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Cannabis Domestication, Breeding History, Present-day Genetic Diversity, and Future Prospects

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Humans and the Cannabis plant share an intimate history spanning millennia. Humans spread Cannabis from its Eurasian homelands throughout much of the world, and, in concert with local climatic and human cultural parameters, created traditional landrace varieties (cultivars resulting from a combination of natural and farmer selection) with few apparent signs of domestication. Cannabis breeders combined populations from widely divergent geographical regions and gene pools to develop economically valuable fiber, seed, and drug cultivars, and several approaches were used with varying results. The widespread use of single plant selections in cultivar breeding, inbreeding, and the adoption of asexual reproduction for commercial drug production, reduced genetic diversity and made many present-day cultivars susceptible to pathogens and pests. The great majority of drug Cannabis cultivars are now completely domesticated, and thus are entirely dependent on humans for their survival. Future ramifications remain to be realized.
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Critical Reviews in Plant Sciences
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Cannabis Domestication, Breeding History,
Present-day Genetic Diversity, and Future
Prospects
Robert C. Clarke & Mark D. Merlin
To cite this article: Robert C. Clarke & Mark D. Merlin (2016) Cannabis Domestication, Breeding
History, Present-day Genetic Diversity, and Future Prospects, Critical Reviews in Plant Sciences,
35:5-6, 293-327
To link to this article: http://dx.doi.org/10.1080/07352689.2016.1267498
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Cannabis Domestication, Breeding History, Present-day Genetic Diversity,
and Future Prospects
Robert C. Clarke
a
and Mark D. Merlin
b
a
BioAgronomics Group Consultants, Los Angeles, California, USA;
b
Botany Department, University of Hawaii at Manoa, Honolulu, Hawaii, USA
ABSTRACT
Humans and the Cannabis plant share an intimate history spanning millennia. Humans spread
Cannabis from its Eurasian homelands throughout much of the world, and, in concert with local
climatic and human cultural parameters, created traditional landrace varieties (cultivars resulting
from a combination of natural and farmer selection) with few apparent signs of domestication.
Cannabis breeders combined populations from widely divergent geographical regions and gene
pools to develop economically valuable ber, seed, and drug cultivars, and several approaches were
used with varying results. The widespread use of single plant selections in cultivar breeding,
inbreeding, and the adoption of asexual reproduction for commercial drug production, reduced
genetic diversity and made many present-day cultivars susceptible to pathogens and pests. The
great majority of drug Cannabis cultivars are now completely domesticated, and thus are entirely
dependent on humans for their survival. Future ramications remain to be realized.
KEYWORDS
Afghanistan; Africa;
cannabinoids; Europe; ber;
hashish; hemp; India; indica;
marijuana; New World;
ruderalis; sativa; seed oil;
sinsemilla; terpenoids
I. Cannabis botany and ecology
The ecological requirements and genetic inheritance of
plants determine where they grow naturally. Cannabis
plants require well-drained soils, adequate sunlight,
warmth, and moisture, so most naturally growing popu-
lations are found seasonally across accommodating
northern temperate latitudes. Under natural conditions,
Cannabis grows well along exposed riverbanks, lakesides,
the margins of agricultural land, and other areas dis-
turbed by humans (Merlin, 1972; Clarke, 1977,1981;
Clarke and Merlin, 2013; Small 2015). Based on ecologi-
cal constraints, Cannabis evolved somewhere in temper-
ate latitudes of the northern hemisphere, and Eurasia is
favored as its primary region of origin (Clarke and
Merlin, 2013). Seminal uses, early cultivation, worldwide
dissemination, and eventual domestication of Cannabis
all began within this natural biogeographical range.
Cannabis plants are usually dioecious and produce
either male (pollen) owers or female (seed) owers. An
individual Cannabis plants gender is determined by X
and Y chromosomes, and monoecious plants of this
genus producing owers of both sexes occur only rarely
in nature. Cannabis also relies on air currents to spread
pollen grains from male plants to female seed plants.
Wind pollination, dioecious sexuality, and X/Y sexual
inheritance are each relatively rare in plants, yet all three
are characteristics of Cannabis.
Cannabis plants grow and develop within their annual
life cycle of sexual reproduction. Moistened seeds germi-
nate as spring weather warms, and juvenile plants grow
rapidly through the summer. Fast-growing juvenile
plants appear much alike, but as autumn day length
decreases, populations begin to ower and plants express
individual phenotypic differences. Male plants within a
single population are often slightly taller than female
plants. Male owers hang from branches with few leaets
and are exposed to the wind (which facilitates pollen dis-
persal), whereas owers on female plants are tightly clus-
tered with small leaets to trap male pollen grains that
fertilize the female ovules. Soon after males shed their
pollen they die. Before the arrival of killing frosts, the fer-
tilized female plants ripen viable seeds. These dissemi-
nules fall to the ground via the wind or feeding birds and
other animals that disperse the seeds inadvertently; and
then they overwinter in the soil ready to initiate another
life cycle the following spring. During their fertilized
development, each enlarging seed is surrounded by a
bract covered with thousands of secretory hairs called
glandular trichomes. These hairs produce a resinous
blend of cannabinoids (chemically related compounds
found in Cannabis) and aromatic compounds (mostly
terpene compounds common in many plants); and these
secondary metabolites are believed to protect the devel-
oping seed by repelling pests and pathogens (Clarke,
CONTACT Robert C. Clarke Rob@BioAgronomics.com BioAgronomics Group Consultants, Los Angeles, CA.
© 2016 Taylor & Francis
CRITICAL REVIEWS IN PLANT SCIENCES
2016, VOL. 35, NOS. 56, 293327
http://dx.doi.org/10.1080/07352689.2016.1267498
1977,1981; McPartland et al., 2000). Psychoactive delta-
9-tetrahydrocannabinol (THC) and nonpsychoactive
cannabidiol (CBD) are the primary cannabinoid constit-
uents in almost all Cannabis. THC and aromatic psycho-
active secretions are evolutionarily signicant as they
attracted early human attention, and at least in modern
times they remain the primary impetus for the continued
breeding and worldwide dispersal of Cannabis cultivars
(Pollan, 2001; also see Merlin, 1972; Clarke, 1998; Clarke
and Merlin, 2013).
Cannabis favors a mild climate with sufcient water
and sunlight, and early humans spread it into a range of
favorable temperate and sub-tropical niches where it
became naturalized (feral) throughout Eurasia, in parts
of Africa, and more recently in the New World. Canna-
bis thrives on the nutrient-rich dump heaps near human
occupation and has readily apparent agronomic traits,
and it was therefore preadapted to cultivation (Ander-
son, 1967; Merlin, 1972; Clarke and Merlin, 2013).
Annual plants of this genus branch freely when culti-
vated in open areas; when grown in dense stands, they
suppress branching, forming a single central stalk (Iltis,
1983). This naturally adapts Cannabis to high density
sowing for ber production, as well as low density plant-
ings that encourage branching and ower formation for
seed and drug production.
II. Cannabis diversity
Environmental factors, in concert with a plants genotype
or set of genes, more or less determine its phenotype, or
the visible expression of its genotype. Genetically and
phenotypically diverse varieties of Cannabis evolved
under the pressures of natural selection within the
diverse environments into which they were introduced,
and were further selected by humans to provide ber,
seed, or drug products. More than two centuries of stud-
ies to characterize Cannabis diversity and bring order to
this multipurpose genus resulted in a series of taxonomic
systems circumscribing one, two, or three species.
Twenty-rst-century taxonomic research by Karl Hillig
(2004a,b,2005) encompassed a wider geographical range
and diversity of population states than previous efforts,
and set the stage for understanding Cannabis evolution.
Recently proposed taxonomic systems (McPartland
et al., 2000; McPartland and Guy, 2004; Small, 2015)
share many commonalities with Hilligs conclusions, and
the evolutionary hypotheses of Clarke and Merlin (2013)
are deeply rooted in his work. Cannabis is presently con-
sidered by many, but not all taxonomists, to be a poly-
typic genus consisting of two extant species Cannabis
sativa and Cannabis indica each circumscribing several
biotypes or subspecies (see Small, 2015 for a recent,
detailed discussion of his long-held single species
hypothesis). Taxonomists also recognize three popula-
tion types for Cannabis based on natural origins and
associations with humans; those that are truly wild, those
that are cultivated, and feral escapes from cultivation that
grow spontaneously in areas associated with and often
disturbed by humans (Clarke and Merlin, 2013; Small,
2015). It is important that taxonomic systems reect
both evolutionary history and the relationships between
differing gene pools. Based largely on archaeological and
historical records backed by Hilligs chemotaxonomic
research, Clarke and Merlin (2013) published names and
acronyms circumscribing geographical and cultural
groupings as an aid to understanding the roles of differ-
ing gene pools in the domestication history of Cannabis
(see Figure 1 for acronym denitions used throughout
this review.)
Northern temperate origin, annual life cycle, dioe-
cious sexuality, camp-following and weedy tendencies,
and readily observable traits of potential value (large
seeds, strong bers, and sticky sparkling resin glands)
predisposed Cannabis to utilization, selection, and
breeding by early humans. The following section of this
review focuses on the impact of the humanCannabis
relationship upon the evolution of Cannabis as a crop
plant (Figures 1 and 2).
III. Early domestication history
The evolutionary history of Cannabis and human inter-
actions span millennia and its ongoing domestication
will continue into the future. Whether early humans rst
used Cannabis as a source of ber, food, or mind-altering
compounds, they eventually developed differing agricul-
tural techniques to increase the yield and quality of all of
these products, which led to the twentieth-century breed-
ing of Cannabis cultivars specically for ber, seed, or
drug production based on local cultural preferences. Phe-
notypic variation between Cannabis populations is well
documented in surveys of the wide range of cultivated
ber, seed, and drug varieties (e.g., Serebriakova, 1940;
Clarke, 1981). Nature and humans have worked together
to control domestication in Cannabis, but at times they
have presented opposing forces that engendered differing
outcomes. For example, isolation, articial selection, and
inbreeding imposed by humans during domestication
have limited genetic diversity, whereas natural outcross-
ing and genome mixing have encouraged genetic diver-
sity. The question remains: How did Cannabis evolve
toward domestication during cultivation and breeding,
as opposed to remnant wild populations and feral
escapes that evolved under natural selective pressures
alone? (Figure 3)
294 R. C. CLARKE AND M. D. MERLIN
Wild Cannabis populations readily yielded seed, ber,
and drugs useful to early humans. In addition to being a
multi-use plant, Cannabis is easy to grow, and must have
been included among the rst plants brought into culti-
vation, almost certainly in the regions where people orig-
inally encountered this versatile resource. As human
populations settled and expanded, they probably
depleted nearby natural stands of Cannabis, and conse-
quently early farmers began to grow plants closer to their
homes. Soon these cultivators began to sow seeds from
plants expressing traits that differed from the norm such
as larger seeds, taller stalks, and/or more resin produc-
tion (e.g., enhanced trichome production, increased tri-
chome size, elevated THC content). As a result of their
articial selection, they unwittingly initiated the
processes of domestication. As humans spread Cannabis
beyond its original wild range into recently settled agri-
cultural centers, the selective pressures of new habitats
and human requirements for varying products became
more evolutionarily important. Landraces are not simply
the products of human selections, as natural selective
pressures constantly impose their effects, but cultivation
favors the outcomes of articial human selection over
those of natural selection. Farmers and breeders select
plants with useful traits, resulting in different frequencies
of traits (both qualitative and quantitative) between wild
populations and their cultivated and increasingly domes-
ticated descendants. Qualitative morphological changes
(e.g., fruit size, stalk height, ower form and color, pres-
ence or absence of secondary metabolites, etc.) are often
Figure 1. Four groups of naturally occurring Cannabis landraces are extant today, along with sinsemilla drug cultivars and industrial
hemp cultivars (the latter are not shown). Presented here are proposed taxon names, acronyms representing their biotype groupings,
region of origin, routes of diffusion, and primary uses.
CRITICAL REVIEWS IN PLANT SCIENCES 295
Figure 2. The various Cannabis taxa (indicated by their acronyms, see Figure 1) have been spread by humans into nearly every part of
the world; PA? = putative Cannabis ancestor (from Clarke and Merlin, 2013).
Figure 3. Humans have found uses for nearly every part of the Cannabis plant and a number of traditional cultures harvested bers,
seed, and drugs from the same crop. Modern breeders developed special purpose cultivars by selecting certain plant parts (e.g., stalks,
seeds, and owers) for increased yield along with other economically valuable traits (from Clarke and Merlin, 2013).
296 R. C. CLARKE AND M. D. MERLIN
controlled by two alleles or gene states operating at a sin-
gle locus or gene position along the chromosome, and
are inherited by simple dominant versus recessive Men-
delian genetic mechanisms. Quantitative physiological
traits (e.g., increased yield, variations in amounts of sec-
ondary metabolites, etc.) are usually controlled by a suite
of alleles at several loci, their inheritance is more com-
plex, and consequently it is more difcult to improve
these traits through phenotypic selection.
Many times throughout history humans most likely
carried only a few seeds to a new settlement, and
often only a limited number of individual plants
(sometimes only one female exhibiting specicfavor-
able traits) were selected as parents. This produced
genetic bottlenecks that gave rise to populations with
restricted genomes in new areas of Cannabis growth.
When limited numbers of seeds were dispersed into
isolated regions where Cannabis was not already
growing, and the resulting small populations were
brought under new selective pressures, strong founder
effects inuenced the evolution of these new Cannabis
crops and in some cases their feral escapes. Only a
few plants of limited genetic diversity established new
populations which eventually evolved into local
landraces.
There are striking examples of founder effects in drug
Cannabis breeding. Early-maturing Afghan broad-leaet
drug (BLD) landraces were introduced into Europe and
North America in the late 1970s and early 1980s, and a
handful of psychoactively potent and early-maturing
individuals were selected and crossed with the narrow-
leaet drug (NLD) cultivars already growing in the West
to make NLD/BLD hybrids. NLD/BLD hybrid cultivars
were strongly selected for high THC content along with
early maturation and large female inorescences; this
produced potent plants when grown outdoors across
northern temperate latitudes, and thus revolutionized
domestic marijuana production. However, the major
horticultural drawback to NLD/BLD hybrids is their sus-
ceptibility to fungal infections. Their large, dense, tightly
packed female inorescences (commonly called buds)
hold moisture and make perfect microclimates for bud
rot caused by the gray mold (Botrytis cinerea Pers.),
known to ne wine makers as the noble rotof grapes.
NLD/BLD cultivars have little natural resistance to fun-
gal infection because the BLD founder plants were natu-
rally adapted to the arid conditions of Afghanistan
where pathogenic fungi cause little threat (McPartland
et al., 2000). Many NLD landraces originated from
regions with relatively more humid conditions (e.g.,
Colombia, India, Jamaica, Thailand, etc.) and evolved a
natural resistance to fungal infection. Bud rot was almost
unheard of when only NLD varieties were grown, and
now it accounts for signicant annual agricultural loses
(Figure 4).
There are no natural sterility barriers between Canna-
bis plants and crosses are generally fully fertile. There-
fore, in order to reinforce and preserve the genetic
integrity of the cultivars desirable trait expressions,
plants with variations in favorable economic traits must
rst be genetically isolated, and then continued selection
and sexual reproduction must be maintained in isolation
to reinforce and preserve those traits. Wind pollination
between separate male and female plants favors hybrid-
ization (recombination of separate gene pools through
sexual reproduction) because male and female gametes
usually must come from different parents, and thus out-
crossing is obligatory. In traditional cropping regimes,
only seeds from favorable female plants were preserved
for sowing, but the seeds on each female plant were fer-
tilized most likely by a wide range of males, thereby
restoring genetic diversity in the offspring population.
