2.3.4Estimation of defense enzymes in tomato
Plant samples (roots) were collected from same treatments of gene expression trial at an interval of 0, 2, 4, 8 & 16 days after inoculation and immediately placed in the liquid nitrogen and stored at -80°C until processing of the sample. The activities of enzymes were determined using assay kit (Himedia Pvt. Ltd) by following the manufacturer’s protocol. Fresh 1 g tissue of tomato roots were powdered using liquid nitrogen and suspended in 0.9 mL of 10 mM chilled phosphate-buffered solution (PBS) with a pH of 7.4 and homogenate was centrifuged 10,000 rpm for 10 min at 4°C. The 200 µl of supernatant was collected and loaded in 96 well micro-titre plates along with blank solution. Absorbance was recorded at 450 nm wavelength in GEN5 micro-plate reader (Molecular Devices) for determination of enzymes activities viz ., super oxide dismutase (SOD), Polyphenol oxidase (PPO), Phenylalanine ammonia lyase (PAL) and Peroxidase (PO) as for standardized protocols (Rajan and Pushpa, 2015).
2.4 Statistical data analysis
Data were analysed for significant mean differences by one-way ANOVA using statistical software Statistical Tool for Agricultural Research (STAR).The multiple mean comparisons were performed using Tukey’s Honestly Significant Difference test (Tukey’s HSD).