Stimulation of furanochromone accumulation in callus cultures of Ammi visnaga L. by addition of elicitors.

In order to check the possibility of producing secondary metabolites, in vitro cultures of A. visnaga callus were established. The best growth of A. visnaga callus was obtained on Murashige and Skoog medium (MS) containing 6-benzyladenine (BA) and alpha-naphtaleneacetic acid (NAA). The study was concentrated on the induction of production of secondary metabolites by exposing callus to abiotic elicitors: benzo(1,2,3)-thiadiazole-7-carbothionic acid S-methyl ester (BION) and a suspension of silica (SiO2) and biotic elicitors: autoclaved lysates of Enterobacter sakazaki and scleroglucan. GC analysis indicated that not-elicited callus of A. visnaga grown in darkness accumulated 2 times more visnagin than the one which was grown under a 16-h photoperiod. The highest accumulation of visnagin was observed in the callus culture elicited with scleroglucan or BION. Scleroglucan induced also the accumulation of khellin in A. visnaga callus. The presented work shows that biosynthesis of pharmacologically important secondary metabolites in A. visnaga cultures could be stimulated by application of elicitors.

Establishment of hairy root cultures of Ammi majus.

Axenically grown Ammi majus plantlets were inoculated with seven different Agrobacterium rhizogenes strains. Hairy root lines were established only after inoculation with the two agropine strains: A4 and LBA9402. The growth rate of hairy root cultures was about thirty times faster than that of callus and cell suspension cultures. Polymerase chain reaction with primers for the genes rolB and rolC confirmed the integration of the T-DNA fragment of Ri plasmid of A. rhizogenes to the genome of hairy roots obtained after transformation by both Agrobacterium strains. The furanocoumarins (psoralen, xanthotoxine, bergapten and imperatorin) usually found in seeds of A. majus were not detected in callus, cell suspension and hairy root cultures using Gas chromatography-mass spectrometry (GC-MS). However, umbelliferone, a precursor of furanocoumarins, was detected in callus, cell suspension and hairy root cultures. The umbelliferone content in extracts of hairy root cultures, obtained after transformation by A4, was similar to that determined in A. majus seeds (19 µg/g DW) and higher than those obtained for cell suspension and callus cultures (2 and 9 µg/g DW, respectively).