Outcrossing favors dominant alleles, leading to rapid
evolution of landraces and cultivars. Natural introgres-
sion (the exchange of genes between populations via a
hybrid intermediate) relies on backcrossing to one (or
both) of the parental populations. Plant breeding
Figure 4. The domestication process starts with individual plants
collected from naturally selected wild populations that are
brought into cultivation, and are then increasingly subjected to
human selection until they reach the cultivar stage. Some culti-
vars may escape human selection pressures, become feral, and
return to natural selection (from Clarke and Merlin, 2013).
CRITICAL REVIEWS IN PLANT SCIENCES 297
transfers genes between populations in the same manner,
and articially controlled introgression is the basis for
breeding hybrid cultivars.
When two previously isolated varieties interbreed they
form a hybrid population with more vigorous growth, a
wider variety of genetic combinations than either of the
parents, and unique phenotypes with conferred survival
advantages that drive natural evolution. During the
domestication process, hybrid offspring are articially
selected which leads to novel combinations of traits. For
example, broad-leaet hemp (BLH) landraces introduced
from East Asia into Europe and North America were
crossed with European narrow-leaet hemp (NLH) land-
races to create improved industrial hemp cultivars, while
NLD varieties introduced into North America in the
1960s and 1970s were crossed among themselves to cre-
ate improved NLD hybrid drug varieties. However, accel-
erated evolution occurred via articial selection in the
1980s when domestic NLD hybrids were crossed with
imported Afghan BLD landraces to create the lineages of
hybrid sinsemillacultivars that are widely grown today
(sin semilla in Spanish literally means without seed).
IV. Phenotypic changes during domestication
There are many examples of variability between cultivars
selected (or not) for differing traits that changed signi-
cantly with domestication. For instance, plants of hemp
ber cultivars (both NLH and BLH) produce longer
internodes and fewer branches, even when grown in
open environments, whereas seed (both NLH and BLH)
and drug varieties (both NLD and BLD) exhibit shorter
internodes and more prolic branching even when
closely sown. Traditional drug landraces produce
5.020.0% THC, whereas European hemp cultivars
contain less than 0.3% THC (Small and Marcus, 2003).
Domestication begins with the wild condition of a trait
as the norm. Unidirectional or one-way evolution pro-
ceeds by articial selection away from the wild character
state in a continuum of intermediate stages leading to a
domesticated form or function (e.g., from small to large
seeds or from low to high content of stalk ber); how-
ever, we only have sufcient archaeological evidence to
draw rm conclusions for a few physical stages of
domestication (Fleming and Clarke, 1998). Seeds are the
only propagules of sexually produced landraces. Mor-
phological changes in seed traits accompanying domesti-
cation were largely controlled by unidirectional
evolution of qualitative characteristics. When early
humans collected seeds for food or sowing, they uncon-
sciously selected for nonshattering inorescences. Persis-
tent seeds remain on harvested plants and are taken
home, whereas naturally dispersing seeds fall to the
ground and are lost. Within self-sowing spontaneous
populations there is no selection for nonshattering phe-
notypes. On the other hand, selection for nonshattering
is strong in hemp cultivars because growing ber elds
requires sowing thousands of seeds, which must be har-
vested the previous year. Traditional Nepalese landraces
are grown to produce drugs, ber, and seed. In the case
of seeds, most are eaten, with some reserved for sowing
(Clarke, 2007), and nonshattering is favored in all Nepal-
ese cultivars.
Landraces, in general, also have larger and lighter col-
ored seeds than wild or feral populations growing nearby;
in addition, the seeds of landraces also generally lack the
horseshoe-shaped base and mosaic-patterned perianth
associated with freely-shattering, camouaged, wild-type
seeds (Vavilov, 1931). The East Asian BLH gene pool
exhibits the greatest diversity in seed coloration and size,
resulting from millennia of natural and human selection
within a large geographically and culturally diverse region.
Chinese wild and feral seeds range in size from 100 to
500/g (approximately 2800 to 14,000/ounce), whereas
large snack food seeds might have as few as 15/g (approx-
imately 420/ounce), representing more than a twenty-fold
range in seed size (Clarke and Merlin, 2013)(Figure 5).
Subtle physiological changes, although less obvious,
are probably more common and possibly also more evo-
lutionarily signicant than morphological changes. In
fact, increased seed size and loss of seed dehiscence char-
acteristics are among the few morphological clues of sub-
stantial articial selection reported; however, changes in
seed physiology also accompany domestication. For
example, mechanisms for perennation (inhibition of
seed germination that allows seeds to survive through
winter before germination occurs in the spring) are often
lost. Cannabis farmers traditionally collect seeds and
store them, ensuring their survival until the following
spring, and, as a result, perennation is no longer of natu-
ral evolutionary advantage. Seeds of naturally selected
feral populations evolved in response to climate uctua-
tions and germinate slowly and unevenly, whereas culti-
var seeds are adapted to agricultural norms and must
germinate quickly and uniformly. Densely sown hemp
eld conditions automatically select for uniform and
rapid germination because late-germinating seedlings
cannot compete with already established seedlings and
are crowded out, thus allowing for more uniform treat-
ment of the crop. Cultivars that have lost their dehis-
cence mechanisms, as well as physiological means of
delaying germination, depend upon human intervention
to perpetuate thema strong sign of domestication.
Cannabis seeds also vary widely in fatty acid content
(M
olleken and Theimer, 1997) offering physiological
traits for further selective breeding.
298 R. C. CLARKE AND M. D. MERLIN
Plant architecture or gross phenotypic expression has
also been affected by domestication. Both NLH and BLH
are selected for herbaceous unbranched stalks and long
internodes associated with longer and more exible
bers, traits expressed by both male and female plants.
Hemp cultivars have also been selected for total ber
yield, and exceptional cultivars (e.g., Kompolti TC) can
produce over 40% dry weight of this product (B
ocsa,
1994). Seed-propagated cultivars (NLH, BLH, NLD, and
BLD), which are selected for maximum ower yield, are
more profusely branched with relatively woody stalks
and shorter internodes, and consequently have shorter
and more brittle bers. Articial selection of economic
traits in Cannabis is usually limited to females, which
has had a marked effect on the evolution of the genus
under domestication. However, the selection of female
plants also affects the phenotypes of their male offspring.
Indeed, males are heavily inuenced by the selective
pressures exerted on their female parent, and, during
domestication, changes in male morphology (e.g.,
shorter height, compact inorescences, and more ow-
ers) paralleled changes in female morphology.
The morphology of Cannabis inorescences also
reects extreme selection during domestication. Both
increased seed and resin gland yields rely on increased
female ower production, and both seed and drug culti-
vars have larger inorescences containing more owers
than ber cultivars, which in turn have larger inores-
cences than the wild and feral types from which they
originated. NLD/BLD hybrid sinsemilla cultivars were
selected for mind-altering potency (e.g., higher THC per-
centage, increased cannabinoid synthesis, more and
larger glandular trichomes) along with immense female
inorescences providing increased surface area for the
Figure 5. Cannabis seeds, stalks, and owers were selected for a wide number of traits that were unintentionally as well as intentionally
altered during human selection and cultivar development. Quantitative and qualitative changes occur in morphological as well as physi-
ological traits (from Clarke and Merlin, 2013). F DFruit, S DSeed, D DDrug, U DUnintentional, I DIntentional.
CRITICAL REVIEWS IN PLANT SCIENCES 299
elaboration of cannabinoid-producing glandular tri-
chomes. Because sinsemilla cultivars are reproduced
asexually by vegetative cuttings, there is no need to allow
space or divert energy to maturing seeds, and their
inorescences are more densely packed with owers and
small leaves than NLD and BLD landraces (Clarke and
Merlin, 2013)(Figure 6).
During domestication, traits more frequently diverge
from the wild condition by bidirectional or two-way evo-
lution via disruptive selectionagain resulting in a con-
tinuum of phenotypes, but with the median wild
condition lying between two domesticated extremes
(e.g., stalk internode length and branching pattern, varia-
tions in cannabinoid content, and timing of maturation).
Disruptive selection occurs when natural selection of
wild populations favors certain environmentally adaptive
traits, whereas articial selection of cultivated crops
favors a certain plant product, and leads to strong evolu-
tionary consequences during the domestication process.
Variation in the timing of oral maturation offers an
example of natural bi-directional evolution. Long before
the appearance of modern humans, the primordial Eur-
asian Cannabis gene pools were likely divided and
reformed several times during the Pleistocene as ice
sheets advanced and receded across southern Europe
and southeastern Asia. Differing photoperiod responses
naturally evolved through geographical isolation and
selection, and ecotype differences were established.
Extremely early owering, northern temperate NLH cul-
tivars (e.g., Finola) and much later owering tropical
NLD landraces (e.g., those from Colombia, India, and
Thailand) evolved under natural selection from separate
ancestral genomes that shared ancient roots in central
Eurasia. In other words, these divergent landraces
evolved by following individual routesto domestica-
tion at differing latitudes both north and south.
Another example of bidirectional evolution in Canna-
bis results from articial selection for THC levels. Early
Eurasians were presented with several non-Cannabis
choices of alternative species producing strong bers and
nutritious foods, but relatively few other known species
with psychoactive options. Revering our ancestors, creat-
ing deities, and opening cognitive channels of communi-
cation with them predate agriculture. Possibly Cannabis
was rst appreciated by early humans more for its
entheogenic (e.g., spiritual perception) or other mind-
altering powers, while its uses for ber and food became
more important later as settled populations grew. Only
in modern times has Cannabis been selected for
extremely low THC content. As noted above, the pri-
mary psychoactive constituent of Cannabis is THC, and
its content varies widely between feral as well as domesti-
cated hemp and drug populations. How did such diver-
sity in cannabinoid content evolve? Why are ber
varieties so low in THC, whereas sinsemilla cultivars
produce the most psychoactively potent marijuana the
world has ever known? (Figure 7)
Feral populations, as well as hemp and hashish culti-
vars, that are unselected for cannabinoid content express
a bell curve of CBD:THC ratios ranging from mostly
Figure 6. Both qualitative as well as quantitative traits have been strongly selected in BLD X NLD sinsemilla hybrids (from Clarke and
Merlin 2013).
300 R. C. CLARKE AND M. D. MERLIN
THC with almost no CBD (Type I) through mostly CBD
with almost no THC (Type III). However, the vast
majority of hemp and hashish populations contain
approximately equal amounts of CBD and THC (THC:
CBD 1 or Type II), which, in the absence of human
selection, may be the naturally selected and evolution-
arily neutral character state of major cannabinoid pro-
duction (Clarke and Merlin, 2013). Within intermediate
Type II (average THC:CBD 1) landrace populations,
seeds selected from the lowest THC:CBD ratio plants, as
well as those seeds selected from the highest THC:CBD
ratio plants, when sown in isolated gardens, will produce
offspring populations with median cannabinoid ratios
skewed in favor of either the high-THC (Type I) or high-
CBD (Type III) parents. When these selection processes
are repeated for a few generations, the following two dis-
tinct chemotypes or chemical phenotypes result: one
with a narrower range of cannabinoid ratios and higher
average THC content (Type I); and a second with a nar-
rower range of cannabinoid ratios and higher average
CBD content (Type III). Since psychoactive THC and
terpenes are easily detected by simple human assay, early
Cannabis farmers readily developed more potent landra-
ces by annually selecting higher potency plants and sow-
ing their seeds. Because CBD is not psychoactive, it can
only be detected by laboratory analysis, and its levels
must be increased by controlled selection and breeding.
The recent public availability of analytic laboratories has
revolutionized CBD breeding.
Hemp ber cultivars are bred to produce tall,
unbranched stalks; consequently, owerandseedproduc-
tion are relatively less important. Hemp landraces evolved
in northern latitudes with a short summer growing season
and long photoperiod that did not naturally favor long
owering periods and THC production. Natural selection
maintained THC at relatively low levels in both East
Asian BLH and European NLH landraces. Modern Euro-
pean NLH cultivars were articially selected for extremely
low levels of total cannabinoids, and especially lower
amounts of THC. Disruptive selection increased THC and
lowered CBD in drug varieties, and lowered THC produc-
tion in ber varieties, resulting in bi-directional evolution
of cannabinoid phenotype over time.
Strong heritability of alleles for THC and CBD pro-
duction (as well as nonproduction) allows for rapid
increases or decreases in potency through selection, and
there is evidence that both marijuana and hemp have
been intensely selected for varying cannabinoid levels
(McPartland and Guy, 2010). Inbred lines produce pre-
dominately either THC (Type I) or CBD (Type III), and
crosses between pure-breeding lines produce offspring of
an intermediate chemotype with approximately equal
amounts of both THC and CBD (Type II); inbreeding of
the F
1
intermediate generation produces F
2
offspring in a
Figure 7. Cannabis BLH and BLD landraces are utilized for a wide range of purposes, are generally unselected for cannabinoid ratio, and
synthesize approximately equal amounts (on a population average) of both CBDA and THCA. However, there are usually a few plants
that produce much more THCA than CBDA, and a few others that produce much more CBDA than THCA. If the high-CBDA plants and
high-THCA plants are isolated in separate breeding populations, and selection for either high-CBDA or high-THCA is continued for sev-
eral generations, descendant populations will develop either high-CBDA or high-THCA proles (from Clarke and Merlin, 2013).
CRITICAL REVIEWS IN PLANT SCIENCES 301
simple 1:2:1 ratio including »25% that produce nearly
pure THC proles (Type I), »50% of intermediate can-
nabinoid ratio (Type II), and »25% that produce almost
entirely CBD (Type III). Marijuana-type (drug) plants
inherit a highly expressed THC-acid or THCA synthase
allele; hemp-type (ber) plants inherit a highly expressed
CBD-acid or CBDA synthase allele; and intermediate
types inherit both active synthase genes. Mendelian phe-
notypic segregation in the F
2
generation indicates a pat-
tern of inheritance where THCA and CBDA synthases
are co-dominant allelic variants (B
T
and B
D
) at a single
enzymatic locus (de Meijer et al., 2003). However, subse-
quent genome research suggests that THCA and CBDA
synthases are controlled by a number of homologous
alleles at separate linked loci (Kojoma et al., 2006; van
Bakel et al., 2011).
Recent research by Weiblen et al.(2015) sheds new
light on the inheritance and evolution of cannabinoid
biosynthesis. Cannabis plants synthesize the carboxylic
acid derivatives of cannabinoids, THC-acid or THCA
and CBD-acid or CBDA, from the same precursor mole-
cule, cannabigerolic acid (CBG-acid or CBGA). How-
ever, CBDA synthase has a higher afnity for CBGA
molecules than THCA synthase; therefore, when both
synthases are active, more CBGA substrate is converted
to CBDA than THCA. Low-THC hemp cultivars do
have an active THCA synthase allele, but because they
also have an active CBDA synthase allele they produce
predominately CBD and little THC. On the other hand,
high-THC marijuana drug cultivars have both an active
THCA synthase allele and a nonfunctional CBDA syn-
thase allele (e.g., Skunk No. 1), but because the CBDA
synthase gene is nonfunctional there is no competition
between the differing alleles for substrate, and conse-
quently nearly all the CBGA is converted to THCA. This
explains why these cultivars are high in THC with little if
any CBD. Also, according to Weiblen et al.(2015), the
marijuana-type THCA synthase allele may be dominant
over the hemp-type allele, and the functional CBDA syn-
thase allele may be dominant over the nonfunctional
allele.Heterozygosity at separate loci, gene duplication,
mutation, and divergence of homologous alleles facili-
tated positive selection of cannabinoid synthase genes,
both functional and nonfunctional (especially the non-
functional CBDA synthase allele). This articial selection
precipitated key evolutionary steps leading toward
potent high-THC marijuana. Hybrid vigor, intensive
selection for more and larger glandular trichomes (Small
and Naraine, 2015), allele mutation, gene duplication,
and selection of potent alleles at an unlinked locus con-
trolling cannabinoid quantity likely also played impor-
tant roles in increasing the psychoactive potency of
sinsemilla cultivars.
