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Abstract
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Glycyrrhiza glabra L., belonging to the Fabaceae family, is an important source of pharmacologically active compounds,
especially glycyrrhizin. Hairy root culture is an efficient approach for the stable biosynthesis of secondary metabolites.
In this study, three experimental sets were performed to optimize hairy root establishment. Initially, the effect of explant
type and Agrobacterium rhizogenes strain on the effectiveness of hairy root induction was evaluated. The maximum
transformation frequency (86%) was obtained from leaf explants infected with the MSU strain. Subsequently, the bacterial
cell density and infection time on transformation frequency and some morphological features were investigated, achiev-
ing optimal results (80%) at the optical density of 0.650 with 25 min of exposure time. Finally, the effects of culture
media and carbon source on the frequency of transformation and some morphological traits were investigated, achieving
an optimal transformation employing leaf explants cultured in 1/2 MS medium supplemented with sucrose and NAA
(1.5 mg/L). To investigate metabolic-response, the hairy roots were treated with elicitors. The maximum glycyrrhizin con-
tent (30.2 ± 0.01 mg/g DW) and fresh growth index (22.42 ± 0.16) were obtained after elicitation with methyl jasmonate.
In contrast, the simultaneous application of methyl jasmonate and mechanical shear stress resulted in the highest total
phenol (25.39 ± 0.22 mg GA/g DW) and flavonoid (12.51 ± 0.22 mg QUE/g DW) contents. These findings prove that the
G. glabra hairy root system was a promising biotechnological approach for biosynthesis of high-value glycyrrhizin and
laid a foundation for future metabolic engineering strategies in plant biotechnology.
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