**2. Materials and Methods**

#### *2.1. Experimental Design*

A greenhouse experiment was conducted during the winter season of 2017/2018 at the Faculty of Science (Girls Branch), Al-Azhar University (latitude 30◦03 22.3" N longitude 31◦19 25.4" E), Nasr City, Cairo, Egypt. The study aimed to test the effects of pretreatment with γ-radiation (0, 25, and 50 Gy) and foliar application of stigmasterol (0, 100, and 200 ppm) on growth, biochemical constituents, and yield characters of wheat plants (*Triticum aestivum*, L.). Wheat grains (Cultivar Sids 12) were received from the Agriculture Research Center (latitude 30◦01 13.4" N longitude 31◦12 24.2" E), Giza, Egypt. Stigmasterol, purity ≥ 95, soluble in chloroform, 50 mg/mL, MP Biomedicals, LLC, France, was used. The seeds were irradiated by gamma 60Co at different doses including 0 (non-irradiated), 25, and 50 Gray (Gy) at the Egyptian Atomic Energy Authority (latitude 30◦02 41.8" N longitude 31◦20 41.0" E), Cairo, Egypt. Wheat grains were sown on November 21 in earthenware pots (no. 50) filled with sandy soil with six replicates for each treatment. The soil texture was sandy, field capacity 11.5%, pH 8.7, EC 0.35 dSm−1, Cl<sup>−</sup> 1.7, HCO3 − 1.10, Na+ 1.2, K+ 0.25, Ca2+ 1.27%, and Mg2+ 0.58 meq L−1. Phosphorus fertilizer was added before sowing at a rate of 6.0 g per pot of calcium superphosphate (15.5% P2O5). Nitrogen fertilizer was applied in two equal portions at a rate of 0.60 g/pot for each in the form of ammonium nitrate (33.5%N) at 30 and 60 days after planting. Potassium fertilizer was applied as a soil application at the rate of 2 g/pot in the form of potassium sulfate (48–52% K2O) 45 days after planting. The foliar spray of stigmasterol treatments was applied at the vegetative stage (45 days after sowing).

#### *2.2. Growth Parameters*

At 65 days after sowing, three representative samples were collected from each treatment for detecting the tested growth traits (shoot length (cm), root length (cm), leaves (no. per plant, flag leaf area), as well as fresh and dry weights of shoot and root per plant).

#### *2.3. Photosynthetic Pigments*

According to the procedure in [26], photosynthetic pigments—chlorophyll-a (Chl *a*), chlorophyll-b (Chl *b*)—and total carotenoids in samples of wheat fresh leaves tissues using 85% acetone with 0.1 g fresh weight (FW) of wheat leaves after grinding in the solvent was measured. The homogenized samples were centrifuged at 3000 rpm, and the filtrate was topped up to 10 mL with acetone (85%). The absorbance was recorded at 663, 644, and 452 nm using a spectrophotometer (VEB, Carl-Zeiss-Promenade, Jena, Germany) using acetone as a blank. The concentration of the pigment fractions (Chl *a*, Chl *b*, and carotenoids) was accounted for as μg/mL using the following equations:

$$\text{Chl } a = \text{[(10.3 \times E663) - (0.918 \times E644)]} = \mu\text{g } \text{mL}^{-1} \tag{1}$$

$$\text{Chl } b = \text{[(19.7 \times E644) - (3.870 \times E663)]} = \text{\textdegree{\#m mL}}^{-1} \tag{2}$$

$$\text{Caroteroides} = \left(4.2 \times \text{E452}\right) - \left[\left(0.0264 \times \text{Chl } a\right) + \left(0.426 \times \text{Chl } b\right)\right] = \mu\text{g }\text{mL}^{-1} \tag{3}$$

The concentrations of chlorophylls and carotenoids were expressed as mg g−<sup>1</sup> fresh weight (FW) of plant material. Pigment contents are represented as mg g−<sup>1</sup> FW.
