) F-C reagent, vortex thoroughly, and then 800 μl of 700 mM Na₂CO₃ was added. Samples were then incubated at room temperature for 1 h. Blank corrected absorbance of samples was measured at 765 nm. A standard curve was prepared using different concentration of gallic acid and a linear regression equation was calculated. Phenolic content (gallic acid equivalents) of samples was determined using linear regression equation.

For ascorbic acid determination, the 2,6-dichloroindophenol (DCIP) method described by Hameed et al. (2005), which measures reduced ascorbic acid only was used. Briefly, each molecule of vitamin C converts a molecule of DCIP into a molecule of DCIPH₂, and that conversion can be monitored as a decrease in the absorbance at 520 nm. A standard curve was prepared using a series of known ascorbic acid concentrations. A simple linear regression equation was calculated to find the ascorbate concentration in unknown samples.

The α-esterases and β-esterases were determined according to the method of Van Asperen (1962) using α-naphthyl acetate and β-naphthyl acetate as substrates, respectively. The reaction mixture consisted of substrate solution [30 mM α or β-naphthyl acetate, 1% acetone, and 0.04 M phosphate buffer (pH 7)] and enzyme extract. The mixture was incubated for exactly 15 min at 27°C in dark and then 1 ml of staining solution (1% Fast blue BB and 5% SDS mixed in ratio of 2:5) was added and incubated for 20 min at 27°C in dark. Amount of α- and β-naphthol produced was measured by recording the absorbance at 590 nm. Using standard curve, enzyme activity was α or β naphthol produced in μM min⁻¹ per g seed weight.

For estimation of protease activity, seed samples were extracted in 50 mM potassium phosphate buffer pH 7.8. Protease activity was dictated by the casein digestion assay described by Drapeau (1974). By this method one unit is that amount of enzyme, which releases acid soluble fragments equivalent to 0.001 A280 per min at 37°C and pH 7.8. Enzyme activity was expressed on seed weight basis.

The alpha amylase activity of wheat seeds was determined by the modified method as reported by the (Varavinit et al., 2002).

Total oxidant status (TOS) was determined by using (Erel, 2005) formulated method which is based on the oxidation of ferrous ion to ferric ion by oxidants present in the sample in an acidic medium and the measurement of ferric ion by xylenol orange (Harma et al., 2005). The assay mixture contained reagent R₁, reagent R₂ and sample extract. After 5 min. the absorption was measured at 560 nm by using spectrophotometer. A standard curve was prepared using hydrogen peroxide. The results were expressed in μM H₂O₂ equivalent per L.

The level of lipid peroxidation in the seed flour was measured in terms of malondialdehyde (MDA, a product of lipid peroxidation) content determined by the thiobarbituric acid (TBA) reaction using method of Heath and Packer (1968) with minor modifications as described by Dhindsa et al. (1981). A 0.25 g seed sample was homogenized in 0.1% TCA. The homogenate was centrifuged at 14,462 × g for 5 min. To 1 m aliquot of the supernatant 20% TCA containing 0.05% TBA were added. The mixture was heated at 95°C for 30 min and then quickly cooled in an ice-bath. After centrifuging at 14,462 × g for 10 min, the absorbance of the supernatant at 532 nm was read and the value for the non-specific absorption at 600 nm was subtracted. The MDA content was calculated by using extinction coefficient of 155 mM⁻¹ cm⁻¹.

Level of reducing sugars in seeds was determined by the dinitrosalicylic acid method (Miller, 1959). Total sugar contents were estimated by the phenol–sulphuric acid reagent method (Dubois et al., 1951). Non-reducing sugars were calculated by the difference.

For protein estimation, seeds were homogenized in a medium composed of 50 mM potassium phosphate buffer (pH 7.0). Estimation of quantitative protein was executed by previously described method (Bradford, 1976). For protein estimation in samples, 5 μl of supernatant and 0.1 N NaCl were mixed with 1.0 ml of Bradford dye and the mixture was allowed to stand for 5 min to form a protein dye complex. Absorbance was calculated at 595 nm by using spectrophotometer.