Traits apparently correlated with potency, such as
later maturation, larger inorescences, and/or increased
resin production, provided clues for early humans, indi-
cating which plants were most psychoactive. It is easy to
observe which inorescences sparkle most in sunlight
and feel the stickiest, and both of these traits indicate
higher resin production and potential mind-altering
potency. Many times in various locations potent wild
and feral plants were selected as a source of seed for sow-
ing; this propelled differing gene pools on separate,
divergent evolutionary paths upon which they remained
more or less genetically isolated from other cultivated
gene pools, as well as from wild and feral populations.
Continued farmer selections for increased potency led to
higher THC content in NLD and BLD landraces. More
recently, hybridization between potent NLD and BLD
landraces resulted in modern sinsemilla cultivars with
increased vigor and potency.
Present-day North American and European drug cul-
tivars are hybrid descendants of two genetically divergent
drug Cannabis gene poolsNLD and BLD. Traditional
Asian NLD landrace farmers from Thailand, India, and
South Africa, as well as New World farmers in Mexico,
Colombia, and Jamaica, maintained varietal purity by
sampling individual plants and selecting seeds from the
most avorful and potent ones to sow the following year.
Consequently, original NLD landraces imported into
North America had a relatively high THC content with
little if any CBD. On the other hand, imported BLD
landraces from Afghanistan were relatively high in both
THC and CBD. Afghan sieved hashish (a mechanically
concentrated resin gland preparation) was collected
from entire populations, so individual plant selection for
potency was difcult and rarely practiced. As a result,
cannabinoid chemotypes of individual BLD plants varied
widely from producing nearly all THC to nearly all CBD,
with a bell curve distribution in between focused on a
median ratio of 1:1, much like unselected feral East Asian
BLH populations, but with a higher total cannabinoid
content resulting from selection for the readily apparent
traits of larger inorescences and more resin glands,
especially on the small leaves subtending the owers
(Figure 8).
Dried sinsemilla inorescences can contain more than
20% THC and/or CBD by weight. How did modern drug
cultivars become such prolic cannabinoid producers?
This unprecedented production results from a combina-
tion of simple heritability of cannabinoid prole, hybrid
vigor between evolutionarily distinct and genetically dis-
tant gene pools (NLD and BLD), and subsequent, highly
focused, human articial selection for potency. Early
NLD £BLD crosses expressing great vigor and diversity
were used to breed seed cultivars, from which present-
302 R. C. CLARKE AND M. D. MERLIN
day production clones were selected. Sinsemilla cultiva-
tion provided a rigorously selective regimen resulting in
cultivars with extremely high THC levels from their
NLD ancestry, and little if any CBD from their BLD
ancestry. Seed-propagated drug cultivars must be rigor-
ously selected to maintain their potency. Feral drug Can-
nabis populations lose potency quickly because open
pollination does not impose selective pressures favoring
high THC content, low CBD content, or favorable aro-
matic proles. The atavistic lowering of potency in natu-
ralized populations may indicate that THC is not of
great natural adaptive signicance. Apparently,
enhanced THC synthesis is primarily an artifact of
human selection, and represents another key aspect of
the ancient humanCannabis relationship (Clarke and
Merlin, 2013).
Natural evolutionary determinants were important
before humans started using and spreading Cannabis,
and natural selection will always play a role in its evolu-
tion. Domestication may have had more profound effects
on the evolution of the functional physiology of Canna-
bis rather than its anatomical physical traits, and perhaps
plants of this genus were so well preadapted for agricul-
ture that few morphological changes were required. Nev-
ertheless, human selection during domestication has had
by far the greatest inuence on changes in Cannabis phe-
notypes, both morphological and physiological. This
process accelerated during the last half of the twentieth
century as industrial hemp and marijuana breeders
developed new cultivars through vigorous articial
selection.
V. Twentieth-century cannabis breeding
Cannabis produces copious quantities of both pollen and
seed, yet it is not an easy plant to improve by selective
breeding. Cannabis populations are almost always dioe-
cious, with male and female owers occurring on sepa-
rate plants which are therefore normally incapable of
selng (self-fertilizing). Selng is the most effective sexu-
ally reproductive means of xing desirable traits, because
selected genes are more likely to be represented in both
the male pollen and the female ovule if they come from
the same plant. In traditional Cannabis breeding, reces-
sive alleles controlling a selected trait locus (aa) must be
present in two separate individuals, one male or pollen
parent (either Aa, aA, or aa) and one female or seed par-
ent (also either Aa, aA, or aa). As a result, in open-polli-
nated outcrossers such as Cannabis, qualitative traits are
usually controlled at single loci by dominant allelic forms
(AA, Aa, and aA), which are much more common than
recessives (aa) (3:1 ratio) and heritability is high. Quanti-
tative traits are more often controlled by differing allelic
forms at various loci; as a result, heritability is low, which
makes improvement by breeding more difcult. Female
plants supply most of Cannabiseconomically valuable
products, including bers, seeds, or drugs, whereas male
plants merely fertilize the females and are occasionally
harvested for their ber. This makes it difcult for plant
breeders to recognize potentially favorable traits in a
male parent, as these traits must ultimately be expressed
in female offspring. All Cannabis plants are wind-polli-
nated, which allows them to intercross freely; therefore,
in order to avoid random fertilization and seed set,
selected female seed parents must be isolated from males
until they are to be pollinated by a selected male parent.
Successful breeding of open-pollinated cultivars requires
the identication of plants with favorable traits, and then
creating breeding lines via recurrent selection, making
hybrid crosses between these lines, and eld testing their
progeny (Posselt, 2010).
Figure 8. Cannabis NLD and BLD plants vary in appearance
throughout their growth cycles. In addition to their narrower leaf-
lets, juvenile NLD plants are taller and more laxly branched than
the shorter, more compact Afghan BLD plants (from Clarke and
Merlin, 2013).
CRITICAL REVIEWS IN PLANT SCIENCES 303
It is a costly and lengthy process to develop true-
breeding Cannabis seed cultivars. In addition to a high
degree of planning, attention, and patience, a breeder
needs organization, infrastructure, and stability to pursue
crop improvement goals; and then must successfully
maintain commercial cultivars through continued selec-
tion of elite lines. Large population sizes are required in
order to select very few individual breeding plants with
favorable combinations of economically interesting char-
acteristics. Much of recent drug Cannabis domestication
has taken place secretly without legal sanctions, using
small populations with a high percentage of select survi-
vors. Under these circumstances, clandestine breeders still
made lucky crosses; however, progress is much faster with
more plants to choose from, more focused selection crite-
ria, and lower selection percentages. Commercial drug
Cannabis seeds have been widely available for more than
30 years, but continued improvements relied on the many
hobby breeders who made interesting crosses and distrib-
uted the seeds to other breeders. Although breeders oper-
ated on limited scales and in near secrecy, the sum of
their creations overshadows their individual roles; indeed,
their cumulative efforts and early crowd sourcing laid the
foundations for the plethora of asexually reproduced sin-
semilla cultivars available today.
Sexually reproducing and outcrossing Cannabis may
(by the nature of its genome randomizing reproductive
system) be resistant to morphological changes associated
with domestication in other crop plants. Until the recent
popularity of asexual propagation to ensure the perpetu-
ation of consistently high-yielding, high-potency drug
crops, Cannabis cultivars had not become completely
domesticated, but now the asexual sinsemilla varieties
survive entirely at the discretion of humans. The discus-
sion that follows documents the twentieth-century
breeding of ber, seed, and drug cultivars; this is where
we outline the pedigree and lineage of example cultivars,
emphasizing the roles of individual plants from diverse
gene pools in cultivar domestication and evolution via
human selection.
VI. Industrial hemp breeding
European NLH landraces were introduced to the New
World by sixteenth- and seventeenth-century colonists,
and eventually they became naturalized to a broad range
of local climates and soil regimes. Even though ber
yields of the early introductions were relatively low, they
were well-suited for seed production, and were the only
hemp grown in North America until the early 1890s.
During the late nineteenth and early twentieth centuries,
Japanese hemp was introduced into California, and Chi-
nese hemp into Kentucky. Both of these early
introductions from East Asia (likely broad-leaet hemp,
i.e., BLH varieties) were from a gene pool that evolved
independently from European NLH varieties. The Japa-
nese introductions were lost, whereas descendants of
Chinese introductions into Kentucky became known as
Kentuckyhemp. By the turn of the twentieth century,
the United States Department of Agriculture (USDA)
hemp breeding experiments revealed that the introduced
Chinese varieties would lose their favorable characteris-
tics of high ber quality and yield when randomly repro-
duced by farmers. Soon after seed production was
purposefully restricted to only a few isolated farms under
the attention of plant breeders, the commercial Ken-
tuckycultivar became relatively uniform and genetically
stable (Boyce, 1900). Later hybrid crosses involved the
Ferraracultivar from Italy (possibly an NLH/BLH
hybrid), Kentuckyhemp (NLH?/BLH), and additional
Chinese BLH landraces. Subsequently, further selections
were made from these complex hybrids to produce even
higher ber yield. Sadly, no known North American
hemp cultivars remain alive today, only their feral
offspring.
During the twentieth century, European hemp
breeders improved locally available, native landraces and
also made crosses with alien imports to create higher
yielding ber and seed varieties. It is evident from their
breeding histories and chemotaxonomic proles that
many present-day European cultivars share common
ancestors (de Meijer, 1995) and are less genetically
diverse (Hillig, 2004a) than their related feral popula-
tions and landraces. Crosses between select individuals
from NLH landraces belonging to the Mediterranean
and Central Russian ecotype groups (Serebriakova,
1940) formed the basic breeding lines of European hemp
cultivars. Brief summaries of their individual and shared
breeding histories are provided below: these outline the
breeding strategy employed, with an emphasis on the
interactions of differing gene pools and the effects of
individual plant selections. For a more detailed discus-
sion of European hemp cultivars, see de Meijer (1995)
and Clarke and Merlin (2013).
Open-pollinated, outcrossing plants like Cannabis are
naturally heterozygous having a higher frequency of dif-
fering alleles at the gene loci, especially for many domi-
nant traits (Aa and aA); however, they lose vigor when
they are inbred to achieve homozygosity with a higher
frequency of identical allele pairs at recessive gene loci
(AA and aa). The primary objective of breeders is to
xselected traits through homozygosity while main-
taining overall heterozygosity and hybrid vigor in the
cultivar. There are several techniques used to breed Can-
nabis cultivars. Early hemp and drug landraces were
developed through recurrent mass selection. Farmers
304 R. C. CLARKE AND M. D. MERLIN
favor homozygosity by sowing seed collected each year
from plants with agriculturally valuable traits, whereas
natural selection for health and vigor encourages hetero-
zygosity in the remaining traits. Mass selection is most
effective in improving simple qualitative characteristics
with high heritability, although persistent recurrent
selection can lead to changes in more complex quantita-
tive characters with relatively low heritability. Mass
selection is used effectively in hemp breeding to enhance
highly heritable traits such as ber and cannabinoid con-
tent (Hennink, 1994).
Mass selections or phenotypic selections are usually
made by choosing the best plants in the eld before har-
vest, and saving their seeds for sowing. Both mass family
and single plant selections are made to produce more
genetically homogenous lines for breeding. This is essen-
tially how local feral populations become landraces. Indi-
vidual female plants are selected, but a single female
plant is usually fertilized by a random assortment of
many unselected male plants, and, as a result, most of
the offspring are half siblings, rather than full siblings. In
an outcrossing plant such as Cannabis, mass selection is
more efciently imposed by identifying superior male
plants in a population prior to pollen dehiscence,
destroying all males that do not t selection criteria, and
then collecting seed only from select females. This was
how the dioecious European hemp cultivars were
selected and maintained (e.g., Carmagnola,”“Kom-
polti,”“Lovrin,and Novisadska). Recently, Chinese
hemp cultivars (e.g., YunMa 1and YunMa 5) were
mass selected from local Yunnan province landraces
(Salentijn et al., 2014).
As scientic breeding progressed, monoecious hemp
cultivars based on the Fibrimoncultivar were developed
by crossbreeding selected individuals from different land-
races and cultivars to increase variability and vigor. Cross-
breeding to combine desirable traits is achieved by
fertilizing a favorable female plant with pollen from a sin-
gle favorable male plant. The seeds are grown and the F
1
population is open-pollinated so each female is fertilized
by many males. Each selected F
1
female is evaluated by
growing out her offspring (F
2
), and favorable lines are
continued (F
2
,F
3
,) by further half-sibling selections. A
select female can also be fertilized by only one select male
from each population and then offspring will be full sib-
lings. This promotes homozygosity while further narrow-
ing genetic variability in the offspring. Single crosses
between a female and a male parent were also used to
establish most sinsemilla cultivar lineages (see below).
Monoecious plants with male and female owers, and
sub-dioecious female plants with male owers, allow
self-fertilization and inbreeding. Monoecious individuals
were used to breed monoecious hemp cultivars (e.g.,
Fibrimon) and female unisexual cultivars (e.g.,
Uniko-B). Female unisex cultivars could serve a similar
purpose as male-sterile lines if they produce no viable
pollen, have very high seed yields, and can be used to
facilitate hybrid seed multiplication. Recessive traits are
xed by serially selng sub-dioecious females, and vigor
is restored by combining two or more lines in the nal
hybrid cultivar (de Meijer, 2004). Hybrid single cannabi-
noid medical cultivars have been developed by the fol-
lowing methods: (1) selecting individual female plants
containing the target cannabinoid (or other compound)
from several genetically diverse origins; (2) using hor-
monal stimulants to form pollen-bearing male owers
on each female plant; (3) selng each female line in isola-
tion for several generations (S
1
,S
2
,S
3
,) to encourage
homozygosity and xproduction of the target com-
pound; and nally, (4) crossing genetically less related
individual inbred lines to restore vigor in the hybrid cul-
tivar (de Meijer, 2014).
The majority of present-day European hemp cultivars
are monoecious. The French cultivar Fibrimonwas
selected directly from an inbred monoecious line created
from a selfed, individual plant with both male and female
owers (monoecious or sub-dioecious) that occurred
spontaneously in a NLH population cultivated in Central
Russia. Pseudo-monoecious cultivars were developed by
crossing monoecious Fibrimonwith dioecious female
high-ber German NLH selections (also originally from
Central Russia), or with late-owering NLH landraces
from Italy or Turkey, and then backcrossing to Fibri-
mon.Genetic homogeneity of monoecious cultivars
must be maintained each year by careful selection of
monoecious parents and the creation of elite monoecious
seed. Second-generation crops sown from elite seed con-
sist almost entirely of monoecious plants, and are multi-
plied by open pollination to produce commercial sowing
seed for ber cropping. Third-generation ber crops
include up to 20% true male and female individuals
resulting from inbreeding and natural genetic drift in the
absence of human selection. Traditional dioecious land-
races were easily maintained by local farmers who pro-
duced their own seed, but today farmers must purchase
sowing seed each year because monoecious cultivars
immediately decline without careful selection and breed-
ing (de Meijer, 1995); this creates a market situation sim-
ilar to seed production of hybrid maize (or corn, Zea
mays).
