Abstract
Shoot-differentiating callus culture of Ruta graveolens ssp. divaricata (Tenore) Gams was maintained on four variants of Linsmaier-Skoog (L-S) medium containing different concentrations of plant growth regulators, 1-naphthaleneacetic acid and 6-benzylaminopurine, ranging from 0.1 do 3.0 mg/l. Methanolic extracts of biomass cultured in vitro were used to determine the contents of free phenolic acids with an HPLC method. Out of eight compounds under analysis, six were shown to be present in the samples: caffeic acid, chlorogenic acid, p-coumaric acid, p-hydroxybenzoic acid, protocatechuic acid and syringic acid. Total contents of the compounds under study ranged from 124.9 to 138.7 mg/100g d.w., depending on the L-S medium variant. p-Coumaric acid was the main compound in the extracts from biomass cultured on all tested medium variants and its amounts were interesting from practical perspective (97.3-112.9 mg/100g d.w.). This compound was isolated, purified and its identity was confirmed by spectral methods. The contents of other compounds did not exceed 11.1 mg/100g d.w.
Extracts of the above-ground parts of plants growing in vivo (stems, leaves, herb), analyzed for comparison, contained three phenolic acids: protocatechuic acid (52.0-88.2 mg/100g d.w.), vanillic acid (2.6 – 17.8 mg/100g d.w.) and trace amounts of syringic acid.
This is the first report on the effect of growth regulators on the accumulation of free phenolic acids in biomass of R.graveolens cultured in vitro. This is also the first report documenting the isolation of p-coumaric acid from biomass of this subspecies cultured in vitro and also from any plant in vitro culture. In addition, for the first time in the above-ground parts of the plants growing in vivo phenolic acids were analyzed.