8.3.1 Cannabinoid Production in C. sativa
There are many ways to increase cannabinoid production inC. sativa. Varieties of
this plant with high cannabinoid contents can be achieved through breeding and
advanced cultivation techniques. Cascini ( 2011 ) reported that marijuana samples
with unusually high cannabinoid content are the products of breeding experiments
rather than of genetic modification. To our knowledge, transgenic cannabis is
difficult to construct. However, protocols to establish transgenic cannabis have been
described elsewhere, suggesting that it is possible to produce transgenic cannabis
with high concentrations of bioactive ingredients.
Plant cell cultures have been used widely for the production of bioactive com-
pounds (Mora-Pale et al. 2014 ). Specifically, a suspension culture ofC. sativahas
been established as a production platform. Elicitation by both various biotic and
abiotic elicitors (e.g., microorganisms and their cell wall fragments as well as
salicylic acid, methyl jasmonate, and UV) has been tested in cell suspension cul-
tures to enhance bioactive compound production (Flores-Sanchez et al. 2009 ;Peč
et al. 2010 ). Although metabolite profiles of treated and control suspensions of cells
were clearly different, cannabinoid production was not detected. Cannabinoid
biosynthesis is very tightly regulated due to its general toxicity. CBGA, THCA, and
CBCA have been shown to induce cell death in tobacco and even inC. sativa
(Sirikantaramas et al. 2005 ; Morimoto et al. 2007 ). These compounds are mainly
produced in the storage cavity of the glandular trichome. The reason thatC. sativa
suspension cells do not produce cannabinoids could suggest the absence of a
transcription factor involved in pathway regulation. The MYB transcription factor
has been shown to be involved in the production of many important bioactive
compounds including those in the phenylpropanoid pathway and glucosinolates
(Hirai et al. 2007 ; Liu et al. 2015 ). A suspension ofArabidopsiscells, which do not
generally produce glucosinolates, can produce glucosinolates upon overexpression
of a specific MYB transcription factor (Hirai et al. 2007 ). Interestingly, two putative
MYB genes preferentially expressed inC. sativaglands were identified (Marks
et al. 2009 ). Involvement of these transcription factors in cannabinoid biosynthesis
needs to be clarified.
TheAgrobacterium tumefaciensstrain EHA101 encoding a gene for phospho-
mannose isomerase was used to transformC. sativasuspension cultures. The
expression of this gene was then successfully detected (Feeney and Punja 2003 ).
A rapid shoot regeneration protocol from the cotyledons ofC. sativahas been
reported by the addition of TDZ to MS medium with an induction frequency of
51.7% (Chaohua et al. 2016 ). These reports suggest a possible method to construct
transgenicC. sativaplants. Using hypocotyl of intact seedlings, hairy root cultures
ofC. sativacan be established afterAgrobacteriuminfection (Wahby et al. 2013 ).
The production of cannabinoids in this culture was not discussed. However, Farag
and Kayser ( 2015 ) reported low levels of cannabinoid (THCA, THC, and CBDA)
production (<2lg/g dry weight) in hairy root cultures that were established from
grown callus culture. It could be possible that low levels of cannabinoids are not
198 S. Sirikantaramas and F. Taura