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).