Department of Analytical Chemistry, University of Granada) (Wahby et al. 2006 ).
With this methodology, atropine, choline and muscarine have been detected at
quantitative levels inA. rhizogenes-transformed roots and in untransformed plant
material of C. sativa(Table14.3) with atropine and muscarine having been
reported for thefirst time so far in this species. Further, the three compounds are
present in hemp hairy roots at concentrations quantitatively higher than in
untransformed control tissues. Thus, results confirm forC. sativathatA. rhizogenes
transformation effects on the biosynthesis of metabolites present in mother plant
were similar to those observed for many other species (Georgiev et al. 2012 ). The
absence of atropine in untransformed control roots may indicate that the concen-
tration was too low and thus out of the detection limit of the analytical method.
Alternatively, it might also be argued that atropine would be characteristic of aerial
parts inC. sativaand thatrolgenes could have activate, some way, its synthesis in
the transformed roots. Such an effect ofA. rhizogenestransformation has also been
suggested for other instances (Georgiev et al 2012 ; Bulgakov et al. 2013 ).
The differences in concentration between choline and the other metabolites in
our hemp materials are not well understood. However, it would may well reflect the
fact that choline have a central role in primary metabolism, while atropine is a
typical secondary metabolite with unknown specific functions for plant growth and
development. On the other hand, atropine and muscarine concentrations inC. sativa
transformed roots are lower than those reported for some common natural sources
of these alkaloids, i.e.Atropa belladonna,Hyosciamus muticusandFlos daturae
(Kamada et al. 1986 ; Eeva et al. 1998 ; Ye et al. 2001 ), for atropine, andClitocibe
andInocibemushroom species (Bollinger and Eugster 1971 ; Floersheim 1987 ) for
muscarine. Nonetheless, with the amenability for in vitro culture and the high
growth potential showed by hemp hairy roots (Wahby et al. 2013 ), exploring new
Table 14.3 Concentration encountered for choline, atropine and muscarine in extracts from hairy
roots and untransformed tissues ofCannabis sativaL
Plant tissue Choline (mg L−^1 ) Atropine (μgL−^1 ) Muscarine (μgL−^1 )
Hairy root culture
HRC 10 510 ± 13 933 ± 95 367 ± 31
HRC 20 203 ± 12 562 ± 30 –
HRC 30 259 ± 14 633 ± 35 –
HRC 40 379 ± 10 645 ± 59 –
HRC 50 311 ± 9 670 ± 31 –
HRC 60 435 ± 12 715 ± 94 231 ± 31
Untransformed controls
Roots CAN0221 97 ± 6 N.D. –
Roots Delta-Llosa 153 ± 6 N.D. –
Leaves CAN0221 66 ± 4 532 ± 53 –
Leaves Delta-Llosa 84 ± 5 553 ± 49 –
Data, mean of three replicates, are representative of two experiments. (–), not determined;N.D.not
detected
14 Hairy Root Culture as a Biotechnological Tool inC. sativa 309