AbstractBACKGROUNDPrevious studies have found that pesticide double‐stranded (ds)RNA usually has a long‐lasting validity period in plants. However, it is uncertain if any factors in plants could extend dsRNA duration. It has been reported that RNA‐dependent RNA polymerases (RdRP) in plants and some other eukaryotes could catalyze RNA amplification and be involved in RNAi (interference). Thus, this study evaluated the effect of RdRP on the tissue content, activity, and duration of exogenous dsRNA.RESULTSWe found that RdRP knockdown in Arabidopsis thaliana had no significant effect on tissue contents of reporter dsRNA parent molecules (8.91% reduction), but it caused significant decrease in the tissue contents of derived short fragments of 200, 120 and 59 bp tested (51.22%, 52.83% and 59.35%, respectively). Aphid inoculation tests showed that the same dose of insecticidal dsAgZFP exhibited a significantly lower lethal effect (mortality 58.8%) in the plants with RdRP knockdown than in the control plants with normal RdRP (86.0%). For Caenorhabditis elegans, the worms treated simultaneously with dsRdRP and reporter dsRNA had similar body contents to reporter dsRNA parent molecules and its long‐fragment derivative (200 bp) as the control (1.28‐ and 1.07‐fold greater, respectively). However, 120‐ and 59‐bp short‐fragment derivatives were significantly reduced by 28.78% and 59.84%, respectively, which also diminished faster in the descendants.CONCLUSIONSWe conclude that RdRP could significantly enhance the tissue content of dsRNA derivatives by catalyzing amplification, thus improving dsRNA activity and extending its lasting validity period. Otherwise, RNAi by exogenous dsRNA was proven to be noninheritable in A. thaliana. This work confirmed the merit of dsRNA as a plant protectant. © 2022 Society of Chemical Industry.