Establishment of hairy roots culture of <i>Perilla frutescens</i> L. and production of phenolic acids

<italic>Perilla frutescens</italic> (L.) Britt., a plant rich in phenolic acids, contains rosmarinic acid (RA) as its main phenolic component. RA exhibits various pharmacological activities, including anti-allergic, anti-inflammatory, and antioxidant effects. In this study, we established hairy root cultures of <italic>P. frutescens</italic> using <italic>Agrobacterium rhizogenes strains</italic> C58C1, A4, and R1000, and assessed phenolic acid content. Among the strains, C58C1 yielded the highest number of rooted explants and the highest induction rate, with the RA content reaching 19.08 mg/g DW in the fastest-growing lines. We also investigated the effects of salicylic acid (SA) and methyl jasmonate (MeJA) on phenolic acid accumulation in hairy roots. SA treatment promoted RA accumulation, reaching 20.92 mg/g DW on day nine, while MeJA increased RA to 17.36 mg/g DW on day three, followed by a decline to 4.27 mg/g DW by day nine. Furthermore, transgenic analysis of <italic>PfbHLH13</italic> and <italic>PfbHLH66</italic> transcription factors showed that overexpression of these genes significantly increased RA content. Quantitative real-time PCR revealed that <italic>PfbHLH13</italic> enhances phenolic acid accumulation by regulating the phenylpropanoid pathway, while <italic>PfbHLH66</italic> influences both the phenylpropanoid and tyrosine-derived pathways. These findings highlight the potential of <italic>P. frutescens</italic> hairy roots for large-scale RA production.