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\begin{document}

\title{\textbf{The Cream of the Crop: Biology, Breeding and Applications of
\emph{Cannabis sativa}}}



\author[1]{Susanne Schilling*^}%
\author[1]{Caroline A. Dowling*}%
\author[1]{Jiaqi Shi*}%
\author[1]{Louise Ryan}%
\author[1]{David Hunt}%
\author[1]{Eve OReilly}%
\author[1]{Antoinette S. Perry}%
\author[1]{Oliver Kinnane}%
\author[1]{Paul F. McCabe}%
\author[1]{Rainer Melzer^}%
\affil[1]{University College Dublin}%


\vspace{-1em}



  
  \date{March 13, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\emph{Cannabis sativa} is an extraordinarily versatile species. Hemp and
its cousin marijuana, both~\emph{C. sativa}, have been used for
millennia as a source of fibre, oil and for medicinal, spiritual and
recreational purposes. Because the consumption of~\emph{Cannabis} can
have psychoactive effects, the plant has been widely banned throughout
the last century. In the past decade, evidence of its medicinal
properties did lead to the relaxation of legislation in many countries
around the world. Consequently, the genetics and development
of~\emph{Cannabis} as well as~\emph{Cannabis}-derived products are the
subject of renewed attention.Here, we review the biology of~\emph{C.
sativa,} including recent insights from taxonomy, morphology and
genomics, with an emphasis on the genetics of cannabinoid synthesis.
Because the female~\emph{Cannabis~}flower is of special interest as the
site of cannabinoid synthesis, we explore flower development, flowering
time well as the species' unique sex determination system in detail.
Furthermore, we outline the tremendous medicinal, engineering, and
environmental opportunities that~\emph{Cannabis} bears. Together, the
picture emerges that our understanding of~\emph{Cannabis} biology
currently progresses at an unusual speed. A future challenge will be to
preserve the multi-purpose nature of~\emph{Cannabis}, and to harness its
medicinal properties and sustainability advantages simultaneously.%
\end{abstract}%



\sloppy


*: These authors contributed equally to this work and should be
considered joint first authors

\^{}: for correspondence: susanne.schilling {[}at{]} udc.ie;
rainer.melzer{[}at{]}ucd.ie

\textbf{Keywords:} building materials, \emph{Cannabis sativa},
cannabinoids, crop breeding, flower development, flowering time, hemp,
marijuana, sex determination, sustainability

\par\null

\section*{\texorpdfstring{1. Introduction -- \emph{Cannabis} in a
nutshell}{1. Introduction -- Cannabis in a nutshell}}

{\label{250777}}

\emph{Cannabis sativa} is a highly versatile crop with dozens of
different uses (Figure 1). There are a multitude of medical applications
for \emph{Cannabis} secondary compounds\emph{,} which have been shown to
reduce pain, nausea and neurological conditions like seizures (Whiting
et al., 2015), and research on effects on inflammation, depression and
cancer is also being conducted (Atalay et al., 2019; Fraguas-S\selectlanguage{ngerman}ánchez and
Torres-Suárez, 2018; Poleszak et al., 2018; \selectlanguage{polish}Ś\selectlanguage{english}ledzi\selectlanguage{polish}ń\selectlanguage{english}ski et al., 2018,
Russo, 2011). Beyond that, fibre varieties of \emph{Cannabis} have high
carbon sequestering potential because of their rapid growth. They are
therefore utilized for carbon storage in building materials or as
biofuel (Finnan and Styles, 2013). For those different reasons, the
\emph{Cannabis} industry is gaining more traction and the need for
specialized varieties, adapted to local climatic conditions, or suited
for specific applications, is steadily increasing.

\par\null\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure1/Figure1}
\caption{{\textbf{Cannabis is a multipurpose crop.~}The uses for \emph{Cannabis}
are manifold. The trichomes present on female flowers produce
efficacious phytocannabinoids including cannabidiol (CBD), that has a
wide range of medical uses that have been demonstrated in clinical
trials. The stalks of the~\emph{Cannabis} plant can be processed into
textiles, paper and building materials. The roots of the hemp plant have
been used in traditional herbal remedies. The seeds can be processed for
biofuel or pressed for oil for human consumption. Furthermore, the seed
cake remaining after oil is pressed is protein-rich and can be used as
animal feed. See also Figure 9 for photos of hemp products.~
{\label{988932}}%
}}
\end{center}
\end{figure}

\emph{Cannabis} is probably best known for one secondary compound, the
psychotropic substance tetrahydrocannabinol (THC). Depending on the THC
content of the plant or more specifically the dried inflorescence,
\emph{Cannabis} is either classified as marijuana (or drug-type, plants
above 0.3\% THC) or hemp (fibre-type, below 0.3\% THC), which is mainly
a legal and not a strict taxonomic classification. A more refined
classification of \emph{Cannabis} according to the phytocannabinoid
profile into distinct 'chemotypes' can also be useful, with chemotype I
and II being marijuana while chemotypes III, IV and V can be seen as
hemp (see chapter 3).

Many countries have been easing the ban on medical and even recreational
use of THC during the past decade. However, because of the prohibition
of \emph{Cannabis} in many countries throughout the last century, it was
not bred to the same extent as other high-value crops. Hence, hemp and
marijuana lines retain a high level of genetic variability and
heterozygosity, that is not found in other crops (Sawler et al., 2015).

Here, we review the biology as well as the applications and future
perspectives of \emph{Cannabis} research and breeding. We discuss
\emph{Cannabis} taxonomy and cannabinoid synthesis as well as flower
development and flowering time control with an emphasis on sex
determination in this predominantly dioecious species. We also summarize
the currently available genomics resources. Since \emph{Cannabis} is so
versatile, we discuss its applications in medicine as well as in the
building industry. \emph{Cannabis} ' future role in a sustainable
society is summarized as well as the future of cannabinoid production
via cell suspension cultures.

\section*{\texorpdfstring{2. \emph{Cannabis}
systematics}{2. Cannabis systematics}}

{\label{121016}}

\emph{Cannabis} is the botanical name of a genus that historically
includes three species, \emph{C. sativa}, \emph{C. ruderalis}
and\emph{C. indica}. However, since the three species can intercross,
they are also often considered one single species, \emph{C. sativa}
(Small, 2015)\emph{.} Recent genetic data support the single-species
concept and recommend that three subspecies should be recognized:
\emph{Cannabis sativa} subsp. \emph{sativa}, subsp. \emph{indica} and
subsp. \emph{ruderalis} (Q. Zhang et al., 2018).

\emph{Cannabis} is a dioecious species, meaning there are male and
female individuals (Figure 2a-c). However, through breeding, monoecious
lines with male and female flowers on the same plant have also been
generated (Figure 2d) (Moliterni et al., 2004).

\par\null\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=0.98\columnwidth]{figures/Figure2/Figure2}
\caption{{\textbf{Hemp varieties of} \emph{\textbf{Cannabis sativa}}\textbf{.}
\emph{Cannabis} plants of the hemp cultivar `Finola' growing in the
field (a). The cultivar is dioecious with female (b) and male
individuals (c). Monoecious plants of the cultivar `Felina 32' show male
flowers and female flowers in one individual plant (d).
{\label{614886}}%
}}
\end{center}
\end{figure}

The genus \emph{Cannabis} is part of the Cannabaceae, a small family of
flowering plants with 10 genera and some 120 species (Jin et al., 2020;
Yang et al., 2013). The Cannabaceae have been estimated to have
originated ca. 70 to 90 million years ago, and are distributed in
temperate and tropical regions throughout the world (Figure 3) (Jin et
al., 2020; Magall\selectlanguage{ngerman}ón et al., 2015). Most species of the Cannabaceae are
trees or shrubs, \emph{Cannabis} as a herb is, therefore, the exception
rather than the rule in the family. However, a trait \emph{Cannabis}
shares with many other species in the family is the inconspicuous
unisexual flowers (Yang et al., 2013).

The closest relative of \emph{Cannabis} is the genus \emph{Humulus}(Yang
et al., 2013), which consists of three species, among which
\emph{Humulus lupulus}(hop) is economically important for the beer
brewing industry. Both hop and \emph{Cannabis} produce separate male and
female flowers, and the trichomes in the female inflorescences are the
site of secondary compound production that make both of those plants
economically valuable (Page and Nagel, 2006).

Within the angiosperm phylogeny, Cannabaceae are most closely related to
the Moraceae (mulberry or fig family) and Urticaceae (nettle family).
Together with the Ulmaceae (elms and relatives) they form a group known
as the urticalean rosids (Figure 3) (Sytsma et al., 2002). It is
interesting to note that unisexual flowers appear to be prevalent in the
urticalean rosids, whereas bisexual flowers are by far the dominant
system in angiosperms in general (Renner, 2014; Sytsma et al., 2002).
The evolution of sex expression and sex determination in this group is
an interesting area of future research.

\par\null\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=0.98\columnwidth]{figures/Figure3-revised/Figure3-revised}
\caption{{\textbf{Phylogenetic position
of~}\emph{\textbf{Cannabis}}\textbf{~}\emph{\textbf{sativa}}\textbf{.}~\emph{Cannabis}
belongs to the Cannabaceae, which belong to the order Rosales. Some
selected species and their phylogenetic relationship to~\emph{Cannabis}
are depicted. Taxonomic groups shaded in blue are inclusive of the
taxonomic groups shaded in yellow (e.g. Rosales belong to Fabids, which
are eudicots). The timescale at the bottom can be used to infer
approximate divergence times.
{\label{150391}}%
}}
\end{center}
\end{figure}

The urticalean rosids belong to the order Rosales, which are eudicots
(The Angiosperm Phylogeny Group, 2016). Though the Rosales comprise some
7700 species (Zhang et al., 2011), they contain relatively few
well-characterized model plants. The flowering plant super-models
\emph{Arabidopsis thaliana} (thale cress, Brassicales) and \emph{Oryza
sativa} (rice, monocots) are only distantly related to \emph{Cannabis},
the lineages leading to \emph{Arabidopsis} and \emph{Cannabis} separated
some 120 million years ago, those leading to rice and \emph{Cannabis}
some 130 to 140 million years ago (Figure 3) (Magall\selectlanguage{ngerman}ón et al., 2015).
Among the relatively well-characterized plants that are more closely
related to \emph{Cannabis} are many Rosaceae species (rose family,
apple, peach and relatives), for which several well assembled and
annotated genomes exist (Aranzana et al., 2019; Zhang et al., 2019), the
Cucurbitaceae (cucumber, pumpkin and relatives), which serve as an
important model for sex determination and sex expression (Li et al.,
2019; Schilling et al., 2020a; Zheng et al., 2019) and Fabaceae (bean
family) for flowering time regulation (Cao et al., 2017; Schmutz et al.,
2010) .

\emph{Cannabis sativa} itself is phenotypically extremely diverse.
\emph{Cannabis} plants vary in numerous traits including height, leaf
shape, photoperiod response, tetrahydrocannabinol (THC) and cannabidiol
(CBD) content, plant architecture and sex expression (Clarke and Merlin,
2016; Grassi and McPartland, 2017; Raman et al., 2017; Schilling et al.,
2020b). The dioecy of many \emph{Cannabis} lines and thus the relatively
high levels of heterozygosity further contribute to the fact that even
within one cultivar the phenotypic diversity can be substantial (our
unpublished observations).

For breeders and farmers, the high level of genetic and phenotypic
diversity can be problematic, as a crop is usually best to handle when
it possesses a high degree of uniformity in the field. However, at the
same time, the existing diversity can be harnessed by breeders to
produce new lines for a multitude of different purposes. For plant
genetics research, the phenotypic and genetic diversity is a gold mine,
as it provides the possibility to study the genetic basis of many traits
in \emph{Cannabis} . Some developments in this arena are outlined in the
subsequent chapters, but many more are sure to come.

\section*{3. Evermore complex: The genetics of phytocannabinoid
biosynthesis}

{\label{216433}}

One of the commercially most interesting and valuable products that can
be generated from \emph{Cannabis} plants are phytocannabinoids. We use
the term phytocannabinoids here for plant-derived cannabinoids, and to
distinguish them from synthetic cannabinoids or those produced by the
human endocannabinoid system. Phytocannabinoids are of great interest
for medical applications (see chapter 8 for a detailed discussion) as
well as commercial exploitations for recreational use. Hence, one of the
major breeding goals involves the accurate prediction and targeted
manipulation of phytocannabinoid profiles to ensure the optimal
combination of active components in plant extracts (see entourage effect
chapter 8) or legal compliance for non-psychoactive products.

While there are over 100 different phytocannabinoids described (Pertwee,
2014), three phytocannabinoids are usually at the centre of attention
from a medical and commercial perspective: cannabigerol (CBG),
cannabidiol (CBD) and tetrahydrocannabinol acid (THC) (Figure 4).
\emph{Cannabis itself} synthesizes phytocannabinoids in the carboxylated
form with a carboxylic acid group, i.e. as CBGA, CBDA and THCA. However,
to be active in the human endocannabinoid system, phytocannabinoids need
to be consumed in their decarboxylated forms, which are usually
generated by high-temperature treatment (for example during smoking)
(Moreno-Sanz, 2016). Phytocannabinoids are predominantly produced in
female inflorescences, more precisely they are secreted from trichomes
of perigonal bracts, subtending flowers, and leaves (`sugar leaves')
within inflorescences. However, in lower concentrations,
phytocannabinoids can also be detected in vegetative leaves at certain
times during the growth period (Aizpurua-Olaizola et al., 2016).

