ORIGINAL PAPER Genetic transformation of Ruta graveolens L. by Agrobacterium rhizogenes: hairy root cultures a promising approach for production of coumarins and furanocoumarins Matylda Sidwa-Gorycka Æ Aleksandra Krolicka Æ Aleksandra Orlita Æ Edmund Malinski Æ Marek Golebiowski Æ Jolanta Kumirska Æ Agnieszka Chromik Æ Edyta Biskup Æ Piotr Stepnowski Æ Ewa Lojkowska Received: 3 May 2008 / Accepted: 24 December 2008 / Published online: 29 January 2009 Ó Springer Science+Business Media B.V. 2009 Abstract Ruta graveolens L. is a source of pharmaco- logically 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 hypo- cotyls 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 approxi- mately 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: dic- tamnine, 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. Keywords Agrobacterium rhizogenes Á Alkaloids Á Coumarins–furanocoumarins Á Transformation Á Ruta graveolens L Introduction Ruta graveolens L. is a good source of diverse classes of secondary metabolites, including coumarins, flavonoids, furanocoumarins and alkaloids (Oliva et al. 2003). Men- tioned in ancient herbals, this plant has a long history in alchemy and folklore (Wink 1998). Extracts from R. graveolens have been used in the treatment of infections, inflammations and eczema as well as an antidote to snake and scorpion venoms (Wink 1998). Coumarins isolated from R. graveolens, such as xanthotoxin, bergapten and isopimpinelin, have been utilized in the treatment of skin diseases, pigmentation disorders and in the symptomatic treatment of demyelinating diseases (Ekiert et al. 2005). Alkaloids like dictamnine and methoxydictamnine, found in R. graveolens tissues, have antimicrobial activity and are very effective natural fungicides (Oliva et al. 2003). The biological activities of furanocoumarins and fur- oquinolone alkaloids make R. graveolens an attractive plant for pharmaceutical uses. In vitro culture can offer new opportunities by providing plant material indepen- dently of the climatic condition. Due to the fact that usually secondary metabolites are produced only in small quanti- ties in in vitro cultures of non-differentiated plant cells (DiCosmo and Misawa 1985) the culture of fast growing transgenic, hairy roots becomes a promising source of plant material for pharmaceutical use (Altamura 2004, Chang M. Sidwa-Gorycka Á A. Krolicka Á A. Chromik Á E. Biskup Á E. Lojkowska (&) Intercollegiate Faculty of Biotechnology, University of Gdansk & Medical University of Gdansk, Kladki 24, 80-211 Gdansk, Poland e-mail: lojkowska@biotech.ug.gda.pl A. Orlita Á E. Malinski Á M. Golebiowski Á J. Kumirska Á P. Stepnowski Faculty of Chemistry, University of Gdansk, Sobieskiego 18, 80-952 Gdansk, Poland M. Sidwa-Gorycka Gdynia Innovation Centre, Bio-Lab Centre of Pomeranian Science & Technology Park, Zwyciestwa Av. 96/98, 81-451 Gdynia, Poland 123 Plant Cell Tiss Organ Cult (2009) 97:59–69 DOI 10.1007/s11240-009-9498-x