Hairy roots of Hypericum perforatum L.: a promising system for xanthone production

Abstract

Hypericum perforatum L. is a common perennial plant with a reputed medicinal value. Investigations have been made to develop an efficient protocol for the identification and quantification of secondary metabolites in hairy roots (HR) of Hypericum perforatum L. HR were induced from root segments of in vitro grown seedlings from H. perforatum, after co-cultivation with Agrobacterium rhizogenes A4. Transgenic status of HR was confirmed by PCR analysis using rolB specific primers. HR had an altered phenolic profile with respect to phenolic acids, flavonol glycosides, flavan-3-ols, flavonoid aglycones and xanthones comparing to control roots. Phenolics in control and HR cultures were observed to be qualitatively and quantitatively distinct. Quinic acid was the only detectable phenolic acid in HR. Transgenic roots are capable of producing flavonol glycosides such as quercetin 6-C-glucoside, quercetin 3-O-rutinoside (rutin) and isorhamnetin O-hexoside. The HPLC analysis of flavonoid aglycones in HR resulted in the identification of kaempferol. Transformed roots yielded higher levels of catechin and epicatechin than untransformed roots. Among the twenty-eight detected xanthones, four of them were identified as 1,3,5,6-tetrahydroxyxanthone, 1,3,6,7-tetrahydroxyxanthone, γ-mangostin and garcinone C were de novo synthesized in HR. Altogether, these results indicated that H. perforatum HR represent a promising experimental system for enhanced production of xanthones.

Agrobacterium enhances xanthone production in Hypericum perforatum cell suspensions

“…Cyril and Methodius''-Skopje, Republic of Macedonia (MKNH). As for a previous study (Tusevski et al 2013), seeds were washed with 70 % ethanol for 30 s, surface sterilized with 1 % NaOCl for 15 min, rinsed 3 times in sterile deionized water and cultured on MS macro and oligoelements (Murashige and Skoog 1962), B 5 vitamin solution (Gamborg et al 1968), supplemented with 3 % sucrose and solidified with 0.7 % agar. No growth regulator was added.…”

“…Though naphtodianthrones and acyl-phloroglucinols represent the best investigated groups of metabolites in H. perforatum, there is still an increased demand for further research on flavonoids and xanthones as pharmacologically active compounds (Fotie and Bohle 2006;Nahrstedt and Butterweck 2010). In this view, several studies demonstrated the potential of Hypericum in vitro cultures to accumulate xanthones and their production can be manipulated by the culture type (Pasqua et al 2003), hormonal supplementation (Dias et al 2000), genetic transformation (Tusevski et al 2013) and elicitation (Conceição et al 2006;Franklin et al 2009;Tocci et al 2011). Among the studies for elicitation of H. perforatum cells, Conceição et al (2006) reported strong enhancement of xanthone accumulation upon treatments with Colletotrichum gloeosporioides, methyl jasmonate and salicylic acid.…”

“…Similar patterns of xanthone accumulation have been observed in H. perforatum cells co-cultivated with A. tumefaciens (Franklin et al 2009). Even if our recent work was focused on A. rhizogenes-mediated transformation as an efficient strategy for enhanced production of xanthones in H. perforatum hairy roots (Tusevski et al 2013), the role of Agrobacterium as bacterial elicitor supplementation in the culture medium need further investigation. Namely, Agrobacterium being a Gram-negative bacterium acts as a source of lipo-polisacharides, peptide-glycans and other cell-wall components that trigger the process of elicitation (Chodisetti et al 2013).…”

Biolistic-mediated plasmid-free transformation for induction of hairy roots in tobacco plants

“…There are a lot of recent works on hairy roots induction using plasmid constructions with rol genes and Agrobacterium-mediated method (Bertoli et al 2008, Tusevski et al 2013. Though cloning step of all rol genes is very laborious by reason of large insert size.…”

“…Previously a different approach of hairy roots formation through single rol gene or rol genes combination was proposed (Spena et al 1987, Cardarelli et al 1987, Vilaine and Casse-Delbart 1987. These rol genes were cloned in binary vectors and were used to transform Agrobacterium tumefaciens strains (Bonhomme et al 2000, Hong et al 2006 which finally infected plant material; co-cultivation plant material with A. rhizogenes strains (Bertoli et al 2008, Tusevski et al 2013, Thilip et al 2015, Rana et al 2016; or biolistic bombardment (Aguado-Santacruz et al 2009). …”