\par\null\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure4/Figure4}
\caption{{\textbf{Phytocannabinoids, synthases, genotypes and chemotypes of
\emph{Cannabis}.} Phytocannabinoids are synthesised via a multi-step
pathway involving different enzymes. The precursor cannabigerolic acid
(CBGA) is first synthesised by a prenyltransferase from the precursor
molecules geranyl pyrophosphate (GPP) and olivetolic acid (OA). CBGA is
metabolised into tetrahydrocannabinolic acid (THCA) via THCA synthase,
into cannabidiolic acid (CBDA) via CBDA synthase or cannabichromenic
acid (CBCA) via CBCA synthase. The different synthases are encoded by
the B\textsubscript{T} (encoding for an active THCA synthase) and BD
(encoding for an active CBDA synthase) loci.
B\textsubscript{T}/B\textsubscript{T} plants produce mainly THCA
(chemotype I), while B\textsubscript{D}/B\textsubscript{D} plants
produce predominantly CBDA (chemotype III). Presence of
B\textsubscript{T} and B\textsubscript{D} results in chemotype II (THCA
and CBDA intermediate). B0indicates that only non-functional THCA and
CBDA synthases are present, which results in the accumulation of CBGA
(chemotype IV). \emph{Cannabis} varieties with very low overall levels
of cannabinoids are categorized chemotype V, which is caused by a
homozygous recessive allele of locus O. To complicate matters further,
there is also a locus C, which is encoding for CBCA synthase. However,
in almost all varieties, CBCA is only produced in young immature
flowers. Chemotypes I and II can be considered marijuana, while the
other low-THC chemotypes can be considered hemp varieties of
\emph{Cannabis}.
{\label{968798}}%
}}
\end{center}
\end{figure}

Among all phytocannabinoids, THC is the major psychotropic one. However,
chemically all molecules mentioned above are very similar in structure
and are produced from the same precursor molecules (Figure 4). CBDA and
THCA are biochemically synthesized by two closely related enzymes, CBDA
and THCA synthase (Shoyama et al., 2012; Taura et al., 1996). CBDA and
THCA are both synthesized from CBGA, while CBGA is synthesized from two
non-cannabinoids, olivetolic acid and geranyl pyrophosphate by a
prenyltransferase (Fellermeier and Zenk, 1998)(Figure 4).
Cannabichromenic acid (CBCA) synthase converts CBGA to CBCA (Morimoto et
al., 1997) and is closely related to THCA and CBDA synthase (Figure 5),
but the CBCA content of most mature \emph{Cannabis} flowers is low (de
Meijer et al., 2009a). Interestingly, CBDA synthase-like genes have been
found in other plants and fungi (Aryal et al., 2019; Vergara et al.,
2019).

\emph{Cannabis} plants can have very high levels of phytocannabinoids or
close to no phytocannabinoids at all, or anything in between
(Aizpurua-Olaizola et al., 2016; de Meijer et al., 2009a). This has
stipulated the description of different chemotypes that are
characterized by their distinct phytocannabinoid profiles. The
chemotypes are a very useful concept for chemical classifications and
for breeding programmes. It should be kept in mind, however, that they
do not necessarily constitute a phylogenetic classification based on
evolutionary relationships (de Meijer et al., 2009b; Small and
Beckstead, 1973). \emph{Cannabis} plants can roughly be categorized into
five different `chemotypes' (Figure 4). Plants of chemotype I (short
`type I') produce high levels of THCA and only low levels of CBDA and
CBGA (Small and Beckstead, 1973). This means the ratio of THCA/CBDA is
much larger than 1. In type II \emph{Cannabis} plants THCA and CBDA are
both produced in approximately equal amounts (Small and Beckstead,
1973). Both, type I and type II plants, are usually classified as
`marijuana' and can underlie strong regulations, depending on the
country or jurisdiction. These plants are bred to produce up to 20 \% of
their dry mass as phytocannabinoids.

In contrast, type III plants have high CBDA levels and low to very low
amounts of THCA.

Chemotype IV and V refer to \emph{Cannabis} plants which have CBGA as
their dominant phytocannabinoid or very low levels of phytocannabinoids
overall, respectively (de Meijer et al., 2009a; de Meijer and Hammond,
2005)(Figure 4).

In addition to the five different chemotypes, also the hemp-marijuana
distinction is used to characterize different \emph{Cannabis} plants
(Figure 4). If the THC/THCA content in the dry flower mass is below
0.2-1 \%, these plants are usually categorized as hemp, above that as
marijuana (depending on the jurisdiction this threshold can
vary)~(Brunetti et al., 2020; Mead, 2017). The differentiation between
hemp and marijuana can typically also be drawn genetically, with hemp
and marijuana varieties forming two genetically distinct populations
(Sawler et al., 2015). Further, hemp and marijuana can be phenotypically
quite distinct with marijuana plants generally being bushier and with a
dense set of inflorescences while hemp plants tend to be taller, less
branched and with less dense flower structures. However, there are also
plants with low THC/THCA content (type III) which strongly resemble
marijuana in overall plant and inflorescence architecture (Grassa et
al., 2018). Hence, the terms hemp and marijuana do not necessarily
always refer to distinct genetic populations or phylogenetic categories.
As the critical distinction between hemp and marijuana is the THC/THCA
content, they can also be considered broader categories of chemotypes.

The underlying genetics of the different chemotypes have been studied in
quite some detail in the last two decades (de Meijer et al., 2009a,
2009b, 2003; de Meijer and Hammond, 2005; Pacifico et al., 2006; Toth et
al., 2020; Weiblen et al., 2015; Welling et al., 2016). However, the
complex nature of the \emph{Cannabis} genome with its many transposable
elements, low complexity regions and high heterozygosity have made a
conclusive analysis of the loci controlling phytocannabinoid production
challenging (Grassa et al., 2018; Laverty et al., 2019; McKernan et al.,
2018).

Different genetic loci had been postulated which determine a plant's
chemotype, they are encoding for the different types of synthases: at
locus B two codominant alleles were hypothesized to exist, the allele
B\textsubscript{T} encodes for the THCA synthase, B\textsubscript{D} for
the CBDA synthase (Figure 4)(de Meijer et al., 2003). Depending on the
presence of either or both loci, the plant will be chemotype I
(B\textsubscript{T}/B\textsubscript{T}), chemotype II
(B\textsubscript{T}/B\textsubscript{D}) or chemotype III
(B\textsubscript{D}/B\textsubscript{D}) (de Meijer et al., 2003; Toth et
al., 2020; Welling et al., 2016). Additionally, non-functional alleles
of the synthase gene (B\textsubscript{0}) are predicted to be associated
with chemotype IV, where neither CBDA nor THCA are produced and the
precursor, CBGA, accumulates (Figure 4) (de Meijer and Hammond, 2005;
Onofri et al., 2015; Welling et al., 2016).

Further, according to this model, CBCA synthase is encoded by an
independent locus (C) while another independent locus (O) is relevant
for precursor production, with a knockout resulting in overall minimal
phytocannabinoid levels (Figure 4) (de Meijer et al., 2009a, 2009b).

The genetic basis of the chemotypes was analysed in detail by producing
a cross between high-THC Purple Kush (chemotype I) and low-THC Finola
(chemotype III). This resulted in an F1 generation of mainly type II
plants, producing both, THCA as well as CBDA (Weiblen et al., 2015).
This confirmed earlier findings of crosses between type I and type II
plants, resulting in intermediate type II individuals (de Meijer et al.,
2003). The segregation pattern of phytocannabinoid profiles in the F2
generation pointed towards a Mendelian inheritance pattern: type I, type
II and type III plants were all observed in the F2 generation with the
expected distribution of 1:2:1 (de Meijer et al., 2003; Weiblen et al.,
2015). A correlation of the expression of either THCA or CDBA synthase
with the respective chemotype was also observed and the THCAS/CBDAS
locus could be mapped (Weiblen et al., 2015).

However, although these findings were consistent with the idea of
codominant alleles at one single locus, it became apparent that the
situation is more complex (Grassa et al., 2018; Laverty et al., 2019;
Weiblen et al., 2015). New draft genomes generated with third-generation
sequencing technology indicated that the THCA and CBDA synthases do not
seem to be encoded by alleles of one and the same gene, but rather by
distinct loci in marijuana and hemp, respectively, without a clear
counterpart in the other genome (Grassa et al., 2018; Laverty et al.,
2019). Sequencing of the hemp cultivar `Finola' and the marijuana
cultivar `Purple Kush' indicates that a functional CBDA synthase gene is
present only in in the `Finola' genome while the `Purple Kush' genome
only encodes for a functional THCA synthase (Laverty et al., 2019).
While mapping to approximately the same region in both genomes, the DNA
sequences surrounding the respective synthase genes are drastically
different from each other. Further, a low albeit still detectable
recombination rate between the two loci supports the notion that they
are genetically distinct (Laverty et al., 2019). The sequencing of a
different \emph{Cannabis} variety (`CBDRx'), which is a chemotype III
hemp-marijuana hybrid revealed an even more complex genomic arrangement
with a number of pseudo- and functional synthase genes in three
different cassettes on the same chromosome (Figure 5) (Grassa et al.,
2018).

The CBDA and THCA synthase genes themselves seem to be embedded in
cassettes of multiple tandem duplications of putatively non-functional
synthase genes, which are regularly interspersed with long terminal
repeat (LTR) retrotransposons, making the assembly and analysis of these
loci even more challenging (Figure 5) (Grassa et al., 2018; Laverty et
al., 2019). This is also the reason why these complex loci could not be
resolved in the first published \emph{Cannabis} genome, which relied on
short-read sequencing data (van Bakel et al., 2011). This genomic
constitution, where the difference between marijuana and hemp comes down
to a large structural variation is, if true, very unusual. Hence, the
aforementioned locus ``B'' with its different alleles might look very
different from what was previously assumed to be simple isoforms of a
single gene.

\par\null\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=0.98\columnwidth]{figures/Figure5/Figure5}
\caption{{\textbf{Genetic location of genes encoding cannabinoid synthases
in~}\emph{\textbf{Cannabis}}\textbf{.} Preliminary analysis of location
and structure of the genes encoding CBCA synthase, CBDA synthase and
multiple cannabinoid-synthase-like pseudogene copies in the
hemp-marijuana hybrid cultivar `CBDRx' (Grassa et al., 2018), which is
the reference genome for \emph{Cannabis sativa}. The genome has 9 pairs
of autosomes and one pair of sex chromosomes, with the main locus of
cannabinoid synthases located on chromosome 7 (green box, revised
chromosome numbering (NCBI)) (a). Three different cassettes (1, 2 and 3,
yellow regions with stripes) have been identified and mapped to a region
on chromosome 7 between 24.5 and 31.5 Mb (b). The exact chromosomal
arrangement is not clear, since the assembly contains gaps in between
the different cassettes (grey boxes). Three cannabinoid synthase genes
appear to have a full coding sequence: a CBCA synthase, the CBDA
synthase and a CBDA-like synthase (*, black), while the other copies
appear non-functional (grey). All sequences, including pseudogenes, have
unique expression data associated with them (arrows, NBCI genome
browser, unique raw reads). The synthases are encoded by one single exon
(black) and surrounded by long terminal repeat retrotransposons
(orange). Functional CBDA and THCA synthase share 83.86 \% protein
sequence similarity, while similarity amongst the other sequences ranges
from 82 to 92 \% (c). The CBDRx genome does not contain a functional
THCAS, the sequence was acquired from uniprot (Q8GTB6). Gene density in
(a) shows the ratio of the number of genes per million base pairs,
calculated and plotted as an ideogram using RIdeogram (Hao et al.,
2020). Gene annotation for the Y chromosome is not readily available,
but genes are present (Prentout et al., 2020; McKernan et al., 2020).
The CBDRx genome was derived from a female individual (Grassa et al.,
2018), the size of the Y (*) chromosome was approximated from McKernan
et al., 2020.
{\label{165049}}%
}}
\end{center}
\end{figure}

The complexity of phytocannabinoid synthases does not end there, though.
Copy number variation of CBDA and THCA synthase genes might be involved
in phytocannabinoid level and composition (Vergara et al., 2019) and
most likely, the number of synthase (pseudo)genes might be different for
each cultivar sequenced (Grassa et al., 2018; Laverty et al., 2019;
McKernan et al., 2020).

High throughput assays for B\textsubscript{T} and
B\textsubscript{D}markers have been developed and show that many plants
actually contain both loci (Cascini et al., 2019; McKernan et al., 2020;
Toth et al., 2020). Moreover, many B\textsubscript{D}/B\textsubscript{D}
plants, especially those with higher CBDA levels, have THCA levels of
above 0.3 \% of dry flower mass, despite the absence of a functional
B\textsubscript{T} allele (Toth et al., 2020). This residual THCA is
probably at least to some extent a by-product of the CBDA synthase
itself. The THCA and CBDA synthase have a relatively high sequence
similarity (83.85 \%, Figure 5) and process the same precursor molecule,
CBGA (Figure 4). \emph{In vitro} studies have shown that the CBDA
synthase produced CBDA and THCA at roughly a ratio of 20:1 (Zirpel et
al., 2018). This is similar to ratios observed \emph{in planta} in
high-CBD hemp varieties as well (Toth et al., 2020; Weiblen et al.,
2015). This potentially results in the problem that, if CBDA production
is increased, THCA also increases as a by-product, even if plants do not
express a functional THCA synthase. \emph{Cannabis} varieties with very
high CBD levels may thus be at risk of exceeding legal THC thresholds.

