Hypericum perforatum hairy root clones (HR A–HR O) transformed with Agrobacterium rhizogenes A4 were evaluated for growth characteristics, phenolic compounds production, antioxidant/radical scavenging activity, antioxidant enzymes, and oxidative stress markers. The screening of growth characteristics revealed that HR clones were characterized with higher biomass accumulation, root elongation, and lateral branching in comparison to non-transformed roots. Significant increase of phenolics production in HR clones was related to phenylalanine ammonia lyase activity indicating an up-regulation of phenylpropanoid/flavonoid metabolism. Positive correlation of phenolics, flavonoids, and tannins with antioxidant assays indicated that these secondary metabolites significantly contributed to the antioxidant/radical scavenging properties of HR cultures. Regarding the enzymatic antioxidant state, an enhancement of superoxide dismutase activity in HR lines coincided with decrease in O₂˙⁻ production rate, while ascorbate peroxidase and catalase greatly contributed to the reduction of H₂O₂ levels. The substantial accumulation of malondialdehyde in HR clones indicated an efficiency of antioxidant enzymes to reduce O₂˙⁻ and H₂O₂ at levels that are not deleterious for membrane lipids. These results confirmed the involvement of an efficient antioxidant defense system in the response adjustment of H. perforatum HR cultures to transformation process. Two superior clones denoted as HR B and HR F were selected as fast-growing and high biomass yielding lines with up-regulated phenolic compounds’ production, antioxidant, and radical scavenging activity. Altogether, the best performing H. perforatum HR clones could be proposed as a prospective biotechnological system for scale-up production of antioxidant phenolic compounds.
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