Shikonin and luteolin production in hairy root culture of Perilla frutescens (L.) Britton

Perilla frutescens (L.) Britton is an important medicinal plant with considerable potential for the production of valuable bioactive metabolites. This study investigated the induction and growth of hairy roots through two sequential experiments designed to optimize hairy root induction and biomass production. In the first experiment, the effects of explant type (leaf, cotyledon, hypocotyl, and stem internode) and co-cultivation period (48 and 72 h) on hairy root induction were evaluated using the R . rhizogenes ATCC 15834 strain. Cotyledon explants co-cultivated for 48 h achieved the highest transformation efficiency (66%). In the second experiment, the effects of R. rhizogenes strains (A4 and ATCC 15834), explant age (14, 21, and 28 days), and culture medium (MS and ½ MS) were investigated. Cotyledon explants inoculated with the ATCC 15834 strain and cultured on ½ MS medium exhibited the highest transformation efficiency (66.0 ± 6.1%), the shortest rooting time (7.33 ± 0.49 days), the greatest number of roots per explant (10.33 ± 1.58), and the highest number of secondary hairy roots (6 per explant). In a separate experiment, the effects of the culture system and light conditions on hairy root biomass were evaluated. Hairy roots cultured in a temporary immersion bioreactor under dark conditions produced the highest biomass (20.96 ± 0.61 g), whereas those grown in shake flasks under light conditions produced the lowest biomass (4.14 ± 0.50 g). PCR analysis confirmed the integration of the rolA , rolB , and rolC genes in R. rhizogenes -induced hairy roots, while the absence of virD amplification confirmed that the cultures were free of residual bacterial contamination. HPLC analysis showed that hairy roots accumulated luteolin at levels comparable to those of greenhouse-grown plants, whereas shikonin accumulation was higher in hairy roots. These findings demonstrate that hairy roots of P. frutescens provide a promising platform for the sustainable production of valuable secondary metabolites.