Understanding the exact genetics underlying the different chemotypes
will be important for future targeted breeding approaches. Tight
restrictions across the world make it difficult for farmers to grow
chemotype III, IV and V varieties, because the presence of residual THC
creates regulatory problems and uncertainties. Especially type III
plants often have THCA/THC levels slightly above the legal THC limit
(Aizpurua-Olaizola et al., 2016; Toth et al., 2020). Hence, one
important breeding goal is going to be the generation of zero-THC lines
which still produce high levels of CBD in the range of 15 to 20 \% of
dry flower mass. Whether this is possible to achieve is difficult to
say, since even in the absence of a THCA synthase, CBDA synthases
produce THCA as a by-product (Toth et al., 2020; Zirpel et al., 2018).
This will, therefore, require identification of a CBDA synthase that
does produce only very low or no amounts of THCA.\emph{In vitro}
experiments show that point mutations can alter the amount of
by-products (Zirpel et al., 2018). Natural variation in synthase genes
exists and have been linked to altered phytocannabinoid compositions
(Onofri et al., 2015). Hence, naturally occurring or artificially
generated CBDA synthase varieties could be used for targeted breeding in
this direction.

In addition, \emph{Cannabis} varieties used for fibre or seed production
could be selectively bred and genotyped to have 0 \% overall
phytocannabinoids (chemotype V), as currently even the farming of these
kinds of varieties is heavily restricted in many countries.

Other phytocannabinoids like CBG(A) and CBC(A) as well as the manifold
variants of terpenes produced in \emph{Cannabis} flowers are
increasingly coming into focus in the medical research fields (reviewed
in Booth and Bohlmann, 2019; Deiana, 2017; Pollastro et al., 2018),
hence generating lines with specific phytocannabinoid profiles might be
of interest in further research.

\section*{\texorpdfstring{4. A hairy topic: Flower development and
morphology in
\emph{Cannabis}}{4. A hairy topic: Flower development and morphology in Cannabis}}

{\label{228988}}

The flower is the reproductive structure of flowering plants
(angiosperms), which represent one of the most successful and diverse
groups of organisms on this planet (Krizek and Fletcher, 2005). While
the characteristic shape of the \emph{Cannabis} leaf is often used as a
symbol for the whole plant, \emph{Cannabis} female flowers are of
particular interest because they are the main site of production of
pharmacologically active compounds (phytocannabinoids) (Spitzer-Rimon et
al., 2019). Understanding the morphology of \emph{Cannabis} flowers and
their developmental genetics is therefore especially important.

The typical angiosperm flower consists of four different organ types,
which are organized in concentric whorls: sepals, petals, stamens and
carpels (Endress, 1992; Krizek and Fletcher, 2005). Sepals are in the
outermost whorl and usually green and leaflike in appearance. Petals are
in the second whorl and often coloured to attract pollinators. Petals
together with sepals are termed the perianth and constitute the
non-reproductive part of a flower. Stamens are typically located in the
third floral whorl. They are the male reproductive organs and are
composed of an anther and a filament. The anthers grow on top of the
stalk-like filaments and are the site of pollen production. Finally,
carpels develop in the fourth and central whorl of a typical flower.
Carpels are the reproductive organs that contain an ovary inside which
ovules develop. The tip of the carpel, the stigma, receives the pollen.
The style connects the stigma to the ovary (Becker, 2020; Endress, 1992;
Krizek and Fletcher, 2005).

Notably, the number, arrangement, and morphology of the floral organs
varies substantially between different species of flowering plants
(Endress, 2011; Theissen and Melzer, 2007). Most flowers contain, as
described above, both carpels and stamens, and are therefore termed
bisexual flowers (Renner, 2014). However some 15 \% of flowering plant
species are monoecious or dioecious and have unisexual flowers that
develop only stamens or carpels (Renner, 2014). In dioecious plants,
female and male flowers develop on separate individuals. In contrast, in
monoecious plants male and female flowers develop on the same individual
(Renner, 2014).

\par\null\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=0.56\columnwidth]{figures/Figure6/Figure6}
\caption{{\textbf{Mature flowers of~\emph{Cannabis sativa}.} Male~\emph{Cannabis}
flowers have sepals and stamens (a) while female~\emph{Cannabis} flowers
consist of two carpels enclosed by a perigonal bract (c). Schematic
depiction of the structure of~\emph{Cannabis} male flower (b) and female
flower (d). The bar = 1 mm. an, anther; b, perigonal bract; fi,
filament; s, sepal; ov, ovary; p., perianth; stg, stigma; sty, style.
{\label{621138}}%
}}
\end{center}
\end{figure}

\emph{Cannabis} is primarily dioecious (Moliterni et al., 2004). The
male \emph{Cannabis} flower is green-yellow in appearance and has a
perianth of five sepals, while petals are completely absent. Further, an
individual male flower contains five free stamens, and no female
reproductive organs (Figure 6a and b) (Leme et al., 2020; Spitzer-Rimon
et al., 2019).

On the other hand, the female flower is enclosed within a green leaflike
perigonal bract. The perigonal bract is sometimes also described as a
sepal, but morphological studies agree that it is a bract (Leme et al.,
2020; Spitzer-Rimon et al., 2019). As such, it is not strictly a part of
the flower. Between the perigonal bract and the carpel is a membranous
and hyaline perianth which tightly embraces the ovary (Leme et al.,
2020; Reed, 1914; Spitzer-Rimon et al., 2019). It is worth noting that
this inconspicuous perianth sometimes is not mentioned in the structure
of female \emph{Cannabis} flowers or is considered missing as it is not
visible from the outside of the flower\textbf{.} Most likely, these
membranous structures are homologous to sepals (Leme et al., 2020). At
the top of the ovary are two filamentous styles. The stigma is
brush-like and has epidermal cells elongated into hair-like projections
(Reed, 1914; Leme\emph{et al.}, 2020) (Figure 6c and d).

The commercially interesting phytocannabinoids and terpenes are
predominantly produced on the perigonal bracts of female flowers, more
specifically in glandular trichomes that cover those bracts. Glandular
trichomes can be categorized into sessile, stalked and bulbous trichomes
(Hammond and Mahlberg, 1973), with bulbous trichomes being metabolically
less active (Livingston et al., 2020). \emph{Cannabis} plants also have
non-glandular trichomes: hair-like uni- or multicellular trichomes which
protect them from biotic and abiotic stresses (Andre et al., 2016;
Dayanandan and Kaufman, 1976). However, glandular trichomes are the main
site of phytocannabinoid synthesis (Furr and Mahlberg, 1981).

Because phytocannabinoids are cytotoxic in higher concentrations, they
have to be secreted and are not stored within cellular compartments.
Phytocannabinoids along with other secondary metabolites are secreted
from glandular trichomes with a globose head-like structure (Figure 7).
This head is formed by an enlarged secretory cavity which is surrounded
by a culticule that encapsulates the secreted secondary metabolites
(Hammond and Mahlberg, 1973). At the base of the head is a layer of
secretory cells (Kim and Mahlberg, 1991; Livingston et al., 2020). The
head can be sessile, directly on the epidermis and often be found on
vegetative leaves (sessile trichomes), or pre-stalked or stalked with
the head being elevated above the epidermis (pre-stalked and stalked
trichomes), which are mainly found on female inflorescences (Kim and
Mahlberg, 1991; Livingston et al., 2020). Additionally, these structures
can be distinguished by different levels of autofluorescence, cell
numbers as well as phytocannabinoid and terpene profiles (Livingston et
al., 2020; Turner et al., 1978). Stalked trichomes seem to be developing
from pre-stalked trichomes and contain a terpene profile distinct from
true sessile trichomes (Livingston et al., 2020). Transcriptome analysis
of floral trichomes of a CBD hemp (`Finola') confirmed high expression
levels of genes involved in the synthesis of phytocannabinoids, terpenes
and their respective precursor molecules in glandular trichomes, with
expression differences between bulbous, sessile, and (pre-)stalked
trichomes (Livingston et al., 2020).

\par\null\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=0.98\columnwidth]{figures/Figure7/Figure7}
\caption{{\textbf{Different types of trichomes on} \emph{\textbf{Cannabis sativa}}
\textbf{hemp varieties.} Trichomes can be found on the majority of
female \emph{Cannabis} plant epidermises of the cultivars `Finola' (a-f)
and `Felina 32' (g, h). Vegetative leaves (a) have mainly non-glandular
hair-like trichomes (b). Subtending (``sugar'') leaves within an
inflorescence (c) do have both, non-glandular trichomes as well as
glandular trichomes (d). The perigonal bract of a female `Finola' flower
(e) is covered in glandular stalked trichomes (f), while the majority of
trichomes found on the bract of `Felina 32' flowers (g) are
non-glandular and sessile trichomes (h). White arrows: non-glandular
trichomes; white arrowheads: sessile trichomes; red arrowheads: stalked
trichomes.
{\label{420694}}%
}}
\end{center}
\end{figure}

It is not clear why predominantly female plants produce glandular
trichomes within their inflorescence structures. Illuminating the
genetic underpinnings of this sexual dimorphism remains a challenge for
further research. Glandular trichomes also develop on male flowers (Leme
et al., 2020), albeit at lower density and probably with less
phytocannabinoids. Understanding which genetic factors restrict the
development of glandular trichomes largely to female inflorescences
during flower development would provide a valuable resource for an
increase of phytocannabinoid production.

\section*{\texorpdfstring{5. The battle of the sexes: Sex determination
in
\emph{Cannabis}}{5. The battle of the sexes: Sex determination in Cannabis}}

{\label{981852}}

\subsection*{5.1. The genetics of sex
determination}

{\label{154980}}

The dioecy of \emph{Cannabis} is genetically controlled (Figure 2). Hemp
is diploid (2n = 20), with nine pairs of autosomes and one pair of sex
chromosomes. Female plants are homogametic with XX chromosomes and male
plants are heterogametic with an XY sex chromosome pair (Moliterni et
al., 2004). \emph{Cannabis} thus represents a rare case among the
flowering plants in which sex chromosomes have been identified
(Charlesworth, 2016).

The diploid genome size of female \emph{Cannabis} plants is estimated to
be 1636 Mbp, that of a male plant 1683 Mbp by flow cytometry (Sakamoto
et al., 1998). The sex chromosomes of \emph{Cannabis} are the largest in
the chromosomal complement, they are estimated to comprise 6.5 \% (Y
chromosome) and 6.1 \% (X chromosome) of the total length of the genome
(Divashuk et al., 2014). Assuming that those estimates roughly
correspond to length in base pairs (which is most certainly an
oversimplification), the X chromosome would be 102.7 Mbp in size, and
the Y chromosome 109.4 Mbp. This is close to the size of the X and Y
chromosomes as determined by genome sequencing (Figure 5, Supplementary
Table 1). However, the flow cytometry results mentioned above (Sakamoto
et al., 1998) indicate that the Y chromosome is 47 Mb larger than the X
chromosome (1683 Mb - 1636 Mb = 47 Mb). Flow cytometry analyses of
other\emph{Cannabis} varieties yielded very similar results (Faux et
al., 2014). It is not clear where the discrepancy between genome
sequencing and flow cytometry measurements is coming from. Structural
genomic variations between different \emph{Cannabis} plants as well as
problems in assembling the sex chromosomes may both play a role.

Detailed analyses of the sex chromosomes revealed that the
pseudo-autosomal region on the X chromosome (i.e. the region still
recombining with the Y chromosome) is ca. 30 Mb in size, whereas the
X-specific region (which is not recombining with the Y chromosome) is
ca. 75 Mbp in size (Prentout et al., 2020). Prentout \emph{et al.} also
identified ca. 500 sex-linked genes, i.e. alleles that are inherited in
a sex-linked fashion (e.g. only from father to daughter, not from father
to son for X-hemizygous alleles). It will be especially interesting to
analyse the X chromosome alleles that have no Y chromosome counterpart
in detail in the future, as they may contribute to sex determination.

Whereas gene density on the X chromosome appears to be similar to
autosomes, about 70 \% of the genes on the Y chromosomes have been
estimated to be lost (Prentout et al., 2020). One explanation for the
relatively large size of the Y chromosome despite substantial gene
losses seems to be the accumulation of transposons and other repetitive
elements (Sakamoto et al., 2000). Prevalence of transposable elements on
the Y chromosome might also help to explain difficulties in chromosome
assembly and discrepancies in size estimates.

Despite progress in identification and sequencing the sex chromosomes of
\emph{Cannabis} , not much is known about the molecular circuits
involved in sex determination. Some confusion exists as to what the
genetic `mode' of sex determination is (Kovalchuk et al., 2020; Vergara
et al., 2016). Some reports suggest the mode is similar to humans and
other mammals, where the presence or absence of the Y chromosome
determines the sex: humans carrying a Y chromosome are almost always
phenotypically male, those without a Y chromosome are female, with
autosomes or extra X chromosomes bearing little consequence on the sex
determination (Gamble and Zarkower, 2012; Sakamoto et al., 1998). An
alternative view is that the X chromosome to autosome ratio determines
the sex (Westergaard, 1958). This would be somehow similar to
\emph{Drosophila}, where the number of X chromosomes determines the sex,
with the presence or absence of the Y chromosome having limited
relevance (Gamble and Zarkower, 2012). In this model, the Y chromosome
essentially becomes a `placeholder', lowering the number of X
chromosomes.

