Gentiana dinarica Beck. hairy root cultures and evaluation of factors affecting growth and xanthone production

Abstract

The induction and establishment of hairy root cultures of Gentiana dinarica using two strains of Agrobacterium rhizogenes (A4M70GUS and 15834/PI) is reported for the first time. Strain 15834/PI had higher induction rate of hairy roots (32.15 \%) than strain A4M70GUS (6.12 \%). Transgenic nature of the roots was confirmed by GUS assay and PCR analysis. Two clones per strain (A4M70GUS-D and -I, and 15834/PI-2 and -3) with marked differences in general morphology and growth rate were further studied. The methanol extracts of hairy root clones were analyzed for xanthones content using HPLC method. The effects of the type of carbohydrate source (sucrose, fructose or glucose) at different concentrations on the growth parameters (growth index, dry weight, fresh/dry weight ratio), phenolic and xanthone production, and free radical scavenging activity of the transgenic clones were evaluated. Statistical two level factorial design was used to define optimal conditions for growth and successful secondary metabolite production in G. dinarica hairy root clones. The results showed that clones A4M70GUS-D and 15834/PI-3 were the superior ones. These two clones had the highest dry weight on 116.8 mM sucrose, producing up to threefold higher amounts of total phenolics and norswertianin-1-O-primeveroside than other clones, untransformed roots and roots of wild-grown plants.