Trehalose and brassinolide enhance the signature ingredient accumulation and anti-oxidant activity in the hairy root cultures of Polygala tenuifolia Willd

Polygala tenuifolia Willd. is a commonly used clinical traditional Chinese medicine with a variety of biological activities. Hairy root cultures of medicinal plants are considered as a source of active constituents for pharmaceutical industries. In this study, P. tenuifolia hairy root cultures (PTHRCs) were established by induction of four Rhizobium rhizogenes, including ATCC15834, LBA9402, C58C1 and ACCC10060. The contents of active ingredients such as polygalaxanthone III, 3′,6-disinapoylsucrose, onjisaponin B, onjisaponin F, polygalacic acid and senegenin were detected in the hairy roots by high performance liquid chromatography (HPLC). Forty-five day-old leaf disc of P. tenuifolia sterile seedlings were prepared for explants, the highest hairy root transformation rate reached 85.83 % by using R. rhizogenes C58C1 co-cultured for 3 d with an optical density of 1.5 at 600 nm, coupled with 200 µM acetosyringone added in the solid culture medium. Meanwhile, the conditions of PTHRCs were optimized for the xanthones, saponins, and oligosaccharides components production. The results showed that the dry weight of the hairy roots reached its maximum of 0.44 g/flask (9.26 fold than that of the control) under the optimal culture conditions: 6,7-V medium, 3 % sucrose (m/v), pH 5.8, and 6 week-old PTHRCs, and the six signature ingredients in the hairy roots accumulated massively under the same condition simultaneously. Furthermore, trehalose and brassinolide were found to significantly promote the biomass and active ingredients biosynthesis in the hairy root cultures. The content of polygalaxanthone III was 8.92 folds higher than the description in the Chinese Pharmacopoeia (2020 year version) induced by 10 µM trehalose, while 1 × 10−12 M brassinolide treatment promoted onjisaponin F accumulation at 33.63 mg/g (dry weight) in PTHRCs. In addition, total phenolics, flavonoids, and antioxidant activity of PTHRCs extracts were significantly increased induced by trehalose (5 µM) and brassinolide (1 ×10−10 M), respectively. This is the first report on the systematic construction of PTHRCs and trehalose and brassinolide as effective elicitors for polygalaxanthone III, 3′,6-disinapoylsucrose, onjisaponin B, onjisaponin F, polygalacic acid and senegenin production in PTHRCs. The results demonstrated that PTHRCs can be used to produce active constituents in pharmaceutical and nutraceutical fields. •An efficient hairy root induction and culture protocol of Polygala tenuifolia