Experimental evidence supporting the one or the other mode of sex
determination is surprisingly scarce. Warmke and Davidson used
colchicine to produce tetraploid \emph{Cannabis} plants (Warmke and
Davidson, 1944). Strikingly, a cross between a tetraploid female and a
diploid male plant yielded female and female-hermaphrodites, but no male
plants, though half of the progeny should have an XXY chromosome
constitution (Warmke and Davidson, 1944). This is evidence that the Y
chromosome does not play a role as prominent as in humans in sex
determination in \emph{Cannabis}, but that an X to autosome ratio model
for sex determination might apply.

It is important to note that, even if an X to autosome ratio model
applies in \emph{Cannabis}, this does not mean that the Y chromosome is
dispensable for the development of male plants. In \emph{Drosophila},
the Y chromosome is not involved in sex determination but encodes genes
required for male fertility, such that XO flies are phenotypically male
but sterile (Gamble and Zarkower, 2012). Likewise, Y chromosome specific
genes in \emph{Cannabis} may well play a critical role in male plant
development, even if not involved in \emph{bona fide} sex determination
(McKernan et al., 2020).

\subsection*{5.2. Monoecy, dioecy and evolutionary
considerations}

{\label{273412}}

To add further complications to the study of sex determination
in\emph{Cannabis} , monecious varieties exist, in addition to the
canonical dioecious varieties. Monoecious cultivars develop male and
female flowers on the same plant and are particularly popular for fibre
production (Figure 2). This is because in dioecious varieties, male
plants flower earlier than female plants, whereas for monoecious
cultivars flowering time is more synchronized, thus facilitating the
determination of an optimal harvest time (Faux et al., 2016).

Genetically, monoecious cultivars carry two X chromosomes and no Y
chromosome, indicating that the XY sex determination system can be
`leaky' in \emph{Cannabis}(Faux et al., 2014; Razumova et al., 2016). In
addition, the monoecious cultivars express `femaleness' and `maleness'
to different degrees, i.e. the ratio of female to male flowers one plant
develops differs between cultivars but also between different
environmental conditions (Faux et al., 2014). At least some genetic loci
relevant for the sex expression in monoecious\emph{Cannabis} plants seem
to be located on the X chromosome (Faux et al., 2016). It will be
interesting to see whether the distinction between dioecious and
monoecious cultivars and between different degrees of sex expression
(femaleness or maleness) in monoecious cultivars can be traced back to
the same molecular circuits.

From an evolutionary point of view, the sexual system in the entire
family of Cannabaceae is complex. In contrast to angiosperms in general,
some 85 \% of which are bisexual (Renner, 2014), true bisexual flowers
are conspicuously rare in Cannabaceae (Yang et al., 2013). Several
shifts of the sex determination system occurred in the Cannabaceae, and
ancestral character state reconstructions indicate that monoecy, or,
with a lesser likelihood, dioecy, is the ancestral state in the family
(Yang et al., 2013). Interestingly, one of the closest relatives
of\emph{Cannabis sativa} , \emph{Humulus lupulus} (common hop), is
dioecious with an XY sex determination system in which the X to autosome
ratio determines the sex (Parker and Clark, 1991). This may be taken as
additional evidence that also\emph{Cannabis} has an X to autosome ratio
sex determination system. However, it should be kept in mind that that
\emph{Humulus} and\emph{Cannabis} may have separated some 25 million
years ago (Jin et al., 2020) and that sex determination systems can
frequently and rapidly change during evolution (Bachtrog et al., 2014).
Nevertheless, available data on the sex determination systems in
Cannabaceae as a whole may be taken as indication that all family
members share an ancestral molecular mechanism for sex determination,
but that this mechanism is relatively labile and manifests differently
in different species.

\subsection*{5.3. Hormonal and environmental factors affection sex
determination}

{\label{251985}}

Beyond genetic consideration, the sex expression in \emph{Cannabis} can
be shaped by environmental factors. It is well established that silver
can be used to induce the formation of male flowers on female plants
(Mohan Ram and Sett, 1982). This treatment is used by breeders to self
female dioecious plants, which results in offspring with exclusively XX
sex chromosomes (Clarke and Merlin, 2016). Because all of the progeny
from such a selfed plant will be female, the `feminized' seeds produced
that way are usually much more valuable than conventionally produced
seeds (Small, 2015). Silver is a known ethylene inhibitor, and there is
also evidence that ethylene induces the development of female flowers on
male plants (McDaniel and Binder, 2012; Ram and Jaiswal, 1970).
Together, this therefore suggests that ethylene is involved in
controlling the sex expression in\emph{Cannabis} .

In addition to ethylene, also other phytohormones have been shown to be
capable of altering the sex expression in \emph{Cannabis}(Ainsworth,
2000) . Auxin, for example, can lead to the development of female
flowers on male plants. Indeed, male flower development can be entirely
repressed by auxin treatment (Heslop-Harrison, 1956). Cytokinin also has
a feminizing effect (Chailakhyan and Timiriazev, 1979), whereas
gibberellic acid triggers the formation of male flowers on female plants
(Ram and Jaiswal, 1972). Together, the hormonal effects are very similar
to what is observed for sex expression in the well analysed
Cucurbitaceae family (Li et al., 2019; Pawe\selectlanguage{polish}ł\selectlanguage{english}kowicz et al., 2019;
Schilling et al., 2020a). Cucurbitaceae are only distantly related to
Cannabaceae, the two lineages separated more than 100 million years ago
(Figure 3) (Magall\selectlanguage{ngerman}ón et al., 2015). Also, many of the studied
Cucurbitaceae are monoecious and thus do not possess sex chromosomes
(Boualem et al., 2015). Nevertheless, we hypothesize that similar
developmental genetic pathways may have been co-opted to control sex
expression in \emph{Cannabis} and the Cucurbits. Melon, cucumber,
pumpkin, and their relatives might thus serve as an excellent model to
unravel the molecular intricacies of sex expression and sex
determination in \emph{Cannabis} .

\emph{Cannabis} is a short-day plant, and flowering is initiated if day
length is below 14 h (see below), but it is well established that sex
expression is also affected by day length. It was reported some 100
years ago already that dioecious plants grown under short day conditions
only (i.e. without an initial period of long day growth) can develop
male as well as female flowers ((Tournois 1911, 1912, as cited by
(Heslop-Harrison, 1957), (Schaffner, 1923)). A lot of other
environmental factors like nitrogen availability or carbon monoxide also
seem to have an influence on sex expression (Freeman et al., 1980;
Heslop-Harrison, 1957; Small, 2015). Together, this yields a very
complex picture of different environmental factors influencing sex
expression in different directions that is far from being completely
understood.

In summary, the picture emerges that, although sex determination is
genetic, hormonal and environmental influences have a significant effect
on sex expression. Because the flowers of female plants are the main
source of phytocannabinoids, a more detailed study of the sex
determination and sex expression mechanism of \emph{Cannabis} is one of
the main areas of future research. For example, creating male sterility
would be very beneficial, as phytocannabinoid production is highest in
unpollinated female plants. Studying both sex chromosomes, their gene
content as well as the molecular intricacies of the sex determination
mechanisms will certainly provide valuable insights for breeders as well
as researchers.

\section*{\texorpdfstring{6. Timing is everything - the complex network
of floral initiation and hints for
\emph{Cannabis}}{6. Timing is everything - the complex network of floral initiation and hints for Cannabis}}

{\label{826979}}

\subsection*{6.1. The evolutionary and developmental importance of
flowering
time}

{\label{667307}}

Flowering time control is essential for reproductive success (Shim et
al., 2017). Mechanisms to control the time of flowering have evolved
given the negative consequences of spontaneous floral initiation:
Premature flowering could coincide with the absence of pollinators or
dispersers, subsequently causing reduced fertilisation rates and
deficient seed dispersal. Conversely, if flowering occurs too late the
plant may fail to set seed before harsh conditions hit at the end of the
growing season (Gaudinier and Blackman, 2020). Moreover, in dioecious
species such as\emph{Cannabis} , the timing of flower emergence is
particularly crucial, because if male and female plants do not flower
concurrently, pollination cannot occur (Hall et al., 2012). Hence it is
evolutionarily beneficial for plants to possess mechanisms to fine-tune
their floral initiation (Gaudinier and Blackman, 2020). Analyses in
various plant species have demonstrated that flowering time in
angiosperms is controlled by internal timekeeping mechanisms as well as
environmental signals. Among the major factors controlling flowering
time are the plant age, the photoperiod, the circadian clock, ambient
temperature, the phytohormone gibberellin and the autonomous pathway
(Fornara et al., 2010; Hill and Li, 2016).

The fine-tuning of flowering time is a major goal for plant breeding and
crop improvement efforts. Floral transition represents the developmental
shift from vegetative to reproductive growth and is a major determinant
of yield potential (Jung and Müller, 2009). Alterations in key flowering
time genes have been crucial to crop domestication, facilitating the
adaptation of crops to local climatic conditions (Gaudinier and
Blackman, 2020; Schilling et al., 2018). The success and worldwide
expansion of staple crops like wheat and rice can partly be attributed
to natural variation in flowering time genes, which enabled local
adaptation for cultivation at a wide range of latitudes (Hill and Li,
2016; Langer et al., 2014). As a quantitative short-day plant,
\emph{Cannabis} flowering time is particularly determined by the
photoperiod. Under long-days \emph{Cannabis} remains vegetative and
flowering is only induced when a number of short-day photoperiods have
passed. Therefore, in order to cultivate \emph{Cannabis} at new lines of
latitude (for example in Ireland where summer daylengths can be over
17h), the adjustment of flowering time genes can be advantageous.
Consequently, comprehensive characterisation of the \emph{Cannabis}
flowering time pathways is crucial to the integration of this crop
species into modern agriculture.

\subsection*{6.2. Flowering time control in Cannabis - what we know so
far}

{\label{660327}}

\emph{Cannabis} has the potential to be a sustainable multipurpose crop.
For virtually all applications of\emph{Cannabis} , a better
understanding of the genetic factors controlling flowering time would be
highly beneficial (Figure 1). The reasons are evident when the flowers
or seeds are the main agricultural product, such as hemp oil from seeds
or CBD production from flowers. But flowering time also determines the
crop purpose in more general terms, with later flowering varieties
favouring vegetative stem growth thus suiting fibre production and
earlier varieties displaying enhanced flower/seed yield (Salentijn et
al., 2019). The interactions between flowering time and fibre quality
are complex (reviewed in Salentijn et al., 2019) and the developmental
stage at harvest has major implications for fibre quality. Additionally,
a better understanding of flowering time is important to generate
varieties that are adapted to local climatic and photoperiod conditions.

While various environmental signals including temperature (Amaducci et
al., 2012, 2008; Cosentino et al., 2012; Nelson, 1944) prompt floral
initiation, \emph{Cannabis} is particularly sensitive to changes in
photoperiod (Hall et al., 2012; Salentijn et al., 2019). In as early as
1912 it was observed that flower induction in \emph{Cannabis} is
influenced by the photoperiod (Tournois 1912 (cited by Heslop-Harrison,
1957)). \emph{Cannabis} is a facultative short-day plant (Salentijn et
al., 2019). This means that while plants will typically flower
eventually under long-day conditions, flowering occurs faster in
short-day conditions i.e. by experiencing a sequence of days each with a
minimum uninterrupted period of darkness. Cultivar-specific variation
for the photoperiod at which flowering is induced has been reported,
with the optimal photoperiod ranging from 9 to 14 h (Lisson et al., 2000
and references cited therein).

A related question is how many consecutive short days are required to
induce flowering. Borthwick and Scully (1954) reported two weeks of a
short photoperiod induced flowering in 3-5-week-old plants, and the
greater the plant age at the time of switching to short-day the faster
the floral transition. Furthermore, Potter (2014) stated flowers can be
visible one week after the reduction in day length. Clearly, more
research is needed in this area, in particular to explore the variation
between cultivars for this trait.

Given that most individuals eventually flower under non-inductive
photoperiodic conditions, a more detailed analysis of the age-related
and the autonomous pathway and their influence on flowering time is
warranted. In addition, it would be especially interesting to observe
stressors that can accelerate flowering under non-inductive
photoperiodic conditions, and detect whether the same signalling
pathways are at play when individuals eventually flower under long days
(Takeno, 2016).

\subsection*{\texorpdfstring{6.3. Model plants and candidate genes:
\emph{Arabidopsis}, soybean and the hunt for flowering time genes in
\emph{Cannabis}}{6.3. Model plants and candidate genes: Arabidopsis, soybean and the hunt for flowering time genes in Cannabis}}

{\label{399065}}

While important efforts to determine the environmental stimuli impacting
floral induction in \emph{Cannabis} have been undertaken, the genetic
pathways and loci underlying the environmental responsiveness still
require elucidation (Salentijn et al., 2019). Huge diversity exists for
flowering time in \emph{Cannabis} with phenotypes generally categorised
as early-, mid- or late-flowering (Salentijn et al., 2019). Furthermore,
photoperiod-insensitive (also known as day-neutral or auto-flowering)
cultivars exist (Small, 2018). A recent study suggests that female
floral initiation occurs independently of the photoperiod in some
\emph{Cannabis} cultivars, while in others shorter photoperiods were
required for flower maturation and development (Spitzer-Rimon et al.,
2019). Further research is required to substantiate the molecular basis
of those observations, and research on model plants may serve as an
important primer to understand the gene regulatory network controlling
flowering time in \emph{Cannabis}.