The first protein extract was obtained by adding 10 ml buffer A (0.01 M Tris-HCl pH 7.5) in 0.02 g seed flour (obtained through dry grinding the seed samples). After 2 h stirring at room temperature, the mixture was centrifuged at 4,000 g for 10 min and the supernatant corresponding to the albumins fraction was recovered. The pellet was then washed with 10 ml of buffer A, centrifuged, and the supernatant corresponding to the albumins fraction was recovered. The pellet was then washed with 10 ml of buffer A, centrifuged, and the supernatant pooled with the previous one. This fraction was albumin extraction (Bradford, 1976).

The pellet was mixed with 10 ml of buffer B (0.01 M Tris-HCl pH 7.5, and 1 M NaCl) and stirred for 2 h before centrifuging at 4,000 g for 10 min, also twice. This fraction was the globulin extraction (Bradford, 1976).

Seeds samples were dry grinded using pestle and mortar and 0.02 g seed flour was added in 2.5 ml of 0.15 M potassium phosphate (pH 7.5), 2.5 ml 5 mM DTT, and stirred 0.5 h at room temperature before centrifuging at 2,000 g for 5 min, three times extracted as before. The protein fraction obtained in the recovered supernatant was referred to as salt soluble protein (Bradford, 1976).

Genotypes were divided in three categories (low, medium, and high) based on comparative values of different studied parameters (Table 1).

For SOD activity in wheat seeds, significant variation was found among wheat genotypes and it was possible to categorize them in low, medium, and high classes. Thirty-eight genotypes were grouped in high category for SOD, in which the values ranged from 171 to 279 Units/g s. wt. Mean values for studied parameters are presented as Figure S1. Among these genotypes, highest SOD activity was found in Manthar-2003 (278.93 Units/g s. wt.). Sixteen genotypes with SOD activity ranging from 151 to 170 Units/g s. wt. were grouped as intermediate class. Twenty-three genotypes were grouped in third category that is genotypes having comparatively low SOD activity. In this class, genotypes having SOD activity <150 Units/g s. wt. were grouped. Among these genotypes having comparatively low SOD activity, the lowest value (76.17 Units/g s. wt.) was observed in Punjab-2011.

Genotypes were divided in three categories based on comparative values of different studied parameters (Table 2). For catalase activity in wheat seeds, significant variation was also found among wheat genotypes and it was possible to categorize them in low, medium, and high classes. Four genotypes were grouped in high category for CAT, in which the values ranged from 368 to 634 Units/g s. wt. Mean values for catalase are presented as Figure S2. Among these genotypes, highest CAT activity was found in Pasban-90 (633.33 Units/g s. wt.). Thirty-eight genotypes with CAT activity ranging from 101 to 367 Units/g s. wt. were grouped as intermediate class. Thirty-five genotypes were grouped in third category that is genotypes having comparatively low CAT activity. In this class, genotypes having CAT activity <100 Units/g s. wt. were grouped. Among these genotypes having comparatively low APX activity, the lowest value was observed in Nesser (46.67 Units/g s. wt.).

Genotypes were divided in three categories based on comparative values of different studied parameters (Table 2). For APX activity in wheat seeds, significant variation was found among wheat genotypes and it was possible to categorize them in low, medium, and high classes. Five genotypes were grouped in high category for APX, in which the values ranged from 1,000 to 1,427 Units/g s. wt. Mean values for APX are presented as Figure S3. Among these genotypes highest APX activity was found in Pavon (1,426.67 Units/g s. wt.). Forty-nine genotypes with APX activity ranging from 401 to 999 Units/g s. wt. were grouped as intermediate class. Twenty-three genotypes were grouped in third category, that is genotypes having comparatively low APX activity. In this class, genotypes having APX activity <400 Units/g s. wt. were grouped. Among these genotypes having comparatively low APX activity, the lowest value was observed in NARC-2011 (173.33 Units/g s. wt.).

