Enhancement of Rosmarinic Acid Production in Hairy Root Cultures of Perovskia atriplicifolia Benth

This study reports the first successful establishment of <i>Perovskia atriplicifolia</i> hairy root cultures using <i>Rhizobium rhizogenes</i> and evaluates their potential for bioactive phenolic acid production, particularly rosmarinic acid (RA). Hairy roots were induced using two <i>R. rhizogenes</i> strains, A4 and ATCC 15834; transformation was confirmed by PCR analysis targeting the rol and aux genes. The A4 strain exhibited higher transformation efficiency (41.3%) than ATCC 15834 (30.2%). Eight transgenic root clones (C1-C8) were established and confirmed as transformed. The clones exhibited significant variation in biomass accumulation and phenolic acid production. RA production was most strongly correlated with <i>PAL</i>, <i>RAS</i>, and <i>CYP98A14</i> expression. Hierarchical clustering clustered the clones into three groups based on growth, metabolite content, and gene expression. Lines C1 and C2 exhibiting the highest RA, total polyphenol content, and the highest productivity were selected for further experiments. McCown Woody Plant (WP) and Schenk and Hildebrandt (SH) media demonstrated the greatest biomass accumulation, with growth indexes exceeding 13. Conversely, Gamborg (B5) medium enhanced RA content, achieving 38.3 and 40.8 mg/g dry weight (DW) for clones C1 and C2, respectively, representing a fourfold increase compared to the least favorable Murashige and Skoog (MS) medium. These findings establish <i>P. atriplicifolia</i> hairy roots as efficient systems for RA biosynthesis and can provide a basis for metabolic engineering and scale-up production of phenolic acids in medicinal plants.