Although the traditional Italian ber cultivar Car-
magnolaand its descendants Fibranova,”“Eletta Cam-
pana,and Superbraare practically unavailable today,
the Italian gene pool formed an important modern foun-
dation for both European and American hemp breeding.
East Asian BLH landrace varieties that were brought to
CRITICAL REVIEWS IN PLANT SCIENCES 305
Italy early on were responsible for much of the heterosis
effect (hybrid vigor) sought by European hemp breeders.
All of the Italian cultivars were dioecious, and most are
now believed extinct. Open-pollinated Carmagnola
was of great antiquity, having originated from a Chinese
BLH landrace, and it formed the basis of Italys famed
high-quality hemp textile production. Carmagnola Sele-
zionatawas selected directly from Carmagnola,while
Fibranovaand Eletta Campanaresulted from crosses
between traditional Carmagnolaand high-ber German
strains obtained from the Central Russian NLH landra-
ces that were also used in breeding Fibrimon.
Traditional Hungarian landraces and cultivars are mostly
dioecious. For example, Kompoltiwas selected for high
ber content from an Italian variety likely related to Car-
magnola.Intentional heterosis breeding resulted in several
hybrid cultivars. Uniko-Bis the progeny of Kompolti
crossed with monoecious Fibrimon,and rst-generation
crops of this cross consist of nearly all female plants, result-
ing in very high seed yields. Unisexual female lines produce
almost no pollen and can be used for seed parents in hybrid
seed production. Second-generation commercial ber and
seed crops produce approximately 30% males and still have
a relatively high seed yield. Kompolti Hibrid TCis a three-
way-cross hybrid in which two selections of Chinese origin
(BLH), one dioecious and the other monoecious, were
crossed to produce a unisexual, almost purely female hybrid
known as Kinai Uniszex.In the second generation, the
unisex line is used as the female parent in the crossing of
Kinai Uniszex£Kompolticultivars. This hybrid
produces commercial sowing seed of the triple-cross (pre-
dominately BLH) ber cultivar Kompolti Hibrid TCwith
a restored 50% female to 50% male sex ratio, hybrid vigor,
and increased ber yield. Kompolti S
argasz
ar
uis a dioe-
cious, chlorophyll-decient, yellow-stemmed Hungarian
paper pulp cultivar obtained from a cross between Kom-
poltiand a yellow-stemmed mutant offspring from a cross
between Finnish early-maturing NLH and Italian late-
maturing hemp (possibly an NLH/BLH hybrid); this cross
was then repeatedly backcrossed with Kompoltito trans-
fer (introgress) the yellow-stemmed trait to homozygosity
in the Kompolticultivar (Figure 9).
Polish hemp cultivars are monoecious. For example,
Bialobrzeskieresulted from serial crossings of dioecious
NLH cultivars of predominately Russian origin that were
nally crossed with Fibrimon,followed by long-term
single line selections for high ber content. Another Pol-
ish hemp cultivar, Beniko,was obtained by individual
progeny selection from a Fibrimonhybrid.
Romanian Fibramulta 151is a dioecious selection
from a single cross between ICAR 42118and German
Fibridia.The parent variety of ICAR 42118was the
progeny of an Italian Carmagnola£Bologna
(another Italian cultivar) hybrid crossed with the Turkish
Kastamonulandrace. The dioecious Lovrin 110culti-
var was bred by selection among family groups from the
Bulgarian SilistrenskiNLH landrace. Monoecious
Secuieni 1was derived from the crossing of Dneprov-
skaya 4£Fibrimon,followed by two semi-back-
crosses with Fibrimon 21and Fibrimon 24,
Figure 9. Four ber hemp cultivars can be visually distinguished in this eld trial. The Hungarian cultivar Kompolti S
argasz
ar
uor yel-
low-stemmed Kompoltiis a reduced stem chlorophyll variety that was bred as feedstock for the paper industry (from Clarke and Merlin,
2013).
306 R. C. CLARKE AND M. D. MERLIN
respectively. The Russian dioecious parent Dneprov-
skaya 4was a descendant from Italian hemp.
Eight cultivars are presently grownincentralandsouth-
ern parts of Ukraine and several regions of Russia, where
the history of traditional and commercial hemp ber and
seed cultivation is very lengthy. Hemp cultivars from the
former USSR are classied into maturity groups or geo-
graphical types (Serebriakova, 1940;deMeijer,1995). Cur-
rent cultivars belong to either the southern, later-maturing
group or to hybrid progenies from an earlier-maturing cen-
tral group crossed with later-maturing southern hemp;
hybrid cultivars of the latter group are intended for sowing
in higher latitudes of central and northern Europe than
those ecologically preadapted to southern Europe. The dioe-
cious southern Russian cultivar Kubanwas obtained by
ten cycles of family group selection in the hybrid population
by the crossing of Szegedi 9£Krasnodarskaya 56culti-
vars. Szegedi 9was selected in Hungary from the native
Tiborsz
all
asilandrace (NLH) and crossed with Krasno-
darskaya 56,which probably came from a cross between a
local Caucasian (NLH) landrace and an Italian cultivar. The
two other cultivars cultivated in areas of Ukraine and Russia
are Zenica,adioecioussouthwesternEurasiancultivar,
and the NLH landrace Ermakovskaya Mestnaya,which is
cultivated on a signicant scale in Siberia and belongs to the
Central Russian maturity group (Serebriakova, 1940).
The remaining Russian and Ukrainian monoecious
cultivars exhibit a southern, later-maturing growth pat-
tern, but are also cultivated at higher latitudes to pro-
mote a longer vegetative growth period, resulting in
taller stalks and increased ber yield. These cultivars
include USO-11bred from three parental populations,
Dneprovskaya 4,”“USO-21,and Dneprovskaya
Odnodomnaya 6.”“USO-11is presently grown in
Canada and New Zealand for oil seed production. The
remaining Ukrainian cultivars were produced by cross-
ing various Russian landrace accessions with cultivars
originating in Italy and France (de Meijer, 1995).
Finolais an early-maturing Finnish variety used for
seed production in more northern latitudes and was reg-
istered in the European Union (EU) in 1999. It is grown
extensively in Canada for seed and seed oil production
(Small and Marcus, 2002). Finolawas selected from
the pooled seed of two nearly identical, early-ripening,
far northern Russian NLH landraces originally obtained
from the Vavilov Research Institute (VIR) in St. Peters-
burg, Russia. The Canadian seed variety Ankawas also
selected from VIR accessions. Finolamatures early and
provides both high seed yields (up to 1.7 metric tons per
hectare or 1500 pounds per acre) and short straw
(stalks); in addition, it can be harvested by combine har-
vesters for both ber and seed yields from the same crop
(Weightman and Kindred, 2005).
European hemp breeders developed more uniform
monoecious varieties with increased resistance to various
pests (McPartland et al.,2000), while also focusing on
further reducing already negligible levels of THC. The
French cultivar Santhicawas selected from a single
plant with no THC and high CBG content; and it may
lack the THC synthase gene (B
T
allele) (de Meijer, 2004),
but it has not been released commercially. Although
research institutes might develop new hemp cultivars for
specic uses, government organizations and/or seed
companies continue to reproduce and sell sowing seed of
existing varieties and traditional farmers continue to
propagate their landrace seed. As long as hemp remains
an economically viable albeit minor crop, hemp cultivars
will be multiplied; however, as soon as economic interest
wanes, they will most likely become extinct.
Present-day European industrial hemp cultivars share
common ancestors. Most ber cultivars originated from
Carmagnola,a traditional hybrid Chinese-Italian BLH/
NLH dioecious cultivar that was combined with various
Central Russian NLH landraces. On the other hand,
while nearly all monoecious varieties are descendants
from French Fibrimon,a single, inbred, monoecious
cultivar that was also derived from a Central Russian
landrace. Genes of the modern industrial hemp cultivars
came from two taxonomically distinct Cannabis groups,
European NLH and Asian BLH, but via only a handful
of individual parents and hybrid crosses. Hemp cultivars
are maintained as select elite populations, which ensures
inbreeding to preserve favorable traits; some outcrossing
is allowed to encourage health and vigor, but all within a
heavily selected and fundamentally restricted genome
preserved by intensive articial selection.
Early in the twentieth century, Chinese BLH landraces
were incorporated into North American hybrid hemp
cultivars, and were later used by European breeders to
make hybrid crosses with NLH landraces of European
origin. The high degree of genetic difference between
East Asian BLH and European NLH gene pools was
illustrated by Hillig (2004a) who placed them in subspe-
cies of different speciesC. indica ssp. chinensis and C.
sativa ssp. sativa, respectively. The heterosis effect or
hybrid vigor resulting from crossing plants of diverse
genotypes was important in the development of all mod-
ern ber cultivars. As we will see below, heterosis breed-
ing was also a key factor in the initial development of
present-day drug cultivars.
VII. Sinsemilla cultivars
Early humans repeatedly introduced NLD landraces that
originated in southern Asia into unusual geographical
regions. Many of these introductions evolved into new, local
CRITICAL REVIEWS IN PLANT SCIENCES 307
landraces that eventually (sometimes centuries later)
entered the international market. In the 1960s and early
1970s, all commercial marijuana imported into North
America and Europe owed its heritage to the vast NLD
group, and seeds of many different landraces were available
in relatively large amounts. Seeds from Colombian and Thai
marijuana grown at more nearly equatorial latitudes rarely
produced plants that matured to the oral stage when culti-
vated outdoors at temperate latitudes before cold autumn
weather killed them, whereas northern Mexican and Jamai-
can varieties often matured earlier, before harsh winter
weather set in. The domesticated NLD hybrid varieties of
the early and mid-1970s originated largely from crosses
between Mexican or Jamaican landraces and more potent,
but later ripening, Colombian, Indian, Panamanian, and/or
Thai landraces (Figure 10).
Traditional Asian and New World drug Cannabis cul-
tures favored and selected potent landrace varieties that in
turn provided North American and European breeders with
the basic genetic building blocks for developing sinsemilla
varieties. In the early 1970s, gardeners began to grow sinse-
milla marijuana by removing all male plants from their
elds, leaving only the unfertilized (therefore seedless)
female plants awaiting pollination, each producing thou-
sands of oral bracts covered by a myriad of resin glands.
Sinsemilla cultivation also brought growers to the realization
that a few select male plants couldbeisolatedfromwhich
they could collect pollen to intentionally fertilize owers on
a few female branches. This in turn produced seeds of
known parentage which essentially gave birth to the inten-
tional breeding of potent hybrid drug Cannabis. Early clan-
destine breeders combined and recombined imported NLD
landraces in multi-hybrid crosses, and by 1980 North Amer-
ican sinsemilla ranked among the worlds most potent drug
cannabis.
Traditionally, both hemp and drug cultivars were
developed by variations on the theme of mass selection,
but modern sinsemilla cultivars evolved in a different
way from hemp cultivars. Sinsemilla cultivars were usu-
ally developed by crossing a single male of one geneti-
cally distinct landrace with a single female of another
landrace (e.g., Mexican NLD £Colombian NLD) to cre-
ate an F
1
hybrid. In the subsequent F
1
generation,
selected male or female offspring were bred by following
one of three basic pathways: (1) they were inbred with
one or more siblings (e.g., Mexican/Colombian £Mexi-
can/Colombian) to establish a heterozygous and rela-
tively inconsistent F
2
population hybrid used for
subsequent mass selection to increase homozygosity and
uniformity; (2) they were backcrossed with an aunt
(seed parent) or uncle(pollen parent) (e.g., Mexican/
Colombian £Colombian) to increase homozygosity and
enhance specic traits before establishing a backcross
population hybrid used for subsequent mass selection;
(3) they were outcrossed with an unrelated plant (e.g.,
Mexican/Colombian £Thai) to create a new F
1
hybrid
serial generation. Strategies one and two were followed
by some early sinsemilla breeders, but pathway three was
by far the most frequently pursued. By introducing a
new exotic landrace or hybrid parent into the breeding
line (e.g., [Mexican/Colombian £Thai] £Jamaican
landrace), heterozygosity and diversity were increased by
creating a new F
1
hybrid in each serial breeding genera-
tion. Although the offspring looked less and less like any
Figure 10. Narrow-leaet drug (NLD) type Cannabis, closely related to this late-nineteenth-century Indian ganja, was spread from South
Asia through Africa and eventually to the New World (from Clarke and Merlin, 2013).
308 R. C. CLARKE AND M. D. MERLIN
of their increasingly distant ancestors, a level of relative
homogeneity between siblings persisted because in each
new generation half of the gene pool was contributed by
a single parent. Although serial hybridization was not an
intentional breeding strategy, the heterozygosity breeders
unwittingly created a cultivation process that has played
an important role in the continued survival of the inces-
tuously recombined drug Cannabis gene pool
(Figure 11).
In all three paths of drug cultivar breeding described
above, landraces were crossed to make hybrid popula-
tions. However, no true hybrid seed varieties were cre-
ated because no pure-breeding lines were maintained to
use each year as parents of the F
1
hybrids. Only more
recently have sinsemilla seed companies multiplied true
hybrids by preserving asexually reproduced vegetative
clones of select male and female parent plants. However,
the clonal parental lines are often highly heterozygous,
and their hybridoffspring are frequently inconsistent
phenotypically.
Sinsemilla breeders have selected primarily for stron-
ger potency (higher THC content) as well as complex
aromas and avors, which are all traits related to ter-
peno-phenolic secondary product metabolism in the
glandular trichomes (e.g., cannabinoids and terpenoids).
Initial serial hybridization followed by careful inbreeding
(pathway 1, described above) and/or backcrossing (path-
way 2) of hybrid crosses resulted in some of the early
NLD varieties that are still popular today (e.g., Original
Haze,”“Big Sur Holy Weed,etc.). During the late
1970s, many clandestine marijuana breeders successfully
developed early-maturing and high-yielding, potent, aro-
matic and colorful connoisseur NLD varieties, and as a
consequence demand for high-quality sinsemilla grew.
Soon an exotic new series of cultivar introductions would
dramatically change the face of sinsemilla breeding and
the evolution of domesticated drug Cannabis.
VIII. Introduction of the Afghan cultivars
Broad-leaet drug (BLD) seeds were introduced from
Afghanistan into North America and Europe several
times from the late 1970s through the 1980s. BLD plants
were very distinctive in appearance; being well-branched
and standing only one to two meters (about three to six
feet) tall at maturity, they were shorter and bushier than
NLD plants with broader, darker green leaets. After
BLD varieties were introduced in the late 1970s, growers
commonly began to call the original NLD varieties sati-
vasbecause their tall growth and narrower, lighter green
leaets more closely resembled NLH ber varieties than
they did the more recently introduced shorter and darker
green Afghan BLD hashish varieties commonly called
indicas.The authors now assume that NLD and BLD
populations belong to different subspecies of C. indica,
subspecies indica (NLD) and subspecies afghanica
(BLD).
