slowly (1 cm on average), were compact in texture and developed green as well as
pale yellow nodule areas on the surface (Fig.14.3g), but neither buds nor shoot
regenerated from these calli. With time, however, the calli developed roots with a
pattern similar to those observed in the absence of PCIB. Callus structure and
appearance is strongly altered by PCIB, but this material would adapt expressing
again previous characteristics, including the HRM response.
Recently it has been reported that the diphenyl urea-type cytokinin thidiazuron
(Lata et al. 2009 , 2010 ) and the natural aromatic cytokinin meta-topolin (Lata et al.
2016 ) could induce high frequency shoot regeneration from nodal explant or leaf
derived calli fromC. sativa. Whether or not these types of cytokinins could
eliminate some barriers to the organogenesis response in calli fromA. rhizogenes
transformed roots ofC. sativaremains to be studied.
Overall,C. sativatransformed roots exhibit a high callusing response with calli
that grew vigorously and developed compact and green nodular areas on the sur-
face, a priori indicative of organogenesis capacity, but being unable of shoot
regeneration. A possible explanation might be the existence of some barriers
pleiotropically exerted that would block downstream organogenesis response.
14.4 Conclusion
In this chapter, we attempted to provide an overview, up to present, of
Agrobacterium-mediated transformation of Cannabis sativaL. This species, a
difficult to transform plant, has been effectively infected with either Ri or Ti
plasmid-bearing agrobacteria and several transformed tissues (tumors and hairy
roots) were established and its transgenic nature confirmed. Hypocotyl of intact
seedlings was the most responsive material and the response depended on both
bacterial strain and plant variety. Transformed tissues were cultured and stabilized
in vitro and showed the characteristic traits of fast and phytohormone-independent
growth as well as high incidence of lateral branching and abundance of root hairs in
the case of roots. The presence of some nitrogen compounds, metabolites of
pharmaceutical implication, has been assayed in these transformed roots. Atropine,
choline and muscarine were detected at quantitative levels in transformed roots and
untransformed plant material ofC. sativa. Further, the three compounds are present
in hairy roots at concentrations quantitatively higher than in untransformed control
tissues. Finally, hemp transformed roots exhibited a high callusing response, with
calli that grew vigorously and developed compact and green nodular areas on the
surface, a priori indicative of organogenesis capacity, but that were unable of shoot
regeneration.
Acknowledgements Financial support was obtained from Junta de Andalucía (PAI Group
AGR-205, Project A42/00).
14 Hairy Root Culture as a Biotechnological Tool inC. sativa 313