Among the model plant species for which comprehensive analyses of
flowering time have been conducted are the long-day
plant\emph{Arabidopsis thaliana} and the short-day plant \emph{Oryza
sativa}(rice). In \emph{Arabidopsis}, the complex flowering time network
is well-characterised with several pathways described including the
vernalisation, autonomous, photoperiod, circadian clock, age, ambient
temperature and gibberellin pathways (Blümel et al., 2015). One of the
key integrators of floral inductive signals in \emph{Arabidopsis} is
\emph{FLOWERING LOCUS T} (\emph{FT} ), the protein product of which is
known as florigen (Turck et al., 2008).

As mentioned previously, \emph{Cannabis} is particularly sensitive to
alterations in the photoperiod and as such the photoperiodic pathway of
\emph{Arabidopsis} warrants some more detailed discussion. The
photoperiodic flowering pathway depends on cross-talk between light
perception and the circadian clock, which coordinate to control the
expression of the main integrator \emph{FT}(Cao et al., 2017). The first
step in the photoperiodic pathway is the perception of light by the
photoreceptors (phytochromes and cryptochromes). Phytochromes exist in
inactive (Pr) and active (Pfr) forms. Pr is synthesised in the dark, and
upon red-light perception is activated to Pfr which translocates to the
nucleus. Pfr can interact with transcription factors and induce
large-scale transcriptional alterations in response to light (Legris et
al., 2019). Pfr then reverts to Pr by far-red light absorption or by
light-independent thermal reversion (Klose et al., 2020). Phytochromes
have a myriad of roles in regulating plant development and several
phytochromes exist in angiosperms. The Brassicaceae possess five
phytochromes: phyA to phyE. In \emph{Arabidopsis} phyA and phyB are
functionally the most important (Legris et al., 2019).

The photoreceptors subsequently transmit signals to the central node of
the photoperiodic pathway: the
\emph{GIGANTEA-CONSTANS-FT}(\emph{GI-CO-FT} ) signalling cascade.
Briefly, the action of the \emph{GI-CO-FT} module in \emph{Arabidopsis}
is as follows: the active Pfr form of phyA promotes the stability of the
nuclear transcription factor CONSTANS (CO) which activates transcription
of \emph{FT}(Putterill et al., 1995; Samach et al., 2000). From the
\emph{FT} locus, florigen is produced, a small mobile protein which
travels via the phloem from the leaves to the shoot apical meristem to
induce the transition from vegetative to reproductive growth (Corbesier
et al., 2007). The circadian clock gene \emph{GIGANTEA} (\emph{GI} )
allows the degradation of transcriptional repressors that repress the
expression of\emph{CO} thus indirectly promoting \emph{FT}(Sawa et al.,
2007). The MADS-box transcription factor gene \emph{SOC1} is indirectly
upregulated by \emph{CO} via florigen. SOC1, in turn, activates the
floral meristem identity gene \emph{LEAFY,} thus promoting flowering
(Lee et al., 2008; Yoo et al., 2016).

Importantly, \emph{SOC1} is a major floral integrator of different
flowering pathways in \emph{Arabidopsis}. For example, another MADS-box
gene, \emph{FLOWERING LOCUS C} (\emph{FLC} ), which is involved in the
vernalization pathway directly binds to the \emph{SOC1} promoter, and
blocks \emph{SOC1} transcriptional activation by CO (Hepworth et al.,
2002). \emph{FLC} also represses \emph{FT} transcription in the leaves
and blocks florigen transport thus inhibiting flowering (Searle et al.,
2006).

\emph{GI}, \emph{CO} and \emph{FT} seem to be conserved in flowering
pathways in many crops, such as wheat, barley, grapevine, pea, tomato,
onion and cucurbits (Watanabe et al., 2011 and references therein).
Thus, these genes are promising candidates for flowering time control in
\emph{Cannabis}. However, the gene functions and mechanisms controlling
the flowering pathways may differ between species, and thus must be
elucidated in \emph{Cannabis}.

Several of these key regulators of flowering time have been demonstrated
to have pleiotropic effects on agronomically valuable characteristics,
further emphasising the importance of elucidating the role of these
regulators in crop species (Blümel et al., 2015).

Given that \emph{Cannabis} is a eudicot, short-day plant, the commonly
used models- the long-day \emph{Arabidopsis} or the monocot rice - may
not be the most applicable for comparative analysis. \emph{Glycine
max}(soybean) is a short-day crop that belongs to the Fabales and is,
therefore, more closely related to \emph{Cannabis} (Rosales) than rice
(Poales) or \emph{Arabidopsis} (Brassicales) (Figure 3). Flowering time
control in soybean is well studied and may provide important clues about
how flowering is regulated in \emph{Cannabis}.

In soybean, the \emph{E} genes and the \emph{JUVENILE} (\emph{J} ) gene
are involved in flowering time control (Figure 8) (Copley et al., 2018
and references therein). \emph{J} is also named \emph{GmELF3}, it is
orthologous to \emph{Arabidopsis EARLY FLOWERING3} (\emph{ELF3} ), which
is an important part of the circadian clock (Lu et al., 2017).
Individuals that carry loss-of-function alleles for \emph{E1}
to\emph{E4} exhibit photoperiod insensitive flowering as higher
transcript levels of the \emph{FT} genes are present (Figure 8) (Xu et
al., 2013).\emph{E1} is a legume specific transcription factor and the
remaining genes are orthologous to those involved in flowering time
control in \emph{Arabidopsis}: \emph{E2} (also named \emph{GmGIa} ) is
an ortholog of \emph{GI,} and \emph{E3} (\emph{GmPhyA3} ) and
\emph{E4}(\emph{GmPhyA2} ) are orthologous to \emph{PhyA.}

\emph{}\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=0.98\columnwidth]{figures/Figure8/Figure8}
\caption{{\textbf{An overview of the working model for the photoperiodic flowering
time pathway in soybean.} Parallel representations of the network under
long-day conditions and short-day conditions are shown as well as the
genes that when mutated infer photoperiod-insensitive flowering. Under
long-day conditions, \emph{Phytochrome A} homologs \emph{GmPHYA3} and
\emph{GmPHYA2} promote \emph{E1} expression and inhibit \emph{GmELF3}
expression. \emph{E1} up-regulates \emph{GmFT4a} (a change-of-function
\emph{FT} that suppresses flowering) and down-regulates \emph{GmFT2a}
and \emph{GmFT5a}, all of which are \emph{FT} homologs (Nan et al.,
2014; Samanfar et al., 2017; Xia et al., 2012; Zhai et al., 2014).
\emph{GmGIa} is a \emph{GI} homolog which inhibits \emph{GmFT2a} (but
interestingly not \emph{GmFT5a}) thus delaying flowering under long-day
conditions (Watanabe et al., 2011).
{\label{636804}}
Under short-day conditions, \emph{GmELF3} is expressed. \emph{GmELF3}
represses \emph{E1} by physically associating with the \emph{E1}
promoter. This leads to the release of the \emph{E1} suppression of the
\emph{GmFT} genes, thus promoting flowering under short days (Lu et al.,
2017; Xia et al., 2012). Natural variation in the \emph{GmFT} gene
family is at least partially responsible for flowering time variation in
soybean, with several polymorphic sites significantly associated with
flowering time variation (Jiang et al., 2019). Soybean plants which
carry loss-of-function alleles for~\emph{E1}, \emph{GmGIa},
\emph{GmPHYA3} and \emph{GmPHYA2} exhibit photoperiod insensitive
flowering as higher transcript levels of the \emph{FT} genes are present
(Xu et al., 2013). Thus, these genes may represent strong candidates for
elucidating photoperiod-insensitivity in \emph{Cannabis}.
{\label{636804}}%
}}
\end{center}
\end{figure}

Natural variation in the \emph{E} and \emph{J} genes are responsible for
the adaptability of soybean to cultivation at various latitudes,
including the tropics, with several polymorphic sites significantly
associated with flowering time (Jiang et al., 2019; Lu et al., 2017).
Thus, these genes may represent strong candidates for elucidating the
natural variation that exists for photoperiod sensitivity in
\emph{Cannabis}.

However, though candidate gene searches can be informative, it is worth
considering that gene functions may not be similarly conserved across
species. As such gene mapping approaches (genome-wide association
studies, quantitative trait loci mapping) and functional analyses are
still required to elucidate the flowering time network in
\emph{Cannabis}. Furthermore, as a wind-pollinated, dioecious species
\emph{Cannabis} is primarily outcrossing (Salentijn et al., 2015). In
self-pollinating species such as rice, \emph{Sorghum} and
\emph{Arabidopsis}, variation in flowering time is controlled by a few
large-effect loci (Gage et al., 2020). However, in the model outcrossing
species maize, several loci all contribute a small amount to phenotypes
of complex traits such as flowering time (Buckler et al., 2009). It
remains to be seen how complex traits will be determined in
\emph{Cannabis}, but taking inspiration from the successful elucidation
of complex traits in maize may motivate the formation of sophisticated
multi-parental mapping populations such as Nested Association Mapping
(NAM) or Multiparent Advanced Generation InterCross (MAGIC) populations
(Gage et al., 2020).

\section*{\texorpdfstring{7. The long read: \emph{Cannabis} genetics and
genomics}{7. The long read: Cannabis genetics and genomics}}

{\label{980399}}

While the medicinal, agricultural, and industrial applications of
\emph{Cannabis} are vast, \emph{Cannabis} genomics has lagged in
relation to that of other crops. In recent years, however, as legal
restrictions have eased and with the advent of third-generation
sequencing, the field has picked up significantly. With this, there is
now a wealth of novel data readily available for analysis. Here, the
currently available genomics and transcriptomics data will be reviewed.

The \emph{Cannabis} genome is diploid (2n=20), consisting of 9 autosomes
and a pair of heteromorphic sex chromosomes (X and Y). The haploid
genome size is predicted to be 843 Mb and 818 Mb for male (XY) and
female (XX) plants respectively, with the larger Y chromosome accounting
for the sex-specific difference in genome size (see also discussion
above on sex determination) (Sakamoto et al., 1998). The \emph{Cannabis}
genome is not large relative to that of other crops like maize and
wheat. However, it has been difficult to resolve due to its high
heterozygosity and the abundance of repetitive DNA sequences. High
heterozygosity levels have been retained in the genome, as
\emph{Cannabis} is dioecious and has not been subject to intense
breeding (Lynch et al., 2016; Sawler et al., 2015). While this genetic
diversity is desirable for selective breeding, it can complicate genome
assembly. Highly variable alleles are often misassembled as segmental
duplications, whereby both haplotypes are incorporated at distinct loci,
inflating the genome assembly size (Claros et al., 2012; Michael and
VanBuren, 2020). Additionally, it is estimated that repetitive sequences
constitute \textasciitilde{}70\% of the \emph{Cannabis} genome (Gao et
al., 2020; Laverty et al., 2019; Pisupati et al., 2018). Misassembly of
multiple repeat elements onto one contig causes assembly collapse,
reducing the genome assembly size (Claros et al., 2012; Michael and
VanBuren, 2020). These features are particularly challenging when
short-read sequencing is applied. While the first draft \emph{Cannabis}
genome, from the marijuana Purple Kush (PK) cultivar, was sequenced in
2011, the short-read sequencing of that time could not resolve
repeat-rich, low complexity regions (van Bakel et al., 2011). This led
to a very valuable yet incomplete genome assembly, consisting of 534 Mb
(van Bakel et al., 2011).

Third generation single molecule (or long-read) sequencing, such as
Pacific Biosciences (PacBio) and Oxford Nanopore sequencing, can
generate long reads which are capable of capturing the regions flanking
repeat sequences and segmental duplications. Thus, long-read sequencing
greatly facilitates the assembly process and has been revolutionary for
plant genomics, enabling chromosome-level assemblies to be achieved
(Jiao and Schneeberger, 2017; Michael and VanBuren, 2020). Recently,
long-read sequencing, in parallel with genetic and physical mapping, has
enabled four chromosome-level assemblies from the CBDRx, PK, Finola
(FN), and a wild \emph{Cannabis} (CR) line (Table 1) (Gao et al., 2020;
Grassa et al., 2018; Laverty et al., 2019). The CBDRx genome (a female
individual) was sequenced using Oxford Nanopore technology and has an
assembly size of 876.148 Mb (Figure 5a) (Grassa et al., 2018). In 2019,
the first genome-wide annotation was made available for this
\emph{Cannabis} genome, making it the reference genome on the NCBI
database (Jenkins and Orsburn, 2019). The PK and FN genomes were
sequenced with PacBio single-molecule sequencing (Laverty et al., 2019).
The PK (female) and FN (male) assembly sizes are 891.965 Mb and 1009.67
Mb respectively, both of which are significant improvements upon the
original draft PK genome from 2011 (Table 1) (van Bakel et al., 2011;
Laverty et al., 2019). The CR variety, which is derived from a wild
\emph{Cannabis} plant, was also sequenced with PacBio, achieving a
genome assembly size of 812.525 Mb (Gao et al., 2020). While linkage
maps were generated to resolve the chromosomes for the CBDRx, PK and FN
genomes (Grassa et al., 2018; Laverty et al., 2019), Hi-C data was used
to create a physical map for CR, enabling the chromosomes to be
assembled (Supplementary Table 1) (Gao et al., 2020).