Afghan BLD plants mature earlier than most NLD
cultivars at northern temperate latitudes (from late
August through September) providing an abundant
source of highly psychoactive resin traditionally used to
make Afghan hashish (Clarke, 1998). Seedless BLD ow-
ers smelled and tasted much like potent hashish, and
connoisseurs paid premium prices for such an exotic
contraband. Breeders crossed BLD varieties with their
sweet-tasting, but later-maturing, NLD varieties to pro-
duce early-maturing BLD £NLD hybrids, commonly
called indica/sativahybrids (in our view, hybrids of dif-
ferent subspecies of C. indica). Hybrid vigor was strong,
and ower yields increased, yet plants rarely exceeded
3 m (10 feet) in height. Afghan £Thai hybrids proved
particularly pungent and extremely potent. By the mid-
1980s, the vast majority of sinsemilla in North America
owed a portion of its heritage to the BLD gene pool.
Thereafter, it became increasingly difcult to nd either
pure NLD (pre-BLD) varieties or the pure BLD introduc-
tions popularized only a few years earlier. Australian and
Pacic Island outdoor varieties were often based on
Figure 11. North American and European marijuana growers
crossed NLD varieties from Mexico (above) with NLD varieties
from many other locations (e.g., Colombia, Jamaica, Thailand,
and India), and these became the rst hybrid home-grownsin-
semilla cultivars (from Clarke and Merlin, 2013).
CRITICAL REVIEWS IN PLANT SCIENCES 309
Southeast Asian landraces introduced in the 1970s, and
until more recently were less affected by BLD/NLH
introductions and indoor surreptitious cultivation
(Figure 12).
During the 1970s and 1980s, relatively early in the
evolution of modern-day sinsemilla cultivars, NLD seeds
inadvertently imported in illicit marijuana were far more
commonly sown than articially selected hybrids pro-
duced by domestic growers. Even after the introduction
of Afghan cultivars, rare, intentionally bred seeds were
sometimes passed from one breeder to another, but their
distribution was quite limited. During the mid-1980s,
seed companies began to distribute selected varieties, but
accidentally produced seeds from illicit commercial sin-
semilla were still much more commonly grown. Seeds
carrying varying proportions of the NLD and BLD gene
pools were grown and crossed repeatedly without con-
scious selection; this inadvertently produced multi-cross
hybrids in which culturally favorable traits were rarely
selected and thus the average psychoactive and aromatic
qualities of domestic marijuana decreased.
Most of the offspring derived from these crosses were
dissimilar to their parents and their siblings, in both
appearance and potency because their genomes consisted
of small random pieces of genetic information inherited
from assorted predecessors, and undesirable
characteristics that previously had been suppressed
through careful selection were often expressed (e.g.,
lower potency, dull psychoactive effects, acrid aroma,
harsh taste, etc.). Many of the early NLD/BLD cultivars
offered hardy growth, rapid maturation, and tolerance to
cold, allowing sinsemilla to be grown outdoors in many
northern temperate climates. This revolutionized the
domestic marijuana market both geographically and cul-
turally by widening the scope and popularity of outdoor
sinsemilla cultivation. NLD/BLD hybrids not only
changed Western outdoor growing, but their rapid matu-
ration time, short stature, and high yields also pre-
adapted them to indoor growing as well. Tall and late-
maturing NLD cultivars were poorly suited for articial
light growing, but shorter and faster ripening NLD/BLD
hybrids were ideal for commercial indoor production.
The combination of increased prohibition pressure,
improved lighting systems, asexually propagated indoor
cultivars, and high marijuana prices fueled the rapid,
very widespread strategy of cultivating sinsemilla indoors
in many regions of the world. The rampant success of
this agricultural model narrowed the selection criteria of
breeders toward developing only indoor cultivars, further
limiting the present-day genetic diversity of drug Canna-
bis. Although consumers and commercial growers
largely accepted NLD/BLD hybrids, serious sinsemilla
breeders began to view them with more skepticism, and
started to develop new drug cultivars for a wider variety
of applications (Figure 13).
IX. Present status of the Cannabis genome
As a result of millennia of dissemination and contact with
differing human cultures worldwide, Cannabis plants,
through natural and articial selection, have evolved huge
genetic diversity which is expressed in a remarkably wide
range of phenotypes. Traditional farmers in geographically
isolated regions selected and maintained genetically unique
landraces that formed the foundation for breeding modern
hybrids. The Cannabis gene pool is amazingly diverse; how-
ever, breederswell-intentioned domestication efforts have
been strongly affected by the repercussions of prohibition
and consequently its genetic diversity is increasingly being
attenuated.
The vast majority of European industrial hemp culti-
vars are based on the same few single plant selections;
and the drug Cannabis gene pool has reached a genetic
bottleneck caused by incest breeding between close rela-
tives, intentional and unintentional selng, highly
directed selection for drug potency, and the preponder-
ance of vegetatively reproduced commercial populations.
The genetic diversity of Cannabis is constantly reduced
by persistent law enforcement seizures, accidental
Figure 12. Broad-leaet drug (BLD) Afghan hashish cultivars
were introduced into Western Europe and North America in the
late 1970s, and hybrid crosses with NLD cultivars changed
domestic drug Cannabis production forever. This large BLD plant
is growing in a family garden in northern Afghanistan (from
Clarke and Merlin, 2013).
310 R. C. CLARKE AND M. D. MERLIN
agricultural mishaps, and the gradual culling of economi-
cally unfavorable clonal lines; commercially valuable
clones are propagated, and the remainders are destroyed.
If we project present trends into the future, genetic diver-
sity will continue to decrease, the lack of sexual recombi-
nation will lower the potential for evolving pest
resistance, and susceptibility to agricultural pests and
diseases will continue to increase. When pathogenic
organisms such as viruses, fungi, mites, and aphids attack
a genetically uniform, asexually reproduced crop, losses
are often extensive. Illegal drug Cannabis cultivation and
breeding have historically suffered from similar problem-
atic pest infestations that hindered and often over-
whelmed the successful cultivation of other monocrops
of commercially valuable plant species.
Devastating agricultural blights have indeed caused
great economic losses in several different crops, and are
directly associated with diminished genetic variability
leading to susceptibility to a pest or pathogen. Susceptibil-
ity to pathogens can arise in two ways. When a cultivar is
introduced to a new region it may be infected by intro-
duced alien organisms for which it has no naturally
evolved resistance (e.g., Afghan BLD cultivars parasitized
by gray mold in humid regions of North America). Alien
pests have also been introduced into new regions where
Cannabis crops have no natural resistance (e.g., the
broomrape parasite introduced to Kentucky from China
where it caused widespread damage to hemp crops, and
hemp russet mites introduced to Indiana along with South
Asian seed stocks). McPartland et al.(2000)provides
more detailed histories of Cannabis pests and diseases.
History presents us with several different serious
blight scenarios. The mid-nineteenth-century Great
Famine of northern Europe was caused by a virulent
strain of Phytophthora infestans, a parasitic and nonpho-
tosynthetic fungus-like organism, which is closely related
to brown algae; P. infestans commonly infects crop
plants, but not Cannabis (McPartland et al., 2000). The
virulent HERB-1 strain of P. infestans, that causes the
potato blight, originated in Mexico; it subsequently
spread into North America where it caused widespread
damage to potato crops, and then, in 1844, it traveled by
ship to Europe where the infection was rapidly dispersed.
Regions where a wider variety of potato cultivars were
being cultivated were not as severely affected, but the
majority of potatoes in Ireland were of the Irish
Lumpervariety, which proved to be particularly suscep-
tible. The Irish potato crop loss in 1845 is estimated at
between one- third and one-half of cultivated acreage,
and in the next year three quarters were lost to blight.
Nearly a million people starved to death, and another
two million emigrated, many to the United States. Potato
cultivars are asexually multiplied by dividing tubers, so
every offspring is genetically identical to its parent plant,
and therefore all share the same susceptibility to pests
and diseases. Periodic failures of the northern European
potato crop persisted until the early twentieth century
when breeders produced potato cultivars resistant to
HERB-1. Drug Cannabis clones are asexually multiplied
by cuttings, and like potato cultivars all the plants within
each clone are equally susceptible to pests and diseases,
which, as we well know, spread rapidly in monoculture
crops (Kelly, 2013).
During the mid-nineteenth century, the Great Wine
Blight affected many vineyards and devastated the Euro-
pean wine industry. During the third quarter of the nine-
teenth century more than 40% of French vineyards were
destroyed, and the suffering economy also accounted for
increased emigration to North America. The blight was
caused by the phylloxera aphid (Daktulosphaira vitifo-
liae) that originated in eastern North America, where it
was a well-known grape (Vitis) root parasite. North
American Vitis rootstocks had been introduced to
Europe years before, but they did not carry the pest. In
the late 1850s, phylloxera-infested Vitis rootstocks were
transported across the Atlantic by steamship, allowing
the pest to reach Europe alive, where it spread rapidly
Figure 13. Female Cannabis owers produce a preponderance of
glandular trichomes or resin glands that secrete a cannabinoid-
and terpene-rich essential oil. Selection for high production of
resin glands and essential oil is the driving force in modern-day
drug Cannabis breeding, to the virtual exclusion of other agro-
nomically valuable traits (Photo by Todd McCormick from Clarke
and Merlin, 2013).
CRITICAL REVIEWS IN PLANT SCIENCES 311
through European vineyards of traditional Vitis vinifera
wine cultivars that had not evolved natural immunity to
a North American pest. Grape breeders crossed suscepti-
ble European wine cultivars with phylloxera-resistant
native North American species, but the offspring were
not particularly resistant to phylloxera and they made
poor wine. The solution lay in reintroducing phylloxera-
resistant rootstocks from North America (Vitis aestivalis
or other American native species) and replacing the sus-
ceptible V. vinifera rootstocks. Following considerable
resistance by traditional French wineries, endangered
French wine grape scions were successfully grafted onto
rootstocks of resistant North American species and the
European wine industry was resurrected (Ordish, 1987).
Traditionally, the vine plants of table and wine grapes
have been vegetatively propagated by grafting shoots, much
the way modern sinsemilla varieties are propagated by root-
ingcuttings.ArecentgeneticsurveybyMyleset al.(2010)
of grape accessions in the USDA collection came to the fol-
lowing conclusion: We propose that the adoption of vege-
tative propagation was a double-edged sword: Although it
provided a benetbyensuringtruebreedingcultivars,it
also discouraged the generation of unique cultivars through
crosses. The grape currently faces severe pathogen pressures,
and the long-term sustainability of the grape and wine
industries will rely on the exploitation of the grapestremen-
dous natural genetic diversity.
In the early 1900s, the American chestnut blight,
caused by a wind-borne fungus (Cryphonectria para-
sitica), decimated American chestnut tree forests before
eventually spreading to Europe. This aggressive fungus
arrived in North America with infected Chinese orna-
mental chestnut trees that were resistant to the disease.
The blight is spread by wind-borne pathogenic spores
that moved the fungus quickly through dense native
chestnut stands, eventually killing an estimated 40 billion
trees; every chestnut tree in the contiguous forests of the
eastern United States succumbed to the disease in less
than 40 years (Freinkel, 2007). The only survivors were
apparently resistant trees or those in small outlying pop-
ulations that the disease did not reach. Subsequently, an
isolated, disease-resistant chestnut tree from Ohio that
had survived was crossed with three naturally resistant
Chinese cultivars. The resulting hybrids remained dis-
ease free, but were shorter than the average American
chestnut trees. Breeding efforts continued by backcross-
ing the hybrid offspring three times to the American
chestnut to transfer the genes for disease resistance to a
cultivar with the taller American chestnut phenotype.
Southern corn leaf blight (SCLB) is a fungal disease
found in many of the worlds maize-growing areas. This
blight is caused by Bipolaris maydis, which occurs in
three genetically variant races. Race T infects maize
plants with Texas male-sterile cytoplasm (Tcms), which
contains a gene causing susceptibility to the race T fun-
gus. Maize plants are monoecious and self-pollinating;
and before this susceptibility was clearly understood,
breeders encouraged a disastrous fungus invasion by
unwitting selection of maize plants infected with Tems
cytoplasm.
During the traditional multiplication of hybrid maize
seed, farmers remove the male owers (tassels) from the
female seed plants by hand so they will not self-fertilize,
and therefore will only receive pollen from monoecious
plants grown in the same eld. Removing tassels is time-
consuming and as a result it provides summer employ-
ment for students, but it is costly and prone to human
error; consequently, in the early 1960s, breeders devel-
oped lines with Tcms to be used as male-sterile seed
parents in hybrid maize seed production. Hybrid sowing
seed rapidly gained popularity, and by the late 1960s an
estimated 90% of the American crop contained Tcms
cytoplasm, making it extremely vulnerable to SCLB. By
1970, SCLB reached epidemic proportions destroying
15% of Americas corn production with losses estimated
at one billion dollars. The SCLB epidemic occurred
because female seed lines carried an unknown male-
linked trait that conferred disease susceptibility to a huge
percentage of the crop. As a result, farmers returned to
detasseling hybrid seed crops until breeders selected
SCLB resistant cultivars and found sources of male steril-
ity other than Tcms (Agrios, 2005). Male-
sterile seed lines could be used in monoecious hemp
breeding, and growing cuttings of sterile high-THC
females would guarantee that drug crops would remain
seedless wherever they are grown, offering another
advantage to sinsemilla growers.
In 1890, a wilting disease of banana was observed in
the Gros Michelplantation crops of Costa Rica and
Panama that reached epidemic levels throughout the
early 1900s. This precipitated catastrophic damage, forc-
ing banana plantations to move to new regions where
the disease had not yet spread. In 1910, the causal agent
of Panama diseasein Cuban bananas was linked to the
soilborne wilt fungus, Fusarium oxysporum f. spp.
cubense (Foc wilt). Cannabis is also susceptible to closely
related Fusarium wilts (McPartland et al., 2000). The
entire dessert banana trade was based on the vegetatively
reproduced Gros Michel(Big Mike) cultivar that had
no resistance to the Foc wilt, and by the 1960s was
almost entirely wiped-out. A search for banana varieties
resistant to Foc wilt revealed specimens from botanical
gardens in the United Kingdom and the United Fruit
collection in Honduras. These were vegetatively repro-
duced by tissue culture, and by the late 1960s two closely
related Cavendishclones, Dwarf Cavendishand
312 R. C. CLARKE AND M. D. MERLIN
Grand Nain,were widely spread throughout the com-
mercial banana producing areas of Central America and
beyond. Presently, these two clones account for nearly
50% of global banana production (http://www.promusa.
org/TropicalCraceC4C-CTR4). Also in the 1960s, Pan-
ama disease was rst noticed in Taiwanese Cavendish
bananas. Subsequently, the Panama disease became a
very widespread and serious problem in various areas of
the world; it destroyed Taiwans banana industry, spread
through southern China, wiped out plantations in Indo-
nesia and Malaysia, threatens the Australian and Philip-
pine industries, and has also been identied in Middle
Eastern and East African plantations. Unfortunately,
Panama disease spreads easily and potentially endangers
monoculture banana production everywhere. Regretta-
bly, the banana industry has been slow to respond to the
renewed threat of Panama disease, and presently there is
no market replacement for the Cavendishcultivars.
Another pest infestation threatening plantations of
bananas is the virulent Foc wilt strain known as Tropical
Race 4 (TR4) that was not identied until 1994. TR4 is
now spreading rapidly, infecting susceptible plantations
of Cavendishclones, all of which are asexually repro-
duced and have no chance to develop resistance to TR4
(http://www.promusa.org/TropicalCraceC4C-CTR4).
