SYNTHESIS OF BIOLOGICALLY ACTIVE COMPOUNDS IN HAIRY ROOTS OF ASTRAGALUS PENDULIFLORUS LAM.

Abstract

Agrobacterium rhizogenes – mediated genetic transformation of medicinal plant A. penduliflorus Lam. using A4-RT, R-1601, 15834 SWISS strains was performed. The competences for transformation of three types of explants: hypocotyls, cotyledons, and primary shoots were tested. The virulent strain (15834 SWISS) and types of explants for transformation (primary shoots and cotyledons) with high growth index (I) were determined. The frequency of transformation of cotyledons by strain 15834 SWISS after 4 weeks of cultivation was 15.4% (I = 59.6), hypocotyls – 9.1% (I = 7.3) and primary shoots – 37.5% (I = 21.0). After 8 weeks of cultivation I increased 4.5 times for primary shoots (I = 94.5 ± 0.20) and cotyledons (I = 265.8 ± 0.35), for cultures from hypocotyls – 5.97 times (I = 43.6 ± 0.30). The roots’ transgenic status and the absence of agrobacterium contamination were confirmed by PCR analysis using rolB-, virC-specific primers. The lines of hairy roots characterized by active increases of biomass with high content of biologically active metabolites were selected, moreover, the content of metabolites in hairy root cultures exceeded their content in the roots of introduced plants. The maximum accumulation of compounds was found in hairy roots obtained from primary shoots (pectins – up 7.8%, protopectins – up 15.3%) and cotyledons (tannins – up 16.1%, triterpenic saponins – up 30.5%) after 8 weeks of cultivation. High perfor-mance liquid chromatography (HPLC) analysis demonstrated that hydrolysates of extracts of hairy roots from primary shoots contained 2 flavonol aglycones – quercetin and isorhamnetin whereas ethanol extracts were characterized by presence of quercetin and 4 flavonoid components. In hairy roots from cotyledons the maximum of phenolic compounds (PСs) content did not differ significantly at 8 and 12 weeks of cultivation (1.38 ± 0.01 and 1.49 ± 0.06% of dry weight, respectively). The content of PCs in hairy roots from primary shoots increased two-fold from 4 to 12 weeks of cultivation (up 1.24 ± 0.18%). To the best of our knowledge, this is the first efficient protocol reported for the establishment of hairy root cultures in A. penduliflorus using A. rhizogenes.