Establishment of high-productive Isatis tinctoria L. hairy root cultures: A promising approach for efficient production of bioactive alkaloids

• A high-productive ITHRCs system is first established for production of valuable AK.
• Molecular identification of ITHRCs by PCR amplification of rolB, rolC and aux1 genes.
• Optimizing and modeling ITHRCs system by efficient Box–Behnken design.
• Six main AK constituents were simultaneously determined by accurate LC–MS/MS.
• Total AK production in 23 day-old ITHRCs is more than 2 year-old field grown roots.

For the first time, Isatis tinctoria hairy root cultures (ITHRCs) induced by Agrobacteriumrhizogenes, were established as alternative sources for the production of bioactive alkaloids (AK). The highest transformation rate (76.67%) was obtained when 3 week-old petiole explants were co-cultured with A. rhizogenes for 2 days by the aid of 125 μM acetosyringone and 1.5 mM arginine. Among eight I. tinctoria hairy root lines (ITHRLs), ITHRL III was screened as the lead line and was confirmed by the PCR amplification of rolB, rolC and aux1 genes. Culture conditions of ITHRCs were optimized by Box–Behnken design, and six main AK constituents were qualitatively and quantitatively determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Under the culture temperature (24.7 °C), inoculum size (0.75%), sucrose concentration (3.14%) and initial pH (5.8), ITHRCs (23 day-old) in MS/2 medium gave the maximum biomass dry weight (DW) of 12.85 g/L and the optimal total AK content of 521.77 μg/g DW. Results showed that the proposed ITHRCs system possessed higher ability of AK production as compared to that of 2 year-old field-grown roots (464.69 μg/g DW). Overall, this work offered a promising, sustainable and high-productive biosynthesis platform that was capable of augmentation production of valuable naturally-derived AK.

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