The blights described briey above were caused by
different pests and diseases affecting various wild and
cultivated plant species, but their rampant spread
relied on one key factorthe underlying cause of all
these epidemics has been a lack of genetic diversity.
The Irish Lumperpotato was an asexually repro-
duced clone; French varietal wine grapes shared com-
mon ancestors and the roots of all were susceptible to
phylloxera; genetically similar American chestnut trees
formed huge contiguous forests that all perished; the
American corn blight infected all hybrid maize culti-
vars that shared Tcms cytoplasm; and Cavendish
bananas are all members of two closely related clones.
These historically documented disasters highlight the
need to further understand the diversity and present
status of the Cannabis genome, and should give us
pause for thought.
By the dawn of the twenty-rst century, it was appar-
ent that Dutch sinsemilla varieties were predominantly
based on three founding Pacic coast varieties: North-
ern Lights,”“Skunk No. 1,and Haze(Clarke, 2001).
Dutch, British, and Spanish companies are the largest
suppliers of drug Cannabis seeds today, mostly selling
multi-hybrid combinations of oldvarieties. In the
twenty-rst century, many newvarieties have been
incorporated into the modern collective gene pool; how-
ever, many of them include reassorted alleles from the
seminal Cannabis varieties distributed far and wide
30 years ago. What appear as novel combinations today
reect a breakdown of suites of characteristic landrace
traits, and the persistent reshufing of individual land-
race genes in modern multi-hybrid cultivars. Over many
generations, sinsemilla cultivars have become hybridized
to the point of inbreeding and are increasingly similar
genetically to one another.
Because Cannabis can be a difcult plant in which to
x traits through selective breeding, and only female
plants are of economic importance for drug production,
asexual reproduction or cloningby rooting vegetative
female cuttings provides an easy solution for rapidly
expanding commercial sinsemilla production. Under
this female vegetative planting regime, there are no male
plants to remove, the crop can be treated uniformly, all
the plants ripen at the same time, and they can be har-
vested together. If the electricity grid does not fail, grow
rooms can produce three to four crops per year, and
yield over 450 g (one pound) of dry owers per square
meter (about 10 square feet) each harvest. Male plants in
breeding programs can also be preserved in a vegetative
libraryunder long photoperiod and induced to ower
when pollen is required. Cloning radically changed Can-
nabis agriculture (and selective breeding pressures) by
making sinsemilla growing possible and protable for
hobbyists. However, in one generation, asexual cloning
xes traits forever (apart from articially or naturally
induced mutations) and attenuates the genetic diversity
promoted by sexual seed reproduction (Figure 14).
Reliance on only a few select male and female cuttings
for seed production reduces genetic diversity. The number
of female clones used as seed parents in sinsemilla breed-
ing is now limited, but even fewer pollen parents are used
to make commercial seed. Seed varieties and clones are
most often named after their female seed parent, but
equally important is what plant was used as a pollen par-
ent. Male parents that consistently produce uniform off-
spring are much harder to nd than female parents,
which are often maintained as commercial cultivars. Indi-
vidual male plants are often selected for phenotypic traits
resembling females (e.g., increased vigor, short internodes,
profuse branching, dense inorescences, strong aroma,
etc.). However, in order to identify which male clone will
produce the best offspring, each of these male clones
must be crossed with a range of female cuttings. The seeds
from each of these crosses must then be progeny-tested to
check for quality and consistency. For example, if ten pro-
spective male clones are each crossed with ten individual
female clones, and 200 seeds (approximately 100 will be
female) of each test cross are sown, about 10,000 female
plants will be produced, and then they must be screened
to determine which male and female clone combinations
produced the best offspring.
CRITICAL REVIEWS IN PLANT SCIENCES 313
A brief summary of the breeding of Skunk No. 1will
illustrate what is involved in developing a relatively true-
breeding seed cultivar. Several plants were grown from a
local California skunkvariety, which was likely a
3
/
4
NLD and 1
=
4BLD hybrid (e.g., Colombian/Afghan £
Mexican) and then all the females were crossed with a
single select male. A female identied as skunk plant
number one was selected as the highest yielding and
most potent, and became the founder of all subsequent
generations. For the following nine growing seasons, at
least one branch of each of the (up to 100) female plants
was fertilized by pollen from a male selected from the
offspring of the previous years best female plant. Seeds
from the select females were sown the following spring.
After only two or three generations, Skunk No. 1was
relatively homogenous and true-breeding compared to
other hybrid lines that continued to segregate in the F
3
and F
4
generations, and Skunk No. 1was then deemed
ready for large-scale selection of parent plants with spe-
cic combining ability (SCA) for subsequent breeding.
A total of nearly twenty thousand plants were grown
in a common garden from the seeds of ten females from
the most promising lines selected from the previous gen-
eration, and the ten best females from among all those
lines were selected based on vigor, potency, type of effect,
ower yield, high ower to leaf ratio, resin gland
development, amount of branching and pest resistance,
as well as attractive oral aromas and avors. Ten males
were also selected for their vigor, pest resistance, female-
type growth form, and aroma. All 20 selections (male
and female) were reproduced asexually to preserve their
unique genotypes.
Ten clonal copies were made of each of the ten select
females. Pollen was collected from each of the ten select
male clones, and pollen from a single male was used to
fertilize a single copy of each female clone, resulting in
100 individual crosses. Two hundred seeds of each cross
were sown (20,000 total) and approximately 100 female
progeny of each cross were evaluated for phenotypic
consistency (homozygosity) of their favorable agronomic
traits (see above). Five female and three male clones
were selected and used for Skunk No. 1sowing seed
multiplication as well as hybrid cultivar development
(Figure 15).
Few sinsemilla seed companies invest as much time
and money in selecting superior male plants as they do
for specially selected females. Most simply select a male
plant based on visual characteristics alone without any
chemical analyses, and cross that individual male with
each of their female clones to produce commercial
hybrid seed. These seeds (all containing genes from a sin-
gle male parent) are then widely distributed and grown
to maturity, and from which female cuttings are then
selected for commercial sinsemilla production. Commer-
cial Cannabis seed production can be compared gura-
tively with historical human conquests. A dominant
invading male defeats all his rivals, procreates with select
captive women, selects one son as his chosen heir, and
marries his choicest daughters off to distant lands;
thereby ensuring his genetic dominance at home, while
spreading his genes far and wide.
More recently, all-femalecultivars have been pro-
duced by transforming a female plant (XX) with hor-
mone applications to produce male owers with viable
female (XX) pollen. The offspring of female plants fertil-
ized with female pollen are all female. Every seed produ-
ces a useful female plant and there is no need to cull
male (XY) plants, which provides the advantages of asex-
ual propagation, but in the convenience of a seed. Female
seeds lower genetic diversity for the same reason as
female cuttings, because there are no male plants, and
therefore sexual reproduction and recombination are all
but impossible without making hybrids. Furthermore,
although all-femaleseeds are sexually reproduced, only
a few female plants can successfully be transformed into
high-yielding pollen parents. Often, only one trans-
formed female pollen parent is used to fertilize seeds on
all the recipient females, and thus all of the all-female
offspring share the genes from that single transformed
Figure 14. Uniform modern-day drug Cannabis crops are grown
from transplanted vegetative and asexually reproduced cut-
tingseither inside under lights, in greenhouses, or outdoors
under the sunon various scales (from Clarke and Merlin, 2013).
314 R. C. CLARKE AND M. D. MERLIN
Figure 15. Skunk No. 1was among the rst nearly true-breeding NLD/BLD hybrid drug cultivars developed, beginning in the 1970s.
The Skunk No. 1single line was selected and bred through nine reproductive cycles to increase homozygosity and the resulting unifor-
mity. This was followed by selection in a common garden of individual breeding parents (ten female and ten male) that were asexually
reproduced before being intercrossed, and their offspring were later grown out and evaluated to determine their specic combining
ability (SCA). Five female and three male plants were preserved for further seed production.
CRITICAL REVIEWS IN PLANT SCIENCES 315
pollen parent. For example, in Spain, a single White
WidowNLD/BLD hybrid cutting that transforms easily
(as well as reliably producing all female offspring) is used
to pollinate almost all of the individual female NLD/BLD
clones for which all-femaleF
1
hybrid seed cultivars are
named. However, several seed companies also produce
single-selfed (S
1
) cultivars by crossing each transformed
female plant with other members of the same female
clone, with no apparent loss in vigor.
All-female cultivars have gained popularity in hobby
and indoor growing markets, and are also increasingly
used in commercial cannabis production regions. Well-
intentioned Westerners took modern NLD/BLD seeds to
Morocco in the 1980s and crossed their progeny with the
traditional Rif Mountain kif or local marijuana landrace.
Within ten years, traits from the improvedWestern
varieties were seen in nearly all the Rif populations. Pres-
ently, all-female cultivars are increasingly popular in
Morocco. The females are completely fertilized by male
plants growing nearby and the betterimproved seed is
sown the next year. In this way local landraces are
quickly swamped out by invasive genes, and are therefore
replaced by an unselected crossbred mix of Western vari-
eties that are poorly adapted to local environmental con-
ditions and processing methods. Mexico, Jamaica, and
Thailand have also lost many of their landrace popula-
tions (Figure 16).
In addition to all-femalecultivars, breeders have
developed auto-oweringcultivars that commence
owering when they reach an early stage in vegetative
development rather than waiting for decreasing day
length to trigger owering. In addition, all modern
auto-oweringcultivars likely have a common ances-
tor, possibly the FinolaNLH seed cultivar or another
variety which owers independent of photoperiod con-
straints. Now these two traits are combined in all-
female/auto-oweringcultivars. Although originating
from two diverse gene pools (NLD/BLD and NLH), these
cultivars are presently reproduced using only a few
parents. Auto-owering plants cannot be maintained as
Figure 16. During the 1960s and 1970s, NLD landraces from diverse geographical regions which contained widely differing genetic
combinations were combined to form hybrid NLD sinsemilla cultivars. In the 1980s, several BLD landraces from Afghanistan were intro-
duced and thus added more diversity to the sinsemilla gene pool. By the 1990s, asexual propagation was commonly used to capture
heterozygous genotypes for use as commercial clonal cultivars. The widespread (yet narrowly focused) search for cultivars with unique
aromas and effects, combined with the proliferation of asexually reproduced cutting populations, obviated the need for sexual seed
reproduction, but also lowered the genetic diversity of drug Cannabis as a whole. Recently, inbreeding to produce all-female and auto-
owering seed lines has further restricted genetic diversity.
316 R. C. CLARKE AND M. D. MERLIN
clones because they will ower and die even under long
day length, but could possibly be maintained in vitro and
regenerated into explants.
In the past decade, many novel cultivars have
appeared, and, with the exception of variations in aro-
matic terpenoid proles, they are largely very similar in
the remainder of their phenotypic traits and agronomic
attributes. This has occurred not only because of the
incessant and thorough reshufing of a diverse, yet lim-
ited, set of genes and their respective alleles; more
recently the homogenization of cultivars and their traits
has been produced by accidental incest between closely
related siblings or cousins. Many popular femaleclonal
cultivars will produce a few functional male owers
when they are stressed by any of a wide variety of factors
that are not far outside of optimum growing conditions
(e.g., changes in temperature, overabundance or shortage
of nutrients, uctuations in light cycles, etc.). The male
owers of these popular femaleclonal cultivars pro-
duce viable pollen and result in unintentional fertiliza-
tion of the neighboring femaleplants. Unstable female
sexuality becomes reinforced generation after generation
because plants with unstable sexuality are the pollen
parents. Usually only a few accidentally produced seeds
occur in sinsemilla (because it is usually seedless). How-
ever, among these, a high percentage will be sprouted,
most will be female,and they will strongly resemble
other popular cultivars, but with their own slight differ-
ences; consequently, the accidentally produced seeds that
do mature are likely to be brought into cultivation.
Along with the unfavorable tendency to produce male
owers and unwanted seed, modern varieties have some
other drawbacks: they are increasingly nicky about
growing conditions, are susceptible to pests (e.g., various
mites) and disease (e.g., gray mold and powdery mil-
dew), and are generally difcult to grow. Increasing
numbers of pests and diseases lead to increasing use of
unapproved pesticides and plant growth regulators,
which can directly impact public health.
Without open-pollinated sexual reproduction, there is
much less chance for evolutionary change. In the past, as
early agriculturalists spread across new geographical
frontiers, they exposed Cannabis to new and evolution-
arily challenging environments. The tremendous genetic
diversity of geographically isolated wild populations and
traditional landraces of Cannabis produced by sexual
reproduction and genetic recombination presented novel
phenotypes for natural and human selection. Narrow
selection and strong founder effects have seriously atten-
uated genetic diversity, bringing us closer to potential
disaster. A nal blow may come from commercialization
of a small number of select varieties that satisfy limited
consumer preferences. Under the assumption of eventual
legalization, and modern monocropping paradigms
favored by economically prudentstrategies to maxi-
mize prots, fewer clonal cultivars will pass the con-
sumer lter, and it is likely that only these will be
proliferated. Variety registration to protect breeders
rights will favor asexually reproduced clones because it is
easier to demonstrate uniqueness, uniformity, and repro-
ducibility with clones than it is by breeding consistent
seed cultivars. Given the present-day economic and
political climates, and the increasing trend toward asex-
ual reproduction in both indoor and outdoor crops, this
scenario seems probable. If this outcome comes to pass,
it will further narrow the Cannabis gene pool to include
only the most commercially viable cultivars, and there-
fore, more of less repeating the history of varietal wine
grape selections and potentially leading to another major
crop disaster.
Whether varieties are lost through prohibition, custo-
dial neglect, or economic priorities, they become extinct
and are potentially gone forever. When indigenous farm-
ers maintain localized landraces, they are preserving
genetic diversity because landrace populations reproduce
sexually, and thus allow for genetic recombination,
mutation, and evolution under human selection. The
numbers and ranges of traditional agricultural societies
are diminishing worldwide, and many landraces are
already extinct, which does not bode well for the future
of the Cannabis gene pool. The loss of any genetic diver-
sity threatens unique genes and allelic combinations, as
well as lowering future evolutionary tness for the genus
as a whole and limiting the diversity of desirable prod-
ucts that crop plants may be able to provide.
The status of the worldwide Cannabis genome has
changed dramatically since the middle of the twentieth cen-
tury. The majority of traditional ber, seed, and drug landra-
ces are no longer grown, and many of the few remaining in
situ landraces have been genetically diluted through inter-
breeding with introduced modern cultivars. Also, European
industrial hemp cultivars share much of the same germ-
plasmdioecious cultivars were bred by crossing middle
Russian and Chinese landraces, and monoecious cultivars
all share Fibrimonas a pollen sourceand overall the
genetic diversity of NLH is currently very limited (Hillig,
2005a,b). Commercial sinsemilla cultivars are asexually
reproduced, and almost all share varying amounts of both
NLD and BLD heritage. Founder effects from BLD landrace
selections introduced gray mold and powdery mildew sus-
ceptibility that cause millions of dollars in crop losses annu-
ally. Novel gene combinations still arise because the modern
drug Cannabis gene pool is based traditional landraces from
geographical origins that represent two genetically distinct
primary gene pool, NLD and BLD. Whenever a seed is
planted, it represents the sexual union of two parents, and
CRITICAL REVIEWS IN PLANT SCIENCES 317
creates an opportunity for new genetic combinations to
appear. Asexually reproduced cuttings are potentially
genetic dead endsonly sexual reproduction offers us a
truly brighter genetic future and the possibility for plant
improvement.
