*2.4. Identification of Endogenous Hormones*

A total of 10 g of fresh tissue per sample was homogenized with 80% (*v*/*v*) ethanol and stirred overnight at 4 ◦C. The extract was filtered through a Whatman filter, and the methanol was evaporated under a vacuum. The aqueous phase was adjusted to pH 2.5 with 1 N HCl, then partitioned with ethyl acetate (3 times), and finally passed through anhydrous sodium sulfate. After that, the ethyl acetate phase was evaporated under a vacuum. The dry residue containing acidic hormones (fraction I) was dissolved in 2.0 mL of methanol and stored in vials at 4 ◦C. The phytohormones (auxins, gibberellins, and abscisic acid) were determined by high-performance liquid chromatography (Shimadzu, Tokyo, Japan), isocratic UV analysis, and a reverse-phase C18 column (RP-C18 μ Bondapak, Waters). The column used included octadecylsilane (ODS) ultra-sphere particles (5 μm), and the mobile phases used were acetonitrile/water (26:74 *v*/*v*) at pH 4.00. The flow rate was 0.8 mL min<sup>−</sup>1, and detection was UV 208 nm. The standard solutions of the individual acids (auxins, gibberellins, and abscisic acid (Sigma, St. Louis, MO, 63178, USA)) were prepared in the mobile phase and chromatographed. All solvents were purchased from Aldrich (Munich, Germany).

#### *2.5. Total Soluble Sugars*

Total soluble sugars (TSS) were estimated in dry flag leaves of the wheat plant by the anthrone technique [27]. The TSS was analyzed by reacting 0.1 mL of ethanol extract with 3.0 mL freshly prepared anthrone (150 mg anthrone (Aldrich Chemical Company Inc., Milwaukee, WI 53233, USA) + 100 mL 72% H2SO4) in a boiling water bath for 10 min. The cooled samples were read at 625 nm using a spectrophotometer (VEB, Carl-Zeiss-Promenade, Jena, Germany). Total soluble sugar was calculated using a standard curve of glucose.