Eight additional genomes have been assembled to varying levels of
completeness (Table 1). Amongst these are the genome sequences of a
father-mother-daughter trio from the Jamaican Lion (JL) cultivar, which
was sequenced using PacBio (McKernan et al., 2020). The parental genome
assemblies including gene annotation are available on the NCBI database,
while all three genome assemblies are available on the Medicinal
Genomics website
(\emph{https://www.medicinalgenomics.com/jamaican-lion-data-release/}).
In addition to these three genomes, 40 genomes from a diverse range of
cultivars were sequenced with Illumina short-read sequencing as part of
the Medicinal Genomics `Cannabis Pan-Genome Project' (McKernan et al.,
2020). The whole-genome sequencing (WGS) data generated in this project
are available on the NCBI sequence read archive (Supplementary Table 2).
These genome sequences will be an invaluable resource for characterising
the genetic basis behind the wide phenotypic diversity observed within
\emph{Cannabis}. Specifically, they will facilitate the development of a
\emph{Cannabis} pan-genome, where gene sets unique to specific cultivars
could be defined. Such cultivar-specific genes are often representative
of niche phenotypic adaptations that have evolved in response to
specific environmental conditions (Montenegro et al., 2017; Tao et al.,
2019). Cultivar-specific genes could be key targets for breeding, where
new cultivars could be designed with desirable traits for specific
production purposes (Tao et al., 2019).

There is also a wealth of additional genomics data available. This
includes sequences of organellar genomes, of which there are seven
mitochondrial and nine chloroplast genome assemblies available
(Supplementary Table 3). The organellar genomes are particularly useful
for resolving phylogenetic relationships. The rate of nucleotide
substitution of mitochondrial coding sequences is lower than that of the
nuclear and plastid genomes, making them useful molecular markers for
resolving deep taxonomic relationships (Knoop, 2004; Wolfe et al.,
1987). Despite this high intragenic sequence conservation, angiosperm
mitochondria can exhibit high variation in genome organisation both
within and between species (Cole et al., 2018; Davila et al., 2011;
Palmer and Herbon, 1988). Perhaps taking a comparative genomics approach
to investigate organisational variation within the mitochondrial genome
between different \emph{Cannabis} cultivars would be insightful for
resolving relationships within the \emph{Cannabis} genus. In contrast,
the chloroplast genome is characterized by both stability in genome
organisation and sequence conservation between species (Palmer and
Herbon, 1988). Hence the chloroplast genome is often used to resolve
phylogenies at the ordinal and familial taxonomic levels (Oh et al.,
2016; Vergara et al., 2015; H. Zhang et al., 2018).

Furthermore, genotyping by sequencing (GBS), amplicon sequencing,
bisulfite sequencing and Hi-C data are available for a multitude of
different hemp as well as marijuana varieties (Supplementary Table 2).
GBS is an efficient and cost-effective method to genotype a large number
of samples, providing insight into the population structure and genetic
diversity within a species (He et al., 2014). There have been at least
three population-based studies that have generated GBS data for
\textasciitilde{}400 samples, representing both hemp and marijuana lines
(Lynch et al., 2016; Sawler et al., 2015; Soorni et al., 2017). These
studies find that hemp and marijuana often form distinct populations,
not segregating based only on the B\textsubscript{T} and
B\textsubscript{D} loci, but on a genome-wide level (Lynch et al., 2016;
Sawler et al., 2015; Soorni et al., 2017). Bisulfite sequencing detects
DNA methylation and is useful for understanding epigenetic gene
regulation (Elhamamsy, 2016; Li et al., 2020). Two bisulfite sequencing
datasets are available for analysis (McKernan et al., 2020; Niederhuth
et al., 2016). Given that economically important traits like sex
expression and flowering time are under strong environmental control, it
will be interesting to explore to which extent those traits are
epigenetically regulated. This may open the possibility of breeding
`climate smart' \emph{Cannabis} plants, similarly to other crops where
epigenetically regulated heat, drought or cold adaption are explored for
crop improvement (Varotto et al., 2020).

Lastly, the 3D organisation of the genome within the nucleus can be
mapped with Hi-C data (Rodriguez-Granados et al., 2016). One Hi-C
dataset exists for the JL cultivar and is available on NCBI (Gao et al.,
2020). Additional Hi-C datasets are available for the Jamaican Lion
genomes through the Medicinal Genomics website
(https://www.medicinalgenomics.com/jamaican-lion-data-release/). The 3D
organization of the genome and its implications for gene regulation are
currently being heavily investigated in plants (Santos et al., 2020).
The available \emph{Cannabis} Hi-C data are both useful for facilitating
genome assembly as well as for understanding epigenetic regulation of
gene expression (Burton et al., 2013; Lieberman-Aiden et al., 2009; Xie
et al., 2015).

There have also been many studies that have focused on characterising
the \emph{Cannabis} transcriptomes (Supplementary Table 2). Perhaps most
notably, in 2019, an extensive `transcriptome atlas' was generated for
\emph{Cannabis}(Braich et al., 2019). This study involved RNA-sequencing
of 71 samples taken from multiple tissues of the Cannbio-2 cultivar
(CN2), at various developmental stages. This transcriptome data will be
useful for the annotation of new genome assemblies, as well as for
inferring gene functions based on spatiotemporal gene expression
patterns. Other studies have characterised the transcriptome of hemp
lines grown under salinity and drought stress (Gao et al., 2018; Liu et
al., 2016), as well as during bast fibre development (Behr et al., 2016;
Guerriero et al., 2017). Three further studies have focused on
sequencing the transcriptome of glandular trichomes, with the aim of
profiling the expression of genes involved in terpene and
phytocannabinoid biosynthesis (Booth et al., 2020; Livingston et al.,
2020; Zager et al., 2019). Furthermore, two recent studies have focused
on identifying the sex chromosomes based on characterising the
expression of sex-linked genes in male and female plants (McKernan et
al., 2020; Prentout et al., 2020). The transcriptomes of the PK and FN
cultivars sequenced in 2011 are also available (van Bakel et al., 2011).

While wide-spread illegalization of \emph{Cannabis} has stunted genomics
research in the past, it is clear that there have been major advances in
this field in recent years. With chromosome-level genome assemblies now
available, as well as genome-wide annotations and abundant transcriptome
data, the resources for future research are plentiful.

\section*{8. More than the sum of its parts: Medical applications of
phytocannabinoids}

{\label{227048}}

\emph{Cannabis} plants represent a rich source of biologically active
compounds, including more than 100 plant-derived cannabinoids
(phytocannabinoids) and more than 200 terpenoids (Russo, 2011). Thus
far, research into the medicinal effects of \emph{Cannabis} has largely
focussed on phytocannabinoids. Among these, the most well-studied are
the psychoactive THC, and the non-psychoactive CBD, though other
phytocannabinoids such as CBG and CBC also show therapeutic potential
(Russo, 2011) (see chapter 3 for details on phytocannabinoid synthesis
and genetics).

\subsection*{\texorpdfstring{8.1. \emph{Cannabis}
metabolites}{8.1. Cannabis metabolites}}

{\label{381887}}

Early investigations into the pharmacologic effects of THC led to the
discovery of the human endogenous cannabinoid (endocannabinoid) system,
which includes endogenous cannabinoid ligands, metabolic enzymes, and
the two major cannabinoid receptors, CB1 and CB2 (Izzo et al., 2009).
The modes of action of phytocannabinoids are complex, with individual
compounds capable of acting at multiple molecular targets. THC modulates
the activity of CB1 and CB2 but can also activate the transcription
factor PPAR\selectlanguage{greek}γ \selectlanguage{english}and the TRP ion channel TRPA1 (Izzo et al., 2009). CBD,
meanwhile, has low affinity for CB1 and CB2, but can modulate the
activity of various components of the endocannabinoid system.
Furthermore, like THC, CBD can target PPAR\selectlanguage{greek}γ \selectlanguage{english}and TRPA1, as well as the
G-protein coupled receptors GPR55 and GPR18, the TRP ion channels TRPV1,
TRPV2 and TRPM8, and the serotonin receptor 5-HT\textsubscript{1a}(Izzo
et al., 2009). By modulating various signalling pathways involved in
multiple diseases, phytocannabinoids have the potential to provide many
therapeutic benefits.

The analgesic, antiemetic and anticonvulsant properties of
phytocannabinoids are well-established (Whiting et al., 2015)\textbf{.}
Growing evidence suggests that \emph{Cannabis}metabolites also produce
anti-inflammatory, anti-depressant, anxiolytic and anti-cancer effects
(Atalay et al., 2019; Fraguas-S\selectlanguage{ngerman}ánchez and Torres-Suárez, 2018; Poleszak
et al., 2018; \selectlanguage{polish}Ś\selectlanguage{english}ledzi\selectlanguage{polish}ń\selectlanguage{english}ski et al., 2018). Synthetically produced
cannabinoids can mimic the effects of plant-derived compounds, and many
countries have approved synthetic cannabinoids for medicinal use.
Dronabinol and nabilone, synthetic forms of THC, are approved for the
treatment of chemotherapy-induced nausea and vomiting, and for appetite
stimulation in AIDS-associated anorexia (Freeman et al., 2019). However,
plant-derived medicines have also been developed, namely Epidiolex, a
purified form of CBD for the treatment of severe forms of epilepsy, and
Sativex, a \emph{Cannabis} extract containing THC and CBD for the
management of pain and spasticity in multiple sclerosis (Freeman et al.,
2019).

Additionally, \emph{Cannabis} contains many non-cannabinoid metabolites,
including terpenoids, flavonoids, ligand amides and stilbenes (Pollastro
et al., 2018b). Of these, the terpenoids have been most extensively
studied, and display many therapeutic effects (Russo, 2011). Due to
growing evidence that various \emph{Cannabis} bioactive compounds act
synergistically to produce therapeutic effects (Russo, 2011), a greater
understanding of the pharmacological contributions of
different\emph{Cannabis} metabolites will be needed to develop the most
effective\emph{Cannabis} -based medicines.

\subsection*{\texorpdfstring{8.2. \emph{Cannabis}
synergy}{8.2. Cannabis synergy}}

{\label{342818}}

To date, research into \emph{Cannabis-}based medicines has primarily
focussed on single isolated cannabinoid compounds. However, some studies
show that combinations of various \emph{Cannabis} components display
greater biological activity than single compounds, suggesting that whole
plant extracts may be more effective than purified phytocannabinoids
(Russo, 2011). The increased activity of whole \emph{Cannabis} extracts
may be due to the synergism between various cannabinoid and
non-cannabinoid components, which has been termed `the entourage effect'
(Russo, 2011). Proposed mechanisms underlying the entourage effect in
\emph{Cannabis}include activation of multiple molecular targets,
enhanced bio-availability or solubility of compounds, and neutralisation
of adverse events (Wagner and Ulrich-Merzenich, 2009). Exploiting
\emph{Cannabis} synergy to develop new medicines based on whole-plant
extracts may be beneficial for a range of pharmaceutical applications.

In epilepsy, the addition of non-THC, non-CBD \emph{Cannabis}metabolites
may enhance the anticonvulsant effects of existing treatments. In a
mouse model of epilepsy, the content of minor phytocannabinoid compounds
in a high-CBD plant extract treatment affected seizure incidence and
survival rates, suggesting that specific combinations of
phytocannabinoids may be more effective than single purified compounds
(Berman et al., 2018). Furthermore, a meta-analysis of observational
clinical studies on epilepsy treatment reported that CBD-rich plant
extracts reduced seizure frequency in patients at doses lower than those
used in clinical trials of Epidiolex. The CBD-rich plant extract also
produced significantly fewer adverse effects, likely due to the lower
dose required (Devinsky et al., 2017; Pamplona et al., 2018; Thiele et
al., 2018).

The benefits of phytocannabinoids for pain management have been
well-established. However, the analgesic effects of \emph{Cannabis} may
be enhanced by combining different \emph{Cannabis} bioactive compounds.
The analgesic and anti-inflammatory effects of CBD occur only within a
very limited dose range, but this bell-shaped dose-response was overcome
when CBD was combined with a \emph{Cannabis} extract (Gallily et al.,
2015). Another study showed that a \emph{Cannabis} extract high in THC
provided no benefit for intractable cancer pain, while nabiximols, a
whole extract CBD/THC combination significantly reduced pain in cancer
patients (Johnson et al., 2010).

Whole-plant extracts may also be preferable to single phytocannabinoids
for the treatment of mood disorders. Several phytocannabinoids,
including THC, CBD and CBC, appear to have antidepressant and/or
anti-anxiety effects, possibly due to modulation of the endocannabinoid
system and/or modulation of serotonin receptors (Crippa et al., 2011;
Poleszak et al., 2018; Zanelati et al., 2010). Other non-cannabinoid
\emph{Cannabis} compounds, such as the terpenoid limonene, also exhibit
antidepressant effects, and lemon oil, which contains high quantities of
limonene displays anti-stress and anxiolytic properties (Komiya et al.,
2006; Piccinelli et al., 2015; Russo, 2011). Plant extracts containing
both cannabinoids and terpenoids may be the most effective
\emph{Cannabis-}based treatment option for psychopharmacological
applications.