X. Seed banks
When a Cannabis landrace is not reproduced every ve
to ten years, the stored seeds will most likely die and the
landrace may be gone forever. Seeds must be properly
kept in a gene bank and reproduced periodically under
ideal conditions. The past 50 years have seen the genetic
diversity of the Cannabis genome dwindle away. Indeed,
the vast majority of landraces may already be extinct,
and we therefore must be careful to preserve and multi-
ply what remains. As Watson and Clarke (1997) warned,
Many local landrace varieties, the result of hundreds of
years of selection for local use, have been lost because of
Cannabis suppression and eradication, neglect on the part
of agricultural ofcials and industry, anti-hemp propaganda
and the general trend (until recently) to reduce industrial
hemp breeding and research. Genetic materials are a living
heritage and we are their custodians. We must concentrate
our efforts to collect, preserve, characterize and utilize the
remaining Cannabis genetic resources before it is too late.
As the worldwide reduction in Cannabis diversity
continues, the importance of genetic preservation
becomes more obvious. Unfortunately, no comprehen-
sive Cannabis germplasm collections exist. Most of the
few seed accessions are held by national gene banks that
may or may not share their valuable inventories with
breeders in other countries. The largest collection of
hemp germplasm is maintained by the Vavilov Institute
of Plant Research (VIR) in St. Petersburg, Russia. It pres-
ently numbers 563 seed accessions, including 23 possible
drug accessions from Afghanistan, Kazakhstan, Syria,
Turkey, and Uzbekistan, while the remaining are all
hemp and feral accessions from Armenia, Bulgaria,
Chile, China, Czechoslovakia, Estonia, France, Germany,
Hungary, Italy, Latvia, Moldova, Poland, Portugal,
Romania, Russia, Spain, Sweden, Ukraine, the United
States, and former Yugoslavia (Grigorev, 2015). Since the
late 1980s, political, technical, and nancial difculties in
Russia have resulted in low population sizes and incom-
plete isolation, and consequently there has been consid-
erable loss of genetic diversity and purity in the VIR
collection (Hillig, 2004b). Many accessions may now be
so similar to each other that their importance to future
breeding programs could be diminished.
In 1992, the Cannabis germplasm collection at Wage-
ningen University in the Netherlands contained over 156
accessions originating from 22 countries and largely
sourced from other collections and research institutes
(De Meijer and van Soest, 1992; Gilmore et al., 2007).
Nearly half of these accessions are from the former
USSR and Hungary. The Institute of Natural Fibres and
Medicinal Plants gene bank collections in Poland contain
139 accessions of predominantly European origin, with
accessions from France, Hungary, and the Ukraine
contributing 54.7% of the collection (Mankowska and
Silska, 2015). The Yunnan Academy of Social Sciences
collection in Yunnan province, China holds approxi-
mately 350 accessions mostly of East Asian origin
(Salentijn et al., 2014) and the Ecobre Global
Germplasm Collection in Australia contains additional
Eurasian accessions (Welling et al.,2015). However,
comprehensive accession data are sorely lacking in
several of these collections and this limits their value to
breeders (Welling et al.2016).
In addition, the subterranean Svalbard Global Seed
Vault on the Norwegian island of Spitsbergen about
1300 km (810 miles) from the North Pole has a total of
43 Cannabis accessions that are duplicated in three other
seed banks. Five of these accessions, from North Korea,
Netherlands, Spain, Syria, and Turkey, may possibly be
Cannabis drug populations; 21 others are hemp acces-
sions from Argentina, Austria, China, Croatia, France,
Georgia, Germany, Italy, Poland, Romania, Slovakia,
Spain, and Sweden; and 16 accessions are of unknown
origin (http://www.nordgen.org/sgsv/). The worlds larg-
est seed repository is the Millennium Seed Bank housed
at the Wellcome Trust Millennium Building in West Sus-
sex, near London, which specializes in wild plants and
has only one Cannabis accession, which is from Slovakia
(http://apps.kew.org/seedlist/SeedlistServlet).
Given the importance of Cannabis as a traditional as
well as present-day crop plant, the biodiversity of this
genus (particularly among the drug cultivars) is sorely
under-represented in seed banks, especially in light of
recent research interest in medical Cannabis. If we take
into account this lack of diversity, in light of genetic
impurity and low seed numbers, there really is no reliable
reserve of Cannabis seeds. The primary goal of germ-
plasm preservation is the conservation of the entire
genome of each population. It is especially important in
open-pollinated, cross-breeding plants that the popula-
tion size is large enough to ensure that as many of the
alleles as possible within each gene pool are reproduced
in the seed. A minimum of 1000 plants for monoecious
accessions, and 2000 plants for dioecious accessions,
assures that 99% of the Cannabis alleles will be repro-
duced (Crossa et al., 1993). Unfortunately, the seed
reserves of many of the Cannabis seed bank accessions
consist of less than 1000 viable seeds (often only 500 or
less); therefore, genetic diversity is already limited by the
318 R. C. CLARKE AND M. D. MERLIN
number of archived seeds. The secondary goal of genetic
preservation is to reproduce the accessions in sufcient
quantities to maintain a reserve for future reproductions
and public distribution.
A common goal of Cannabis breeders should be
establishing a more comprehensive core collection of
Cannabis seed accessions that have been exhaustively
characterized agronomically in the eld, and on molecu-
lar levels, genetically and chemically, in the laboratory.
Only then, can we see what diversity really is available
for researchers to work with in the future. This core col-
lection should be maintained with optimal reproduction
and storage methodology, and individual accession eval-
uations should be made accessible to breeders (Watson
and Clarke, 1997).
In the past 20 years the situation has only stagnated.
According to Welling et al. (2016), in their ne review of
the present state of ex situ Cannabis germplasm
collections:
Coordinated and comprehensive conservation and
characterization of ex situ Cannabis resources holds the
promise of preserving genepool diversity and enabling cul-
tivar development. However, the legal constraints imposed
by international narcotics conventions over more than
50 years have been inuential in the fractionation and
erosion of publicly accessible Cannabis ex situ genetic
resources. The restrictions on legal exchange of bona de
research materials continues to limit the establishment of
physical and centralized ex situ core collections.
XI. Present and future directions for Cannabis
breeding start here!
The primary evolutionary process that is presently fur-
thering domestication in Cannabis is basic Mendelian
breeding; this traditional system involves selecting sim-
ply inherited traits and increasing their homozygosity
through sexual recombination. Many of the primary eco-
nomic traits of Cannabis are simply inherited (e.g., stalk
height, seed size, and cannabinoid content). Cannabi-
noid biosynthesis is controlled by a narrow range of
alleles limited to only a few loci; heritability is extremely
high, which has favored successful breeding for high-
THC and high-CBD sinsemilla cultivars as well as low-
THC industrial hemp cultivars. High variability and
strong heritability also inuence both owering and
maturity times, and breeding for earlier-maturing drug
varieties and later-maturing ber varieties continues.
Selection for reduced sensitivity to day length could
improve the versatility of ber hemp varieties making
breeding and seed multiplication easier. Late-maturing
or nonowering ber hemp varieties with low THC con-
tent would signicantly improve biomass yield and
extend the range where ber hemp can be grown eco-
nomically into sub-tropical and equatorial regions
(Salentijn et al., 2014). Indeed, breeders are expected to
make further advances on all fronts as Cannabis cultiva-
tion becomes more widespread for a variety of uses.
There are many achievable goals awaiting twenty-
rst-century hemp breeders. Fiber hemp breeders will
certainly focus on raising ber content and yield (e.g.,
increasing ber percentage, extending vegetative period),
improving stalk and ber quality for specic uses (e.g.,
paper pulp, composites, and ne textiles), altering canna-
binoid content and composition (e.g., low THC percent-
age with high CBD yield), developing cultivars that are
easier to process (e.g., faster retting, easier peeling, and
cheaper dyeing), and increasing resistance to pests
and diseases. Hemp ber is more easily extracted and
processed when the ber bundles are high in cellulose
and low in lignins and pectins that bind the bers
together within the bundles. Sequencing the genes
responsible for the production of these compounds and
the ways they are assembled could assist in cultivar
development for the textile and paper industries
(Mandolino and Carboni, 2004).
Seed hemp breeders could develop cultivars with spe-
cic protein and fatty acid compositions tailored for spe-
cic whole seed, kernel, and oil markets, as well as easily
processed (e.g., harvesting, threshing, and hulling) culti-
vars, and should learn to control monoecy for hybrid
seed production (Salentijn et al., 2014).
Industrial hemp cultivation is presently restricted to
temperate latitudes, but its economic range could be
widely enlarged by developing cultivars adapted to lower
latitudes, or by selecting for day-neutral cultivars that
could be grown at any latitude. All commercial hemp
cultivars are presently adapted to growing at or above 45
degrees latitude, and will yield less ber when they are
relocated as little as two degrees closer to the equator.
Closer to the equator, the day length is shorter earlier in
the summer, and consequently the Cannabis crop will
ower prematurely when plants are small, which lowers
ber and seed yield. European hemp cultivars cultivated
closer to the equator than within their normal latitudinal
farming range are also attacked by a host of alien pests
and diseases for which they have little resistance. As a
result, no hemp varieties approved by the EU can be suc-
cessfully grown closer to the equator than at about 40
degrees latitude. Limited traditional hemp production in
other regions of the world relies mostly on unimproved
local landraces (Clarke, 1995,2007; Watson and Clarke,
1997; Clarke and Gu, 1998).
Further advances in industrial hemp breeding are also
expected in the near future. Economically efcient ber
production relies on increasing the scale of production,
CRITICAL REVIEWS IN PLANT SCIENCES 319
reducing production costs, and tailoring production for
specic markets. These goals could be achieved in part
by breeding for increased yield of biomass along with
increased ber yield and quality, reducing waste such as
dust, and improving ber color. Field experiments on
small test plots indicate that hemp may have the poten-
tial to produce much higher stalk and ber yields than
are presently achieved. Improved yields of both ber and
seed varieties could be attained by continued selection
for optimum owering date, enhanced resistance to pests
and diseases, and improved canopy architecture.
Improvements developed by breeders for specic uses,
such as ber size, aspect ratio, strength, stiffness, density,
surface characteristics, adhesive properties, lignication,
and color, must be carried out in concert with develop-
ment of optimized agronomic regimes and specic proc-
essing strategies for woven, nonwoven, and composite
applications. Ideal hemp ber varieties should have a
high proportion of ne and easily extracted bers that
enhance rapid processing, as well as producing less
waste. However, ber quality and ease of retting are con-
trolled by a complex set of genes which interact with
agronomic and processing conditions that result in a
variety of difculties for ber hemp breeders, including
lower heritability of ber traits. On the other hand, seed
fatty acid and protein syntheses are controlled by fewer
genes encoding for specic products; this makes selection
and breeding for seed traits more straightforward
(Weightman and Kindred, 2005).
One of the primary goals of EU hemp breeders over
the past several decades has been the reduction of THC
levels. In 2001, the EU lowered allowable THC levels to
0.2%, although previously mandated at below 0.3%. This
legal reduction of allowable THC levels created problems
for hemp breeders faced with maintaining cultivar pro-
ductivity while further reducing already negligible psy-
choactive drug content. The lowering of THC levels
encouraged breeders to further increase inbreeding with
monoecious lines and was a major factor leading to
reduced genetic variability in present-day European cul-
tivars. Breeders are now working on medicinal hemp
drug cultivars that are high in CBD while remaining low
in THC. There is an ancient tradition of growing multi-
purpose Cannabis plants in various areas of China. How-
ever, very little had been cultivated during modern times,
until Chinese farmers recently began growing hemp
more intensely for ber and seed as well as by-product
CBD production. Indeed, two high-CBD industrial
hemp cultivars should have been registered, with sowing
seed released by the Yunnan Academy of Agricultural
Sciences in 2016 (Salentijn et al., 2014).
Two predominant trends of Cannabis breeding have
appeared in recent decades. On the one hand, Cannabis
has been modied by industrial hemp breeders who have
lowered THC content and raised ber content
while developing uniform, high-yielding cultivars. On
the other hand, sinsemilla breeders have also altered the
genetic makeup of drug varieties effecting quantitative
traits such as raising THC content, ower to leaf ratio in
the inorescences, and yield of owers. Intense selection
for potency explains in part why drug cultivars are gen-
erally less polymorphic and heterozygous than hemp cul-
tivars(Weiblen et al., 2015). Additional qualitative traits
in drug cultivars have also been altered recently such as
increasing branching, shortening internodes, and select-
ing for rapid maturation and early oral response to
inductive photoperiods.
The following four key evolutionary events differenti-
ate drug cultivars from ber and seed cultivars: (1) ram-
pant and continued blending of the modern domestic,
hybrid drug gene pool; (2) exportation of improved
drug seed; (3) gene ow of modern hybrid traits back
into traditional drug populations; and (4) the establish-
ment of unique, asexually reproduced clonal drug culti-
vars. Recent stages in the evolution of modern drug
Cannabis varieties have few parallels in the simple breed-
ing history and legal distribution of hemp.
Although industrial hemp breeders still develop ber
and seed cultivars, the major thrust of Cannabis breeding
today is directed toward developing drug cultivars, espe-
cially those with enhanced medical efcacy. Recycling of
public domain geneticscontinues, but sinsemilla
breeders are increasingly reexamining original landraces
and older hybrids to revitalize their breeding programs,
while the quest to collect traditional landrace parents
continues.
The present-day NLD/BLD hybrid gene pool, despite
its recent history of incest, continues to produce novel
allelic combinations. Breeders have recently developed
new sinsemilla cultivars with enhanced efcacy in treat-
ing particular medical indications. The worldwide spread
of BLD genes into traditional NLD regions has nearly
brought an end to pure NLD landraces. In the face of
this degradation of in situ conservation, breeders are
developing new cultivars by growing some of the few
remaining pure NLD and BLD landrace seeds. NLD
landraces are valued in hybrid crosses to impart particu-
lar aromas and avors or to enhance potency and diver-
sify effects. Traditional NLD landrace varieties imported
from India, Kashmir, Nepal, Indonesia, Korea, Southeast
Asia and Mexico, as well as areas in western, central and
southern Africa, have regained favor because they
mature relatively early, but express fewer of the undesir-
able traits of BLD landraces (e.g., low ower to leaf ratio,
fungal susceptibility, and unpleasant aromas). However,
many of these traditional NLD landrace varieties have
320 R. C. CLARKE AND M. D. MERLIN
become more difcult to procure in recent years.
Breeders only recently learned that Afghan BLD landra-
ces produce CBD (largely bred out of modern hybrid sin-
semilla varieties) and new accessions from Central Asia
are occasionally also introduced. In addition, breeders
are also developing hashish varieties by selecting traits
such as large and easily removed resin glands, dry rather
than sticky or oily resin texture, and aromatic proles
that persist through various processing protocols.