Genotypes were divided in three categories based on comparative values of different studied parameters (Table 2). For peroxidase activity in wheat seeds, significant variation was found among wheat genotypes and it was possible to categorize them in low, medium, and high classes. Seven genotypes were grouped in high category for POD, in which the values ranged from 20,000 to 42,580 Units/g s. wt. Mean values for POD are presented as Figure S4. Among these genotypes highest POD activity was found in IQBAL-2000 (42,579.6 Units/g s. wt.). Twenty-four genotypes with POD activity ranging from 9,001 to 19,999 Units/g s. wt. were grouped as intermediate class. Forty-six genotypes were grouped in third category that is genotypes having comparatively low POD activity. In this class genotypes having POD activity <9,000 Units/g s. wt. were grouped. Among these genotypes having comparatively low POD activity, the lowest value was observed in Punjab-90 (1,642.8 Units/g s. wt.).

Genotypes were divided in three categories based on comparative values of different studied parameters (Table 2). For total phenolic content in wheat seeds, significant variation was found among wheat genotypes and it was possible to categorize them in low, medium, and high classes. Fourteen genotypes were grouped in high category for TPC, in which the values ranged from 20,233 to 25,383 (μM/g s. wt.). Mean values for TPC are presented as Figure S5. Among these genotypes highest TPC was found in Bhakkar-2000 (25,383.33 μM/g s. wt.). Fifty genotypes with TPC ranging from 9,701 to 20,232 (μM/g s. wt.) were grouped as intermediate class. Thirteen genotypes were grouped in third category that is genotypes having comparatively low TPC. In this class genotypes having TPC <9,700 (μM/g s. wt.) were grouped. Among these genotypes having comparatively low TPC, the lowest value was observed in WL-711 (1,200.0 μM/g s. wt.).

Genotypes were divided in three categories based on comparative values of different studied parameters (Table 2). For ascorbic acid content in wheat seeds, significant variation was found among wheat genotypes and it was possible to categorize them in low, medium, and high classes. Thirteen genotypes were grouped in high category for ascorbic acid content, in which the values ranged from 682 to 713 μg/g s. wt. Mean values for AsA are presented as Figure S6. Among these genotypes, highest ascorbic acid content was found in SH-2002 (713.0 μg/g s. wt.). Fifty genotypes with ascorbic acid content ranging from 641 to 681 μg/g s. wt. were grouped as intermediate class. Thirteen genotypes were grouped in third category that is genotypes having comparatively low ascorbic acid content. In this class genotypes having ascorbic acid content <640 μg/g s. wt. were grouped. Among these genotypes having comparatively low ascorbic acid content, the lowest value was observed in S-24 (439.5 μg/g s. wt.).

Genotypes were divided in three categories based on comparative values of different studied parameters (Table 2). Seventeen genotypes were grouped in high category for esterase activity, in which the values ranged from 708 to 988 μM/min/g s. wt. Mean values for esterase activity are presented as Figure S7. Among these genotypes highest esterase activity was found in Dharabi 2011 (987.80 μM/min/g s. wt.). Thirty genotypes with esterase activity ranging from 503 to 707 μM/min/g s. wt. were grouped as intermediate class. Thirty genotypes were grouped in third category that is genotypes having comparatively low esterase activity. In this class, genotypes having esterase activity <502 μM/min/g s. wt. were grouped. Among these genotypes having comparatively low esterase activity, the lowest value was observed in AS-2002 (358.53 μM/min/g s. wt.).

Genotypes were divided in three categories based on comparative values of different studied parameters (Table 2). For protease activity, in wheat seeds, significant variation was found among wheat genotypes and it was possible to categorize them in low, medium, and high classes. Ten genotypes were grouped in high category for protease activity, in which the values ranged from 8,801 to 11,183 Units/g s. wt. Mean values for protease activity are presented as Figure S8. Among these genotypes, highest protease activity was found in NR-234 (11,183.33 Units/g s. wt.). Fifty-three genotypes with protease activity ranging from 6,001 to 8,800 Units/g s. wt. were grouped as intermediate class. Fourteen genotypes were grouped in third category that is genotypes having comparatively low protease activity. In this class genotypes having protease activity <6,000 Units/g s. wt. were grouped. Among these genotypes having comparatively low protease activity, the lowest value was observed in SA-75 (4,023.33 Units/g s. wt.).

Genotypes were divided in three categories based on comparative values of different studied parameters (Table 2).

