Effect of chitosan oligosaccharides and other biotic elicitors on root cultures and in vitrogrown plantlets systems of Hypericum perforatum L.

Hypericum perforatum is one of the most studied medicinal plants due to its demonstrated antidepressant, anticancer and antimicrobial properties (1) and it has been used for centuries in popular medicine. Its most well-known metabolites, hypericin and hyperforin, are extracted from the aerial part of the plant (2, 3). Although many therapeutic properties of aerial parts have been extensively studied, little information is available on the chemical composition and biological activity of root extracts. Recently, we focused on bioactive metabolites accumulated in the root, with particular interest for xanthones. These compounds exhibit numerous pharmacological activities, including antifungal (4 and references therein), antiradical and anti-inflammatory (5). Xanthones are non-flavonoid phenolic compounds produced by some plants, fungi, lichens and bacteria. As all the other phenols, they are synthesized through the phenylpropanoid biosynthetic pathway. In H. perforatum root, xanthones are accumulated at very low levels, which are not adequate for large-scale production (4). In vitro roots exhibit a higher content in xanthones and the production could be enhanced by treating them with both biotic and abiotic elicitors. In previous works, we demonstrated that chitosan is the most effective elicitor in increasing xanthone production in H. perforatum root cultures. This is a polysaccharide obtained by partial deacetylation of chitin, the main structural element of arthropod exoskeleton and of cell walls in fungi. One of the problems related to the use of chitosan is its insolubility in water at neutral pH and in other organic solvents; therefore it is generally dissolved in water acidulated with acetic acid. Although this does not limit its use for application purposes, it makes difficult to discriminate the effect of chitosan from that of its solvent, and consequently its use to determine elicitation mechanisms (4). Recently, chitosan oligosaccharides (COS), obtained through chemical or enzymatic hydrolysis of chitosan, have gained interest for their high water-solubility at neutral pH, low viscosity, biodegradability, biocompatibility and non-toxicity (6). These features make COS promising elicitors both for application purposes and basic search. This research aims to evaluate the effects of chitosan oligosaccharides (COS) both on in vitro root cultures of H. perforatum and in vitro-grown plants. Chemical analyses have been conducted on both systems to compare their elicitor-responsivity to COS in term of xanthone production. The experiments have been carried out on in vitro-grown plantlets with elicitors such as jasmonates and salicylic acid, which in previous tests resulted ineffective on in vitro cultures of regenerated roots, to verify if shoot organs are involved in the perception of elicitors. The impact of elicitor treatments was evaluated not only in relation of root-specific metabolites (e.g. xanthones), but also on shoot-specific metabolites (e.g. hypericin and hyperforin). Further investigations regarded the expression, in COS-elicited root cultures, of genes involved in the biosynthetic polyphenols pathway, namely phenylalanine ammonia lyase (PAL), benzophenone synthase (BPS) and chalcone synthase (CHS). Previous results shown an antagonistic relationship between genes involved in the biosynthesis of chalcones (mainly flavonoids) and benzophenones (mainly xanthones), suggesting a common gene-regulation system, in agreement with (7)