Genetic transformation of Ruta graveolens L. by Agrobacterium rhizogenes: hairy root cultures a promising approach for production of coumarins and furanocoumarins

Ruta graveolens L. is a source of pharmacologically active compounds such as coumarins, furanocoumarins and furoquinolone alkaloids. Hypocotyls, callus and shoots of R. graveolens were inoculated with bacteria from two Agrobacterium rhizogenes strains. Hairy root cultures were established after inoculation of hypocotyls with wild A. rhizogenes strain LBA 9402. The transgenic nature of the regenerated tissue was confirmed by PCR amplification. Coumarins, furanocoumarins and alkaloids present in the hairy root tissue were identified by GC and GC-MS and compared with those present in in vitro shoot cultures. The level of pinnarin and rutacultin, bergapten, isopimpinelin and xanthotoxin was approximately twofold higher in hairy root than in shoot cultures. Two additional coumarins: osthole and osthenol, never been found in R. graveolens, were identified in hairy root tissue. Besides coumarins, alkaloids were identified: dictamnine, skimmianine, kokusaginine, rybalinine and an isomer of rybalinine. The levels of nearly all coumarins and alkaloids in hairy roots cultured in the darkness were higher than those accumulated under a photoperiod mode.