For alpha amylase activity in wheat seeds, significant variation was found among wheat genotypes and it was possible to categorize them in low, medium, and high classes. Twelve genotypes were grouped in high category for α-amylase, in which the values ranged from 281 to 293 (mg/g s. wt.). Mean values for α-amylase activity are presented as Figure S9. Among these genotypes highest α-amylase activity was found in SH-2002 (292.70 mg/g s. wt.). Twenty-eight genotypes with α-amylase activity ranging from 201 to 280 mg/g s. wt.) were grouped as intermediate class. Thirty-seven genotypes were grouped in third category that is genotypes having comparatively low α-amylase activity. In this class genotypes having α-amylase activity <200 mg/g s. wt. were grouped. Among these genotypes having comparatively low α-amylase activity, the lowest value was observed in Dharabi 2011 (112.58 mg/g s. wt.).

Genotypes were divided in three categories based on comparative values of different studied parameters (Table 2). For TOS in wheat seeds, significant variation was found among wheat genotypes and it was possible to categorize them in low, medium, and high classes. Seventeen genotypes were grouped in high category for TOS, in which the values ranged from 290 to 390 μM/g s. wt. Mean values for TOS are presented as Figure S10. Among these genotypes highest TOS was found in Faisalabad-2008 (390.0 μM/g s. wt.). Thirty genotypes with TOS ranging from 251 to 291 μM/g s. wt. were grouped as intermediate class. Thirty genotypes were grouped in third category that is genotypes having comparatively low TOS. In this class genotypes having TOS <250 μM/g s. wt. were grouped. Among these genotypes having comparatively low TOS, the lowest value was observed in NR-421 (241.0 μM/g s. wt.).

Genotypes were divided in three categories based on comparative values of different studied parameters (Table 2). For MDA content (μM/g s. wt.) in wheat seeds, significant variation was found among wheat genotypes and it was possible to categorize them in low, medium, and high classes. Thirteen genotypes were grouped in high category for MDA, in which the values ranged from 601 to 679 μM/g s. wt. Mean values for MDA are presented as Figure S11. Among these genotypes highest MDA activity was found in Margalla-99 (679.23 μM/g s. wt.). Twenty-eight genotypes with MDA activity ranging from 501 to 600 μM/g s. wt. were grouped as intermediate class. Thirty-six genotypes were grouped in third category that is genotypes having comparatively low POD activity. In this class genotypes having MDA activity <500 μM/g s. wt. were grouped. Among these genotypes having comparatively low MDA activity, the lowest value was observed in PERWAZ (167.23 μM/g s. wt.).

Genotypes were divided in three categories based on comparative values of different studied parameters (Table 2). Twelve genotypes were grouped in high category for reducing sugars, in which the values ranged from 9 to 13 mg/g s. wt. Mean values for reducing sugars are presented as Figure S12. Among these genotypes highest reducing sugars was found in Punjab-96 (12.68 mg/g s. wt.). Twenty-four genotypes with reducing sugars ranging from 5 to 8.9 mg/g s. wt. were grouped as intermediate class. Forty-one genotypes were grouped in third category that is genotypes having comparatively low reducing sugars. In this class genotypes having reducing sugars <4.9 mg/g s. wt. were grouped. Among these genotypes having comparatively low reducing sugars, the lowest value was observed in MEXI PAK (1.27 mg/g s. wt.).

Genotypes were divided in three categories based on comparative values of different studied parameters (Table 2). For total soluble sugars in wheat seeds, significant variation was found among wheat genotypes and it was possible to categorize them in low, medium, and high classes. Twenty-four genotypes were grouped in high category for total soluble sugars, in which the values ranged from 16 to 30 mg/g s. wt.) Mean values for total soluble sugars are presented as Figure S13. Among these genotypes highest total soluble sugars was found in Saleem-2000 (29.86 mg/g s. wt.). Thirty-three genotypes with total soluble sugars ranging from 12 to 15.9 mg/g s. wt. were grouped as intermediate class. Twenty genotypes were grouped in third category that is genotypes having comparatively low total soluble sugars. In this class genotypes having total soluble sugars <11.9 mg/g s. wt. were grouped. Among these genotypes having comparatively low total soluble sugars, the lowest value was observed in Soghat-90 (7.77 mg/g s. wt.).

