2.5.6. Determination of Indole acetic acid (IAA), Gibberellic acid (GA) and Abscisic acid (ABA) Contents

The extraction and purification for above mentioned phytohormones were made following the method of Kettner and Doerffling [34]. Plant leaves (1g) were grinded in 80% methanol at 4 ◦C with butylated hydroxytoluene (BHT) used as antioxidant. The extract was centrifuged and the supernatant was reduced by using a rotary thin film evaporator (RFE). The aqueous phase was partitioned 4 times at pH 2.5–3 with <sup>1</sup> <sup>2</sup> volume of ethyl acetate. The ethyl acetate was evaporated by a rotary thin film evaporator. The residue was re-dissolved in 1 mL of methanol (100%) and examined on HPLC (LC-8A Shimadzu, C-R4A Chromatopac; SCL-6B system controller) using UV detector and C-18 column (39 × 300 mm). The wavelength used for the detection of IAA was 280 nm and for GA was 254 nm. For ABA, the samples were injected onto a C18 column and eluted at 254 nm with a linear gradient of methanol (30–70%), containing 0.01% acetic acid, at a flow rate of 0.8 mL min-1 [35].

### 2.5.7. Determination of Salicylic Acid (SA) Content of Leaves

Enyedi et al. [36] and Seskar et al. [37] method was employed for salicylic acid detection. After crushing the fresh leaves (1 g) of tomato in 10 mL of 80% methanol at 4 ◦C. The sample was kept for 3 days with subsequent change in methanol after 24 h. The methanol was then evaporated using RFE and the residue was dissolved again in methanol, filtered and subjected to high-performance liquid chromatography (HPLC) (Agilent Technologies USA) equipped with S-1121 dual piston solvent delivery system and S-3210 UV/VIS diode array detector. Detection of SA was done at 280 nm by co-chromatography with 2-hydroxybenzoic acid as standard. The peak areas were recorded and calculated with SRI peak simple chromatography data acquisition and integration software (SRI instruments, Torrance, CA, USA).
