To effectively evaluate the efficacy of pyroligneous acid on salt stress, drought stress, and insect resilience in rice cultivars, consider the following suggestions:

Use biochemical assays to measure antioxidant enzyme activities (superoxide dismutase, catalase, peroxidase), chlorophyll content, proline accumulation, and soluble sugars as markers of osmotic and oxidative stress response.

Implement physiological measurements such as germination rate, seedling vigour index, shoot and root length, and dry weight under stress conditions to assess performance enhancement after pyroligneous acid application.

Perform gene expression analysis (qRT-PCR) for stress-responsive genes—such as those encoding antioxidant enzymes, abscisic acid biosynthesis and signaling components, and defense-related proteins to uncover molecular mechanisms of improved resilience.

In the case of insect resilience, combine metabolic profiling of phenolic and flavonoid content with pest bioassays and monitoring expression of defense pathway genes (jasmonic acid, salicylic acid responses).

Consider proteomics approaches to identify key proteins upregulated by pyroligneous acid under stress, as well as metabolomic analysis to assess pathway activation and secondary metabolite changes.

Apply seed priming and foliar spray treatments at different concentrations to standardize protocol, then analyze results statistically for significance and reproducibility.

These integrated approaches will help reveal both the physiological benefits and molecular basis of pyroligneous acid’s efficacy against multiple stresses in rice cultivars.