16.3.1 Plant Regeneration
A major drawback to progress in genetically transforming Cannabis has been the
difficulty in regenerating plants from transformed cells in tissue culture. Cannabis
explants readily form callus and develop roots but have had a very poor ability for
shoot formation (Hemphill et al. 1978 ; Fisse et al. 1981 ; MacKinnon et al. 2000 ;
Feeney and Punja 2003 ). However, shoot tip (Richez-Dumanois et al. 1986 ;
MacKinnon et al. 2000 ; Wang et al. 2009 ) and axillary bud (Lata et al. 2009 ) explants
readily produce shoots that can be multiplied and rooted. While this provides a
convenient system to micropropagate valued plants, these explant types are less
attractive for transformation due to a higher risk of obtaining chimeric plants than
there would be for organogenic- or embryogenic- induced explants (Dong and
McHughen 1993 ; Hansen and Wright 1999 ). In spite of this recalcitrance, advances
are being made in regenerating Cannabis plantlets by indirect shoot organogenesis
(via a callusing stage). Slusarkiewicz-Jarzina et al. ( 2005 ) demonstrated shoot
regeneration in a variety of explant sources fromfivefibre cultivars, but the efficiency
of plantlet regeneration was very low. A much higher efficiency of shoot formation
(83–96%) and plantlet regeneration was obtained from young leaves of a marijuana
variety by Lata et al. ( 2010 ). Rooted shoots were transferred to soil and acclimated
with a 95% survival rate. Established plants showed comparable development,
morphology, cannabinoid profile and THC content to the mother plant. At this time,
there is no established protocol for regeneration of hemp by somatic embryogenesis.
Biotechnological advances with Cannabis will be challenging until an efficient
regeneration procedure is developed together with a transformation procedure to
yield transgenic plants that are able to stably express recombinant DNA
(Sirikantaramas et al. 2005 ; Stout et al. 2012 ; Altpeter et al. 2016 ). The high-efficiency
regeneration procedure put forward by Lata et al. ( 2010 ) is very encouraging and
deserves further investigation for transformation studies. Likewise, development of a
procedure to transfer a gene of interest into Cannabis using engineeredA. rhizogenes
as a vector would become a useful tool for testing gene expression within native root
tissues (Ron et al. 2014 ; Sun et al. 2015 ). Moreover, hairy roots are, in some
instances, capable of inducing plant regeneration in recalcitrant species (Tepfer 1990 ;
Chattopadhyay et al. 2011 ). Wahby et al. ( 2012 ) have demonstrated that hemp and
marijuana cultivars are amenable to hairy root transformation thus presenting an
avenue to explore for the development of transgenic Cannabis plants.
16.4 Stable and Transient Heterologous Expression
of Cannabinoid Genes
Among the many products that can be derived from Cannabis, the cannabinoid
pathway is currently attracting the most attention from the biotechnology com-
munity (Andre et al. 2016 ). Cannabinoids are a unique class of compounds that are
348 M. Feeney and Z.K. Punja