Synergistic effects are also potentially beneficial for cancer
treatment. In breast cancer cell lines and animal models, a
\emph{Cannabis} extract produced enhanced anti-tumour effects compared
to purified THC, possibly due to the presence of other cannabinoid
compounds (Blasco-Benito et al., 2018). Another study reported that
whole plant extracts reduced cancer cell survival and proliferation more
effectively than pure THC, across a range of cancer cell types (Baram et
al., 2019). Notably, one study found that cancer cells were killed most
effectively when treated with phytocannabinoids and terpenoids at ratios
similar to those found naturally in the plant (Namdar et al., 2019).

The above findings suggest that, for the treatment of various medical
conditions, producing a range of \emph{Cannabis} varieties , or
chemotypes, with varying phytochemical contents may constitute a more
effective approach than developing new synthetic cannabinoid-based
medicines. A greater understanding of the synergistic activities of
different phytocannabinoids, terpenoids and other
\emph{Cannabis}components is needed to identify the most effective
combinations for various pharmaceutical applications.

\subsection*{\texorpdfstring{8.3. \emph{Cannabis} breeding for
medicine}{8.3. Cannabis breeding for medicine}}

{\label{933237}}

\emph{Cannabis sativa} is a versatile multi-purpose crop which requires
a simple, low-input cultivation technique, adapts to various ecological
conditions, produces sustainable products, and provides raw material for
a wide range of applications, including medicine (Amaducci et al.,
2015). Research into the synergistic pharmacological effects
of\emph{Cannabis} metabolites suggests that ratios of phytocannabinoids,
terpenoids and other \emph{Cannabis} metabolites influence a plant's
therapeutic potential.

More research is needed to determine the influence of environmental and
genetic factors on the phytochemical profile of the
\emph{Cannabis}plant. Existing studies show that total phytocannabinoid
yields are related to environmental conditions. Phytocannabinoid and
terpene levels are affected by factors such as the humidity, rainfall
and temperature of the growth environment (Meier and Mediavilla, 1998;
Murari et al., 1983; Pavlovic et al., 2019). However, the relative
ratios of the different \emph{Cannabis} metabolites are dependent on the
genotype (see Chapter 3) (Janatov\selectlanguage{ngerman}á et al., 2018; de Meijer et al., 2003;
Toonen et al., 2006; Vanhove et al., 2011). Identifying the
environmental and genetic factors that influence phytochemical
production by \emph{Cannabis} could aid in the development of new
\emph{Cannabis} cultivars with tailored ratios of various metabolites.

The therapeutic effects of a given plant reflects the proportions of the
various pharmacologically active components. The development of
\emph{Cannabis} chemotypes containing high levels of specific
phytocannabinoids can be achieved through breeding. De Meijer \emph{et
al} . produced \emph{Cannabis} chemotypes high in specific single
phytocannabinoids, including THC, CBD, CBG and CBC (de Meijer et al.,
2009a, 2003; de Meijer and Hammond, 2005). Cannabinoid-free chemotypes
were also developed, which could aid investigation into the
contributions of non-cannabinoid bio-actives, such as terpenoids, to the
pharmacological effects of \emph{Cannabis} (de Meijer et al., 2009b;
Russo, 2011). The development of these chemotypes through conventional
breeding demonstrates the high diversity of the \emph{Cannabis} genome,
which may obviate the need for genetic engineering of
\emph{Cannabis}(Russo, 2019).

Understanding the interactions between different
\emph{Cannabis}bio-actives remains one of the key challenges to
harnessing the full medicinal potential of the \emph{Cannabis} plant.
Research into \emph{Cannabis}-based medicines highlights the importance
of various \emph{Cannabis} metabolites in producing therapeutic effects.
Further research is needed to elucidate the mechanisms underlying the
entourage effect observed with whole \emph{Cannabis} extracts, and to
assess the contributions of various \emph{Cannabis} metabolites to
determine the most effective combinations for various pharmaceutical
applications. Applying our knowledge of the entourage effect in
\emph{Cannabis} to the development of tailored chemotypes has the
potential to provide improved \emph{Cannabis}-based therapies for
various medical conditions, which could benefit many patients.

\section*{\texorpdfstring{9. Hemp for houses: \emph{Cannabis} as building
material}{9. Hemp for houses: Cannabis as building material}}

{\label{187412}}

Many hemp varieties of \emph{Cannabis} are fibre crops with multiple
inherent qualities as building material. The high tensile strength of
hemp fibres, traditionally exploited in rope and fabric applications,
also enables mechanical advantages for building construction
applications. Additionally, shiv particles of the woody-core are a
biobased alternative to mineral aggregates for low-impact concrete.
Therefore both the plant fibres and the shiv particles are suitable for
developing biobased, environmentally friendly building materials that
have been shown to have inherent thermal, hygrothermal and acoustic
characteristics (Kinnane et al., 2016; Reilly et al., 2019; Shea et al.,
2012).

Building products that integrate hemp are many, but reductively may be
grouped into two general categories, hemp concrete and hemp insulation
blankets: Hemp concrete are those mixed with a binder to form a porous
concrete composite with thermal insulation qualities. Hemp insulation
blankets are thermo-formed without an added binder to create a low
density, blanket type product. These respectively encompass shiv and
fibres, and are distinguished by their dry densities; typically in the
range 390-670 kg/m\textsuperscript{3} in the case of the hemp-concrete
(Collet-Foucault et al., 2004), and about 38 kg/m\textsuperscript{3} for
the hemp-insulation (`Technichanvre', 2017). The concretes have a wide
density range as they are typically bespoke and include varying levels
of binders, often lime, but also cement. As noted, both products are
recognised for their good thermal properties. The hempcretes have a low
thermal conductivity (\selectlanguage{greek}λ \selectlanguage{english}= 0.12 W/(m*K)) (Walker and Pav\selectlanguage{ngerman}ía, 2014)
relative to other standard concretes (\selectlanguage{greek}λ \selectlanguage{english}= 1-2 W/(m*K)). However, their
conductivity is higher than the former insulation wool blanket product
(\selectlanguage{greek}λ \selectlanguage{english}=0.04 W/ (m*K)) (Collet-Foucault et al., 2004), which contains up to
90\% hemp fibre, are formed in panels or rolls and used for roof, attic
and wall insulation.

Hempcrete, in contrast, is a composite material. It is commonly mixed in
a ratio by weight of 1:2:3 of hemp: binder: water. In contrast to
standard concrete, hempcrete has relatively low mechanical strength. It
is therefore typically cast around a load-bearing timber structure. The
wet mix is poured between temporary shuttering, and the hempcrete is
tamped down to compact it and form the wall. The thickness of these
walls typically ranges from 300-600 mm, to ensure structural stability
and to meet thermal requirements. These dimensions limit the widespread
applicability of hempcrete, particularly in urban infill sites. However,
new innovative products are enabling its wider applicability.
Increasingly, precast hempcrete blocks are appearing on the market.
Exhibiting higher densities, certain of these have load-bearing
capability and they generally enable time and labour efficiencies on
site. They are also popular for renovation and retrofit projects,
primarily because hempcrete is characterised by an open pore structure
which allows for the transmission of moisture. Moisture is often present
in the walls of old buildings, and breathable insulation allows this
water escape, instead of trapping it as do modern-day synthetic
insulations which can lead to mould growth, structural and air quality
issues.

These are just some example of advantages of biobased materials.
However, the construction industry and the agricultural industry
diverged during the modern post-war age of development. Synthetic
products were developed to meet high demand with a price point that
enabled use, and waste, of products during phased redevelopment
(Kinnane, 2020). Today synthetic polymer and mineral wool products
continue to command almost full market share of the insulation industry,
and that demand is increasing as we aim to reduce the operational energy
of buildings. Hemp, and biobased materials more generally, remain niche
products. Today hemp insulation products are almost twice the price of
the mass produced alternatives (Carus et al., 2013), even though plant
fibres have a lower cost of processing than synthetic fibres.

However, the building sector, and its considerable environmental impact,
is increasingly in focus, and the environmental benefits of biobased
materials are giving them greater traction. Hemp, with its fast-growth
cycle and multi-purpose advantages is increasingly proposed as a
low-impact design solution. Although specific quantification of carbon
sequestration remains challenging (Reilly and Kinnane, 2017), authors
report levels of between 1.5-2.1 kgCO\textsubscript{2}per kg of plant
grown and values of energy for production of 0.085-0.19
kgCO\textsubscript{2} per kg of hemp shiv (see S\selectlanguage{ngerman}áez-Pérez et al., 2020
for review). It should be noted however that although hemp exhibits
carbon positive credentials, the embodied carbon of any hempcrete is
high due to the high quantity of binders often used, and this is often
underestimated by proponents of the material.

\section*{\texorpdfstring{10. Also a medicine for the environment?
Sustainability aspects of \emph{Cannabis}
farming}{10. Also a medicine for the environment? Sustainability aspects of Cannabis farming}}

{\label{883973}}

Considering global warming and consequential efforts to divest from
fossil fuels, bioenergy crops and biofuels are gaining increasing
interest (Rogelj et al., 2018). \emph{Cannabis} is a high-yielding,
annual crop, and has much-untapped potential for contributing to carbon
sequestration efforts (Finnan and Styles, 2013). Besides storing carbon
in building materials, alternative uses for this crop exist, and as
such, there is a high potential for carbon to be stored both short- and
long-term in bioenergy, textiles, and paper (Figure 1, Figure 9).
Further contributing to the environmental connotations of this species,
hemp has been employed in phytoremediation efforts to restore land
implicated by heavy metal contaminants (Citterio et al., 2003). Hemp
leaves and seeds also provide the basis for human consumption (Figure
9).

\par\null\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=0.98\columnwidth]{figures/Figure9/Figure9}
\caption{{\textbf{Products made from hemp.} Non-THC \emph{Cannabis} (hemp) can be
processed into a multitude of different products. Hemp husk of fibre
varieties (a) can be processed into hempcrete (b), while hemp fibre (c)
can be made into rope (d) or insulation material (e). Remains of the
plant can be pressed into hemp pellets for heating (f). Composite
hemp-plastic material for 3D printing (g) is more sustainable than
regular printing plastic. Hemp seeds can be dehulled into hemp hearts
for human consumption (h). A variety of hemp products like tea (i), as
well as chocolate (k) and candy (l) are also available. (i) courtesy of
Christine Schilling.
{\label{775704}}%
}}
\end{center}
\end{figure}

Hemp is considerably more efficient (high annual yields with low
agrochemical/fertilizer input) than the traditional annual bioenergy
crops (sugar beet and oilseed rape) and possesses similar greenhouse gas
mitigation potential to the perennial bioenergy crops Miscanthus and
willow (Finnan and Styles, 2013). Annual bioenergy crops like hemp can
be appealing options for farmers to diversify and explore the bioenergy
market without the demands of perennials, namely high establishment
costs and long-term commitment (15--20 years) of their land to bioenergy
(Finnan and Burke, 2013; Finnan and Styles, 2013).

Hemp biomass has good combustion properties and could be used to
generate either heat or electricity (Finnan and Styles, 2013). There are
multiple biofuel options: Biogas, solid fuel briquettes, bales, and
bioethanol (Kraszkiewicz et al., 2019; Prade et al., 2012, 2011).

As hemp is an annual crop it can be readily integrated into crop
rotation cycles, thus not competing with food supplies and can therefore
contribute towards sustainable cropping systems (Finnan and Styles,
2013). Moreover, hemp has been reported to improve yields of crops
subsequently grown thus complementing food production. Winter wheat
planted after hemp had 10--20\% yield increases (B\selectlanguage{ngerman}ócsa and Karus, 1998),
with similar observations recorded for soybean and alfalfa (Adesina et
al., 2020). A low input crop, hemp can produce high yields similar to
switchgrass and sorghum but with lower nutrient and pesticide
requirements (Das et al., 2017). Hemp offers the combined potential of
an effective break crop and an efficient energy crop, thus generating
income while promoting productivity. Break crops like hemp can be used
to disrupt pest cycles and the ability of hemp to tolerate high planting
densities suppresses weed growth, thus pesticide and herbicide
requirements of subsequently, cultivated crops are reduced (Bhattarai
and Midmore, 2014). The hemp root system promotes soil health, as the
large taproots penetrate deep into the soil facilitating aeration, but
simultaneously forms soil aggregates to prevent soil erosion (Amaducci
et al., 2008). Model analysis comparing the relationship between leaf
nitrogen status and photosynthesis rate in hemp, cotton and kenaf
revealed hemp to have a high photosynthetic capacity, even at low
nitrogen levels (Tang et al., 2017). This provides an additional line of
evidence that hemp may fulfil a future niche as a sustainable bioenergy
crop that can be cultivated over a wide range of climatic and agronomic
conditions.