Because commercial-size marijuana shipments do not
often originate in traditionally noncommercial regions,
landrace seeds are usually collected in small numbers by
Figure 17. Cultivar development begins by combining two or more landraces through parental (P) generations to create a diverse
parental population. Select female and male siblings from the parental population are then crossed for several lial (F) generations to
form a synthetic multi-hybrid. The multi-hybrid seeds are subsequently grown out and female and male plants selected for breeding
are asexually reproduced by rooting vegetative cuttings. At this point, ve different intentional and unintentional breedingpaths can
be followed. The most common is to cross a female clone (1.4) with a male clone (2.1) to produce single-cross F
1
hybrid male and female
seed. A related technique using the same parents would be to backcross (BC) a select F
1
female (1.4 £2.1) to the same male parent
(2.1), sometimes for multiple generations, to produce backcross hybrid male and female seeds. These traditional breeding techniques
have been practiced for decades. The next three twenty-rst-century pathways rely on female plants to produce pollen. Plant growth
regulators, such as colloidal silver ions, are intentionally applied to female (XX) plants (1.1) to initiate male owers that will produce via-
ble female (XX) pollen. This pollen can be applied back to the same female clone (1.1) to make selfed (S) (1.1) all-female lines to increase
homozygosity in breeding, or it can be crossed to another female clone (1.2) to make (F
1
) all-female sowing seed. The accidental fth
path mimics the previous two, except male owers infrequently appear spontaneously in femaleclones (1.3), release pollen unnoticed
by the grower, and fertilize the same (1.3) or other clones unintentionally. This is the recent incestuous lineage of many modern-day sin-
semilla cultivars.
CRITICAL REVIEWS IN PLANT SCIENCES 321
travelers, and are relatively rare compared to imported
seeds from the major marijuana producers (Figure 17).
Sinsemilla growers prefer clones with a number of
economically desirable traits: (1) high yield of dry bio-
mass; (2) high proportion of owers as opposed to leaves
and stems; (3) preponderance of large glandular tri-
chomes; (4) high total cannabinoid content in the ow-
ers; (5) reproducible prole of the target cannabinoids
(e.g., CBD, THC, etc.); and (6) desirable suite of aromatic
terpenesthese are the major traits that breeders
attempt to accentuate. Similar to sinsemilla breeding, the
development of cultivars to supply pharmaceutical raw
materials starts with several promising hybrid crosses,
followed by serial inbreeding of select individuals from
the hybrid population and/or backcrosses to a parental
line. When successful, hybrid vigor is restored by making
crosses between selected inbred lines (de Meijer, 2004).
Medisinsis a vegetatively reproduced Skunk No. 1
high-THC pharmaceutical cultivar registered by Horta-
Pharm in the Netherlands in 1998, and Graceis a
high-CBD seed cultivar registered by GW Pharmaceuti-
cals in the United Kingdom in 2004; both of these culti-
vars have been awarded plant breeders rights
(Weightman and Kindred, 2005). Cultivars can also be
developed that produce other cannabinoid compounds
of medical or industrial interest, and secondary metabo-
lite synthesis can be altered to provide enhanced protec-
tion from pests and pathogens.
Developing sterile cuttings of female varieties or seed
varieties that are infertile would guarantee that female
plants will be seedless even when grown near pollen
sources such as hemp seed elds, and offer an additional
advantage to commercial sinsemilla growers. More culti-
vars will be developed that produce economically
valuable amounts of rare cannabinoids (e.g., cannabi-
chromene (CBC), cannabigerol (CBG), etc.). Cultivars
could also be developed that produce predominately one
aromatic terpenoid compound or suites of varying terpe-
noids in xed amounts. These new cultivars may prove
to be of pharmaceutical interest, and competition may
develop between plant breeders that will enhance the
production of these rare cannabinoid compounds. In
addition, the bio-engineering manipulation of cannabi-
noid synthases by microorganisms may lead to innova-
tive production systems of these or other rare
cannabinoids. Among the major attractions of sinsemilla
cultivars are their diverse aromas and avors. However,
breeding for differing parameters may prove difcult
because terpenoid synthesis pathways are controlled by a
suite of genes.
In the future, legal Cannabis enterprises will rely on
the protection of intellectual property rights. Uncommon
and readily apparent biological traits could be added
through breeding or genetic modication (GM, see
below) to identify novel cultivars and protect the intellec-
tual property of the breeder. These traits might include
recessive morphological markers such as abnormal pig-
mentation (e.g., yellow stem, purple owers, or red
leaves); abnormal leaf shapes (e.g., hooked serrations or
webbed leaves); or unique chemical proles (e.g., traces
of a minor unique aromatic terpenes or aldehydes) that
would make the cultivar phenotypically distinct. Genetic
ngerprinting of DNA sequences will also be used to
identify cultivars.
Biotechnology in the form of GM has already
reached Cannabis. Japanese researchers transferred the
THCsynthasegenefromCannabis to cultivated
tobacco (Nicotiana tabacum L.) and induced it to con-
vert CBG (cannabigerol, the precursor molecule to
THC) into THC (Shoyama et al., 2001). German
researchers have engineered a yeast (Pichia)tomake
the same synthetic step and plan to insert more genes
for earlier biosynthetic steps along the pathway to
THC (Zirpel et al., 2015). Other agronomically valu-
able traits may also be transferred to Cannabis such as
enhanced pest resistance, increased yields of medically
valuable compounds, tolerance of environmental
extremes, and sexual sterility.
Recently, breeding lines that are focused primarily on
oleic acid production have been created using gene Tar-
geting Induced Local Lesions in Genomes (TILLING)
techniques; this method allows identication of muta-
tions in a specic gene. Vegetable oils with high oleic
acid content are more stable at room temperature than
those rich in essential fatty acids (EFAs), alpha-linolenic
acid (an omega-3 fatty acid) and linoleic acid (an omega-
6 fatty acid), which are both commonly found in Canna-
bis seed. Bielecka et al.(2014) identied a suite of alleles
for putative desaturase genes representing the four
main activities required for production of polyunsatu-
rated fatty acids in hemp seed oil,and by gene TILLING
a chemically mutagenized population, they were able to
select plants that lacked the desaturase genes. Four back-
crosses and sibling crosses achieved homozygosity.
When grown in the eld, offspring of this population
produced seeds with a 70% oleic acid oil content and
total oil yields similar to parental lines; this lays the
foundation for the development of additional novel oil
varieties in this multipurpose low input [Cannabis]
crop(Bielecka et al., 2014).
The reaction to development and release of GM
organisms has been guarded in general and will be with
Cannabis as well (Russo, 2011). Cannabis presents a par-
ticularly high risk of transmitting GM genes to industrial
hemp crops and weedy Cannabis because it is wind-
pollinated, although so is GM maize that is widely
322 R. C. CLARKE AND M. D. MERLIN
grown. The EU has installed strict regulations to prevent
the accidental release of GM genes, and therefore
production of GM Cannabis in the EU may prove
impractical. However, nonfood industrial ber and phar-
maceutical Cannabis cultivars may not receive as much
resistance from consumers and environmentalists as
food crops. Genes coding for cannabinoid biosynthesis
might also be transferred from Cannabis to organisms
that are less politically sensitive than tobacco such as sin-
gle-cell organisms. Transferring cannabinoid biosynthe-
sis genes from Cannabis into other plants, fungi, and
bacteria opens up the possibility of producing medically
valuable cannabinoids in industrial fermenters and cir-
cumventing Cannabis growing altogether. However,
Cannabis plants already produce extremely high yields
of target compounds. Dried female sinsemilla inores-
cences can yield 20% of THC or CBD, which is very high
for a target compound. With the widespread, recently
increasing legalization of Cannabis cultivation and devel-
opment of high-yielding cultivars for cannabinoid
extraction, genetically engineered microorganisms will
face much competition for market share.
Molecular markers for suites of genes associated with
biosynthesis of individual target compounds as well as desir-
able agronomic traits would allow mass screening of juvenile
populations; this could be of great advantage to breeders (de
Meijer, 2004). Sequencing cannabinoid synthase alleles
would allow marker-assisted breeding for development of
single cannabinoid cultivars and could be especially useful
in recognizing rare cannabinoid variants (e.g., plants with
increased production of cannabigerol (CBG), tetrahydro-
cannabivarin (THCV), or cannabichromene (CBC)) for
pharmaceutical production (Mandolino and Carboni,
2004). Once the entire Cannabis genome is mapped and a
reference genome established, researchers can begin excising
and inserting sequences coding for each trait. Next-genera-
tion genome sequencing technology will allow breeders to
explore the diversity of the Cannabis genome, measure relat-
edness between individuals, identify gene sequences linked
to certain economic traits, screen thousands of individuals
for rare mutations, and use marker-assisted selection to
improve complex traits (Salentijn et al., 2014).
One important insight revealed by recent Cannabis
genome projects is how inbred the drug Cannabis genome
has becomemost clones are closely related to all the
others. It will be benecial to breeders to choose parents that
are less related (i.e., ones with a greater genetic distance
between them) in order to encourage hybrid vigor and
reduce inbreeding depression. Initial studies aimed at foren-
sic identication of illicit cannabis concluded that the
genome is highly heterozygous due to its obligate outcross-
ing with most alleles appearing at low frequency. With the
exception of certain major alleles shared by inbred cultivars
(e.g., Fibrimonrelatives), alleles are shared across a wide
spectrum of phenotypes, and consequently there is a high
degree of variation even within inbred female cultivars.
These are all factors that have so far limited the application
of genome data for taxonomic, evolutionary, forensic, and
plant improvement research (Salentijn et al., 2014). Only
more recently have genetic differences between Cannabis
hemp and drug populations become more clear (Van Bakel
et al., 2011;Sawleret al., 2015).
XII. Summary and conclusions
Cannabis is an annual, sun-loving plant that thrives in open,
nitrogen-rich environments, including rubbish piles created
by humans (Anderson, 1967;Merlin,1972;Clarkeand
Merlin, 2013;alsoseeSmall,2015). Cannabis also has rela-
tively large, numerous, and easily sown seeds, and therefore
was preadapted for cultivation. Close associations between
humans and Cannabis stimulated its early cultivation, and
over time this eventually led to its domestication. As ancient
agricultural strategies took form, humans began to select
plants that provided more and better products. Some of the
earliest seed selections perished and growers must have
constantly collected from spontaneous populations to
replace, supplement, or modify extant varieties, resulting in
evolution of improved landraces. As their familiarity with
Cannabis grew, humans became more discerning with their
seed selections.
Because Cannabis grew well as a camp follower, later
selections were increasingly made from feral populations
near human settlements, rather than truly wild popula-
tions. Early semi-domesticated varieties also became nat-
uralized and interbred with nearby feral and/or wild
populations. Selection for differing economic traits con-
tinued as Cannabis spread well beyond its original puta-
tive range in Central Asia. Backcrosses between
cultivated and truly wild populations became rare,
whereas crosses between cultivated and feral populations
became more common. Eventually, isolation developed
between wild, cultivated, and feral populations which
evolved their own phenotypes. This scenario led to the
extreme variation encountered today in geographically
isolated populations, and accounts for the absence of a
ubiquitous, single spontaneously growing biotype
throughout Eurasia.
Profound evolutionary changes can occur rapidly in
annual plants such as Cannabis, and because it was likely
abundant in its original natural environment, repeated
selection was possible. Under such conditions, early
humans probably did not worry about improving yields,
collecting enough from nearby populations to satisfy
immediate needs. Only along with the advent of Canna-
bis management and eventually cultivation did an
CRITICAL REVIEWS IN PLANT SCIENCES 323
interest arise in increasing yields. As an intimate rela-
tionship between each plant population and the indige-
nous people developed, selection and controlled
breeding began.
The economically valuable traits of Cannabis such as
its strong bast ber, edible seed, and psychoactive resin
are readily noticed and appreciated, and its rst use was
likely very early in human history in the regions where it
was rst encountered. The biology and ecology of Can-
nabis were determined by when and where it evolved
temperate Central Asia during an ancient warm intergla-
cial period being the most likely choice for the origin of
Cannabis, a sun and warmth-loving, water and nutrient
hungry, dioecious, wind-pollinated, short-day annual
plant. Although this genus was undoubtedly useful to
early peoples in its naturally evolved state, human selec-
tion has played the major role in its evolution as a culti-
vated species and allowed Cannabis to thrive almost
everywhere it has been introduced. Selection and breed-
ing of Cannabis are the means by which humans exert
the most control over its evolution as a domesticated
crop plant, and lie at the heart of the humanCannabis
relationship. Articial selection has raised ber content,
increased seed size and yield, and both raised and low-
ered cannabinoid levels. Cannabinoid chemotypes are
characteristic of ultimate geographical origin and usage
groups; and they are correlated with movements of Can-
nabis germplasm by humans to new environments where
they were affected by local cultural selection for particu-
lar products. Cannabis is now grown legally in many
regions of the world.
Presently, few if any imported drug Cannabis ship-
ments are as psychoactively potent as the best sinsemilla
grown in North America and Europe. However, seeds of
improved hybrid cultivars have spread to many Canna-
bis producing nations, and the potency of Cannabis orig-
inating from select locations is steadily increasing.
Because of continuing international pressure against
marijuana growers, and the highly inated price of sinse-
milla, indoor cultivation will maintain popularity world-
wide in any location with a reliable electrical grid.
Instructional information is readily available on the
Internet, and it is easy to install and operate grow rooms
in attics, bedrooms, or basements. Under these crowded
circumstances, there is no tolerance for nonproductive
plants, and the single most productive female clone is
usually selected for all future cultivation. The use of a
single clone improves grow room performance, but pre-
cludes the possibility of seed production. Breeding is no
longer possible and variety improvement ceases entirely.
Vegetative propagation essentially freezes evolution, and
gene diffusion via cuttings has been more localized and
much slower than with seeds. Presently, due to the rapid
spread of indoor cultivation, growers are increasingly
likely to exchange vegetative cuttings of proven clones
rather than carefully bred seeds, and consequently the
genetic diversity in drug Cannabis cultivars has
decreased in the past two decades. As cannabis prohibi-
tion fades, production is rampantly proliferating in exist-
ing agricultural regions, and in the near future only the
most productive and protable clonal cultivars will be
grown. Cloning will have a lasting effect on sinsemilla
production and will further slow the evolution of drug
Cannabis.
Economic competition will increase with legalization
and normalization, and Cannabis will be further domes-
ticated as just another crop, with high yields and ease of
growing taking precedence over other traits. Although
traditional smoked marijuana will always have its fol-
lowers, and some connoisseur cultivars and their bou-
tique growers will prevail as they have in the wine
industry, the bulk of future production will probably be
extracted for use in edible and vaporized delivery sys-
tems. If target compounds in the future are derived from
a limited number of industrial drug cultivars, Cannabis
genetic diversity will once again suffer.
Present-day clonal sinsemilla cultivars remain geo-
graphically and functionally isolated from gene
exchange with their parental populations in Afghani-
stan or other regions of origin. However, many NLD/
BLD hybrid seeds were produced in Western countries
and some did interbreed with original ancestral popula-
tions. Well-meaning travelers visited many regions
where NLD landraces were still growing and gave mod-
ern hybrid seeds to local farmers who crossed them
with their traditional landraces, hoping for economic
benet. In the process, these traditional landraces are
becoming contaminated and eventually displaced by
the introduced hybrids. Original pure NLD landraces
have become rare in all traditional marijuana producing
countries(e.g.,Jamaica,Mexico,Morocco,andThai-
land). This situation makes the collection of traditional
landrace varieties increasingly difcult, and conse-
quently their preservation and reproduction are more
signicant issues than ever before. It is important to
collect landrace building blocks on which future
breeders will depend for creating improved cultivars;
for the sake of genetic diversity, we must escape from
our reliance on clones and their inherent evolutionary
stagnancy. Save and sow your seeds!
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