Genotypes were divided in three categories based on comparative values of different studied parameters (Table 2). For non-reducing sugars in wheat seeds, significant variation was found among wheat genotypes and it was possible to categorize them in low, medium, and high classes. Twenty-three genotypes were grouped in high category for non-reducing sugars, in which the values ranged from 11 to 28 mg/g s. wt. Mean values for non-reducing sugars are presented as Figure S14. Among these genotypes highest non-reducing sugars was found in Saleem-2000 (27.33 mg/g s. wt.). Thirty-five genotypes with non-reducing sugars ranging from 6 to 10.9 mg/g s. wt. were grouped as intermediate class. Nineteen genotypes were grouped in third category that is genotypes having comparatively low non-reducing sugars. In this class, genotypes having non-reducing sugars <5.9 mg/g s. wt. were grouped. Among these genotypes having comparatively low non-reducing sugars, the lowest value was observed in Manthar-2003 (1.31 mg/g s. wt.).

Genotypes were divided in three categories based on comparative values of different studied parameters (Table 1). Eight genotypes were grouped in high category for total soluble proteins, in which the values ranged from 401 to 487 mg/g s. wt. Mean values for total soluble proteins are presented as Figure S15. Among these genotypes highest total soluble proteins was found in Punjab-96 (487.33 mg/g s. wt.). Twenty-nine genotypes with total soluble proteins ranging from 299 to 400 mg/g s. wt. were grouped as intermediate class. Forty genotypes were grouped in third category that is genotypes having comparatively low total soluble proteins. In this class, genotypes having total soluble proteins <298.0 mg/g s. wt. were grouped. Among these genotypes having comparatively low total soluble proteins, the lowest value was observed in MEXI PAK (68.67 mg/g s. wt.).

Genotypes were divided in three categories based on comparative values of different studied parameters (Table 2). Twenty-one genotypes were grouped in high category for albumins, in which the values ranged from 291 to 353 mg/g s. wt. Mean values for albumins are presented as Figure S16. Among these genotypes highest albumins was found in TC-4928 (352.89 mg/g s. wt.). Thirty-three genotypes with albumins ranging from 251 to 290 mg/g s. wt. were grouped as intermediate class. Twenty-three genotypes were grouped in third category that is genotypes having comparatively low albumins. In this class, genotypes having albumins <250 mg/g s. wt. were grouped. Among these genotypes having comparatively low albumins, the lowest value was observed in Manthar-2003 (199.56 mg/g s. wt.).

Genotypes were divided in three categories based on comparative values of different studied parameters (Table 2). Eleven genotypes were grouped in high category for globulins, in which the values ranged from 200 to 253 mg/g s. wt. Mean values for globulins are presented as Figure S17. Among these genotypes highest globulins was found in MEXI PAK (252.67 mg/g s. wt.). Thirty-two genotypes with globulins ranging from 162 to 199.9 mg/g s. wt. were grouped as intermediate class. Thirty-four genotypes were grouped in third category that is genotypes having comparatively low globulins. In this class, genotypes having globulins <161 mg/g s. wt. were grouped. Among these genotypes having comparatively low globulins, the lowest value was observed in Nifa Lalma 2012 (112.44 mg/g s. wt.).

Genotypes were divided in three categories based on comparative values of different studied parameters (Table 2). Seventeen genotypes were grouped in high category for salt soluble proteins, in which the values ranged from 111 to 162 mg/g s. wt. Mean values for salt soluble proteins are presented as Figure S18. Among these genotypes highest salt soluble proteins was found in Faisalabad-2008 (162.44 mg/g s. wt.). Forty genotypes with salt soluble proteins ranging from 71 to 110 mg/g s. wt. were grouped as intermediate class. Twenty genotypes were grouped in third category that is genotypes having comparatively low salt soluble proteins. In this class, genotypes having salt soluble proteins <70 mg/g s. wt. were grouped. Among these genotypes having comparatively low salt soluble proteins, the lowest value was observed in Punjab-90 (61.33 mg/g s. wt.).