\section*{\texorpdfstring{11. Future prospects: Phytocannabinoids without
\emph{Cannabis: In vitro} synthesis using cell
cultures}{11. Future prospects: Phytocannabinoids without Cannabis: In vitro synthesis using cell cultures}}

{\label{464127}}

Phytocannabinoids have high potential for medical but also recreational
use and therefore their production and extraction are of high commercial
interest. However, plant breeding and cultivation come with their own
challenges and phytocannabinoid yield and profiles can highly depend on
environmental factors. Cell cultures methods are a powerful tool for the
production of high-quality plant material in a manner that is time
efficient, seasonally independent, and which can satisfy good
manufacturing practice guidelines (Tekoah et al., 2015). This technology
has attracted a lot of attention as it can allow the harvesting of high
value products produced within cells in suspension or secreted into
their surrounding medium (Weathers et al., 2010). Improvement of culture
growth kinetics and product yield can be achieved via medium
optimisation (Holland \emph{et al.} , 2010; Ullisch\emph{et al.} , 2012;
Vasilev \emph{et al.} , 2013) and by selecting for high-producing cell
populations using techniques such as fluorescent marker-based cell
sorting (Kirchhoff \emph{et al.} , 2012). Cell lines optimised in these
ways can subsequently be cryopreserved to ensure consistent production
going forward (Ogawa \emph{et al.} , 2012).

Secondary metabolites, including pharmacologically valuable compounds
such as paclitaxel and scopolamine or transgenic proteins to be used as
vaccines, antibodies, immunomodulators and other therapeutics are
already produced in cell suspension cultures on a commercial scale
(Mountford, 2010; Paul et al., 2015). Hence, this might be a promising
avenue to produce cannabinoids as well (Figure 10).

\par\null\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=0.98\columnwidth]{figures/Figure10-revision/Figure10-revision}
\caption{{Developments in plant cell and tissue culture methodologies allow the
efficient production of metabolites and recombinant proteins at
commercial scales. Cell suspension cultures and hairy root cultures are
the most commonly used techniques, with protocols described for both in
Cannabis . However, efficient, large-scale production of
phytocannabinoids in vitro has yet to be developed. Image was made using
Biorender (\href{http://www.biorender.com}{www.biorender.com}).
{\label{375608}}%
}}
\end{center}
\end{figure}

Of the various types of plant cell culture available, cell suspension
cultures are the most commonly used due to their scalability and
relatively rapid growth rates (Santos et al., 2016). The use of cell
suspension cultures involves growing dedifferentiated plant cells in
liquid medium supplemented with hormones to induce culture proliferation
(Mustafa et al., 2011). However, the use of suspension cultures for
these purposes faces several barriers to successful execution. Firstly,
due to their genetic instability, cultures can often lose their ability
to produce valuable compounds over time, while low productivity rates
sometimes require large volumes of biomass to be grown, therefore
increasing costs relative to field-grown plants (Moon et al., 2020;
Weathers et al., 2010). Cell suspension cultures tend to form
heterogeneous cell clusters rather than proliferating as single cells in
culture, leading to increased difficulty of use and inconsistent growth
kinetics and product yield. In addition, the scale-up of cell suspension
cultures from laboratory to commercial production scale is often
associated with a decline in cell productivity (James and Lee, 2006).

In relation to \emph{Cannabis,} the use of \emph{in vitro} bioprocessing
techniques has the potential to allow the synthesis of high yields of
cannabinoids in a manner that satisfies good manufacturing practice
guidelines and guarantees a high-quality product. Metabolic engineering
offers the possibility of developing plant or microbial cell lines which
exclusively produce a desired cannabinoid, thus circumventing the high
costs associated with purifying a desired product during downstream
processing. However, achieving these aims poses a number of challenges
to which researchers must still find answers.

A study described by Pacifico et al., (2008) assessed the cannabinoid
content of callus cultures (which are metabolically identical to and
often constitute the starting material for suspension cultures) derived
from five different \emph{Cannabis} varieties . The calli did not show
any detectable levels of phytocannabinoids at any time during culture,
irrespective of the presence or absence of hormones or the
phytocannabinoid content of the original plants from which the cultures
were derived. As such, cell suspension cultures are unlikely to be an
effective biofactory for the production of cannabinoids without some
form of intervention.

One method which may overcome the lack of phytocannabinoid production in
\emph{Cannabis} suspension cultures is the use of elicitors. These are
compounds or a mixture thereof which can be added to the culture medium
to stimulate the transient production of a desired secondary metabolite.
Elicitors can be either biotic (animal, plant or microbial extracts) or
abiotic (metal ions, organic compounds or electric current) and have
been used previously with varying degrees of success (Weathers et al.,
2010 and references therein). However, since many elicitors are either
toxic or stress-inducing, their addition to a suspension culture often
leads to a reduction in the vitality of the culture and can even be
fatal. Such an approach was used by Flores-Sanchez et al. (2009) in
order to try to stimulate phytocannabinoid production in suspension
cultures of \emph{Cannabis}. However, no detectable levels of
phytocannabinoids were found in response to any of the treatments used,
which included a range of biotic and abiotic stimuli. As such, the
search for an elicitor which can induce phytocannabinoid production
remains ongoing.

One other point worth noting is that phytocannabinoids are known to be
toxic to plant cells when they accumulate at high enough concentrations,
which is why \emph{Cannabis} plants use trichomes to compartmentalise
these compounds into storage cavities outside the plant. THCA and its
precursor molecule CBGA are highly toxic to both \emph{Cannabis} and
tobacco cell suspension cultures, inducing 100\% apoptotic-like
programmed cell death after 24 hours in culture at a concentration of 50
\selectlanguage{greek}μ\selectlanguage{english}M (Sirikantaramas et al., 2005). This negative feedback phenomenon is
observed in many plant species which produce secondary metabolites and
is the reason why many metabolites are sequestered in specialised
structures.

However, the development of cell culture methods to avoid cell toxicity
is one area where researchers have relative success. Strategies such as
two-phase cultures have been shown to enhance the production of
secondary metabolites in a range of species (Malik et al., 2013). In
these systems, an aqueous phase is used to support cell growth while a
non-aqueous phase, typically consisting of a solvent or resin, is
employed to act as a sink for the accumulation of the desired product
and in some cases facilitates its subsequent extraction. Two-phase
systems have been shown to greatly increase metabolite yield in both
plant cell suspension and hairy root culture cultures (Chiang and
Abdullah, 2007; Malik et al., 2013; Rudrappa et al., 2004;
Syk\selectlanguage{polish}ł\selectlanguage{english}owska-Baranek et al., 2019; Wu and Lin, 2003), although to the best
of our knowledge such an approach has not yet been attempted in
\emph{Cannabis}.

Hairy root cultures are generated by the infection of plant tissues with
\emph{Agrobacterium rhizogenes}, a species which can modify the plant's
genome by introducing a segment of DNA known as T-DNA which codes for a
number of genes affecting the production and regulation of plant
hormones (Ono and Tian, 2011). This results in the development of
extensive root networks which can be easily cultured \emph{in vitro},
are genetically identical to the mother organ from which they were
derived and can also produce the same phytochemicals. Like cell
suspension cultures, hairy root cultures have already attracted
attention as a means of producing secondary metabolites such as
flavonoids (Gai et al., 2015), isoflavonoids (Jiao et al., 2014),
artemisinin (Patra and Srivastava, 2014) and lignans (Wawrosch et al.,
2014), albeit less commonly than cell suspension cultures due to their
increased difficulty of use.

THCA has previously been produced from tobacco hairy root cultures which
were transformed to express the enzyme responsible for its production,
THCA synthase, under the transcriptional control of the
\emph{cauliflower mosaic virus} 35S promoter (Sirikantaramas et al.,
2004). When these hairy roots were cultured in liquid medium
supplemented with CBGA, the precursor molecule to THCA, 8.2\% of this
CBGA was converted to THCA after two days of culture, approximately half
of which was then secreted into the surrounding medium, thus
demonstrating that CBGA uptake and THCA release from these transgenic
roots was possible \emph{in vitro,} albeit at low levels.

In \emph{Cannabis}, a protocol for the production of hairy root cultures
has already been described which shows that cultures are best
established from the hypocotyl of intact seedlings by piercing the
epidermis with a syringe and inoculating with \emph{A. rhizogenes}(Wahby
et al., 2013). Five varieties of \emph{Cannabis} (three hemp-type and
two marijuana drug-type) were used and all were shown to be responsive
to infection by \emph{A. rhizogenes}, although with varying
morphological responses. Similarly, all eight \emph{A. rhizogenes}
strains used could induce a hairy root morphology, albeit with varying
degrees of frequency (43-98\%, depending on the strain).

An alternative approach described by Farag and Kayser (2015) outlines
how hairy root cultures can be developed from \emph{Cannabis} callus
cultures without the use of \emph{A. rhizogenes} by growing them in B5
medium supplemented with 4 mg/ml of the auxin NAA. Under these
conditions, cannabinoid contents peak at 1.04 \selectlanguage{greek}μ\selectlanguage{english}g/g dry weight for THCA,
1.63 \selectlanguage{greek}μ\selectlanguage{english}g/g dry weight for CBGA and 1.67 \selectlanguage{greek}μ\selectlanguage{english}g/g dry weight for CBDA after 28
days of culture. These low yields highlight the fact that while
phytocannabinoids can be produced from hairy root cultures, significant
improvements in yield will need to be achieved before this methodology
is commercially viable for phytocannabinoid production.

Recent studies have attempted to demonstrate the production of
cannabinoids in non-native hosts, with a particular focus on yeast due
to the relative ease with which metabolic engineering can be achieved in
this model organism. A landmark paper by Luo et al. (2019) described the
complete biosynthesis of CBGA, THCA and CBDA and several unnatural
analogues in yeast via engineering of the native mevalonate pathway and
the introduction of a heterologous hexanoyl-CoA biosynthetic pathway as
well as the \emph{Cannabis} genes responsible for the biosynthesis of
complete cannabinoids. However, the cannabinoid yields achieved from
this system were found to be approximately 100-fold less than those
produced by \emph{Cannabis} plants. As such, the efficient production of
cannabinoids in either plant or microbial cell culture remains a work in
progress.

\section*{12. Conclusions: Stay tuned, there is more to
come!}

{\label{892636}}

The recent progress in \emph{Cannabis} research has been remarkable, and
has revealed exciting challenges ahead: The evolution and genetic
diversity of phytocannabinoid synthases has proven to be a complex field
of research, as is the genetic and environmental control of sex
determination and flowering time. The increasing availability of genomic
resources will undoubtedly facilitate progress in all those areas, but
we predict that experimental analyses, including detailed morphological,
molecular genetics and phenotyping studies will be equally important to
understand the developmental and physiological intricacies of
\emph{Cannabis}.

Unusual in that it is a multipurpose crop, the full sustainability
potential of \emph{Cannabis} can only be fulfilled if it is used as
such. Thus, one major challenge will be to design crop ideotypes that
harmonise traits of medicinal relevance with those important for carbon
sequestration. This will not be an easy task, as the genetic control of
different traits is currently unclear. However, the production of e.g.
large fibre varieties that do nevertheless develop a dense inflorescence
with high CBD content seems not too farfetched. Even if those
hypothetical cultivars may not be able to provide the high yields of
specialized CBD cultivars they may provide farmers focusing on fibre
production with a second source of income.

In summary, the genetic and morphological diversity of \emph{Cannabis}
is a treasure trove that we are only beginning to explore. It is
important that we capitalise on this treasure to construct a
multipurpose swiss knife, and not a series of highly specialised tools.

\subsection*{Acknowledgements}

{\label{733128}}

SS is grateful for support by a Postdoctoral Scholarship of the Irish
Research Council and Greenlights Medicines under the Enterprise
Partnership Scheme (Grant No.: EPSPD/2019/220). CAD is supported by an
Irish Research Council-Environmental Protection Agency Government of
Ireland Postgraduate Scholarship (Grant No: GOIPG/2019/1987). JS is
supported by a Chinese Research Council Postgraduate Scholarship (Grant
No: CSC no. 201908300031). EOR and ASP are supported by the Irish
Research Council and Greenlight Medicines via an enterprise partnership
scheme (Grant no.: EPSPG/2017/376).

RM and SS are grateful to Beate and Peter Melzer, Christine Schilling,
and Andreas Schmidt for childcare and general support during the
pandemic, and to Jonathan and Theodor Schilling for their patience
during those unusual times. This substantially helped with writing this
manuscript.

All authors are members of HempHub, a strategic initiative of the UCD
Earth Institute, and are grateful for general support through this
initiative.\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Table1/Table1}
\caption{{Table 1 - Excel and PDF attached
{\label{742941}}%
}}
\end{center}
\end{figure}



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\par\null

\section*{Tables}

{\label{686786}}

\textbf{Table 1}. Summary of the currently
available~\emph{Cannabis~}genome assemblies. ~



\textbf{Hosted file}

\verb`Schilling2020_Table.xlsx`  available at \url{https://authorea.com/users/360584/articles/483690-the-cream-of-the-crop-biology-breeding-and-applications-of-cannabis-sativa}

\section*{}

{\label{997189}}

\section*{Supplementary Material}

{\label{196220}}



\textbf{Supplementary Table 1. Chromosome lengths for each
\emph{Cannabis} genome assembly.}

\textbf{Supplementary Table 2. Available \emph{Cannabis} sequencing data
from the NCBI sequence read archive.}

\textbf{Supplementary Table 3. Available \emph{Cannabis} organellar
genome assemblies.}

\textbf{}

\textbf{}

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