staining (Fig.14.1f), demonstrating cotransfer of the binary vector and the
expression of the foreign GUS gene. All this provides evidence that hemp is
transformable byAgrobacteriumand extends results of Feeny and Punja ( 2003 ).
Finally, individualrolgenes or their combinations have been able to induce
transformed roots in a number of species (Capone et al. 1989 ; Palazón et al. 1997 ;
Bonhomme et al. 2000 ). In hemp, however, only the combination of the three
rolABCloci evoked biological responses similar to those elicited by the strains with
the complete set of T-DNA genes in the corresponding Ri plasmid, which showed
that the effect of the threerolgenes was synergistic.
14.2.5 Secondary Metabolites inC. sativa
Transformed Root Cultures
Hairy root cultures have become popular as biotechnological matrices for the
production of secondary metabolites synthesized in the plant roots. Moreover, they
are also been considered as potential sources for new natural products, both
metabolites characteristic from aerial part and metabolites not detected in mother
plant (Bulgakow 2008 ; Zhou et al. 2011 ; Georgiev et al. 2012 ; Ludwing-Müller
et al. 2014 ; Matveeva et al. 2015 ), asrolgenes might some way activate secondary
metabolism pathways. Cannabinoids (lastly referred as phytocannabinoids to dis-
tinguish them from endocannabinoids and those of synthetic origin, Chandra et al.
2013 ), the class of terpene phenolic compounds unique toC. sativa, accumulate
mainly in glandular trichomes of the plant, not having been, to our knowledge,
reported in the plant roots. Because of above discussion, however, in our laboratory
to study the biosynthetic capacity of hemp hairy roots, the presence of cannabinoids
(among other compounds) was assayed.
To investigate the presence of phytocannabinoids, samples (1–3 g) of lyophi-
lized transformed root cultures were extracted with 20 mL petroleum ether for
15 min under agitation on a rotary shaker. After centrifugation, anhydrous sodium
sulfatefiltration and evaporation, under N 2 stream, the dry residues were redisolved
in 0.5 mL ethanol and kept at−20 °C. Ethanol extracts were analyzed by gas
chromatography with nitrogen/phosphorous detector (GC/NPD) and HPLC analysis
using known solutions of commercial tetrahydrocannabinol (THC), cannabidiol
(CBD) and cannabinol (CBN) as standards (Dr. A. Plá, Department of Forensic
Medicine, Toxicology and Physical Anthropology, University of Granada). Results
showed no signals corresponding to the used standards or similar chemical struc-
tures. Thus detectable levels of cannabinoids are not present inC sativahairy roots,
at least under the experimental conditions used. However, with a high genetic
variability within the genusCannabis,the possibility that transformed root cultures
derived from additional plant variety-bacterial strain combinations (combined with
improved culture protocols and analytical techniques), could synthesize phyto-
cannabinoids should not be ruled out. On the other hand, the gene for
14 Hairy Root Culture as a Biotechnological Tool inC. sativa 307