Note: Descriptions are shown in the official language in which they were submitted.
HEMP PLANT NAMED `AF14B15-21'
FIELD
[0001] The disclosure relates to a hemp varieties, hemp extracts, CBD-
containing
compositions, and methods of producing and using the same.
BACKGROUND
[0002] Cannabis is a genus of flowering plants that includes at least three
species,
Cannabis saliva, Cannabis indica, and Cannabis ruderalis as determined by
plant
phenotypes and secondary metabolite profiles. Hemp, also known as industrial
hemp, is a
type of cannabis plant grown specifically for the industrial uses of its
derived products. In
the United States, Cannabis is classified as hemp if it accumulates no more
than three-
tenths of one percent (i.e., 0.3%) concentration of tetrahydrocannabinol (THC)
at harvest
maturity. Hemp plants can also accumulate high levels of cannabidiol (CBD),
which is
used in a variety of consumer goods, including food, drinks, dietary
supplements and
cosmetics.
[0003] Hemp production however, remains challenging for farmers. The presence
of a
single male plant in a hemp field can ruin an entire crop by fertilizing the
valuable female
flowers. Large scale hemp cultivation also requires hemp varieties with
uniform growth to
avoid early or late maturation of a significant portion of the crop. Feminized
seeds for high
CBD producing lines with early maturity and dwarfism are highly desirable, but
not yet
widely available.
[0004] Thus, there remains a need for new hemp varieties to meet the growing
demand for
fiber and CBD-based products.
BRIEF SUMMARY
[0005] This disclosure relates to a new and distinctive hemp cultivar
designated as
'AF14b15-21'. In some embodiments, the 'AF14b15-21' is a Cannabis saliva L.
plant.
[0006] The inventors reproduced the 'AF14b15-21' cultivar through strategic
crosses and
selections from proprietary lines. The 'AF14b15-21' plant is maintained as a
hybrid seed
1
Date Recue/Date Received 2022-04-12
produced by crossing two parental lines maintained at the inventors'
greenhouses,
nurseries, fields and/or facilities in Colorado.
[0007] The present disclosure provides a new and distinctive hemp variety
designated as
'AF14b15-21'. The present disclosure relates to the seeds of hemp variety
'AF14b15-21',
to the plants or parts of hemp variety 'AF14b15-21', to the plant cells of
hemp variety
'AF14b15-21', to the plants or plant parts or plant cells having all of the
essential
physiological and morphological characteristics of hemp variety 'AF14b15-21'
and to
plants or plant parts or plant cells having all of the essential physiological
and
morphological characteristics of plant cells listed in Tables 1-5, including,
but not limited
to, as determined at the 5% significance level when grown in the same
environmental
conditions, including when grown side-by-side with a comparison or check
cannabis and/or
hemp plant.
[0008] The present disclosure relates to methods for producing a hemp plant
and/or seed,
by crossing the hemp variety 'AF14b15-21' with itself or another cannabis
and/or hemp
plant. A further aspect relates to hybrid hemp plants, and hemp seeds produced
by crossing
the hemp variety 'AF14b15-21' with a cannabis and/or hemp plant.
[0009] Another aspect of the present disclosure is also directed to a method
of producing
a cannabinoid extract comprising contacting plants of the hemp variety 'AF14b
15-21' with
a solvent or heat, and producing the cannabinoid extract.
[0010] In some embodiments, the present disclosure teaches a seed, plant,
plant part, or
plant cell of hemp plant variety designated 'AF14b15-21', wherein
representative seed of
the variety has been deposited under NCMA No. 202203083. In some embodiments,
the
present disclosure teaches that the plant part is an inflorescence and/or a
flower.
100111 In some embodiments, the present disclosure teaches a hemp plant or a
plant part
or a plant cell thereof, having all of the essential physiological and
morphological
characteristics of the hemp plant variety designated 'AF14b15-21' listed in
Tables 1-5
including, but not limited to, as determined at the 5% significance level when
grown in the
same environmental conditions, including when grown side-by-side with a
comparison or
check cannabis and/or hemp plant. In some embodiments, the present disclosure
teaches a
2
Date Recue/Date Received 2022-04-12
hemp plant, or a plant part or a plant cell thereof, having all of the
essential physiological
and morphological characteristics of the hemp plant of the present disclosure.
[0012] In some embodiments, the present disclosure teaches a hemp plant, or a
plant part
or a plant cell thereof, having all of the essential physiological and
morphological
characteristics of the hemp plant variety designated 'AF14b15-21', wherein a
representative sample of seed of said variety was deposited under NCMA No.
202203083.
[0013] In some embodiments, the present disclosure teaches a tissue culture of
regenerable
cells produced from the plant, plant part or plant cell of the present
disclosure, wherein a
new plant regenerated from the tissue culture has all of the morphological and
physiological characteristics of the hemp plant variety designated 'AF14b15-
21' listed in
Tables 1-5 when grown under the same environmental conditions. In some
embodiments,
the present disclosure teaches a hemp plant regenerated from the tissue
culture of the
present disclosure, said plant having all the morphological and physiological
characteristics of the hemp of the present disclosure. In some embodiments,
the present
disclosure teaches a hemp plant regenerated from the tissue culture, wherein
the
regenerated plant has all of the characteristics of the hemp plant variety
designated
'AF14b15-21', wherein a representative sample of seed of said variety was
deposited under
NCMA No. 202203083.
[0014] In some embodiments, the present disclosure teaches a method for
producing a
hemp seed, comprising selfing the hemp plant of the present disclosure, and
harvesting the
resultant hemp seed. In some embodiments, the present disclosure teaches a
hemp seed
produced by the method of the present disclosure.
[0015] In some embodiments, the present disclosure teaches a method for
producing a
hemp seed comprising crossing the hemp plant of the present disclosure with a
second,
distinct plant. In some embodiments, the present disclosure teaches an Fi hemp
seed
produced by the method of the present disclosure. In some embodiments, the
present
disclosure teaches an Fi hemp plant, or a part or a plant cell thereof,
produced by growing
the seed of the present disclosure.
3
Date Recue/Date Received 2022-04-12
[0016] In some embodiments, the present disclosure teaches a method of
producing a hemp
plant derived from the variety 'AF14b15-21', comprising: a) crossing the plant
of the
present disclosure, with itself or a second plant to produce progeny seed; b)
growing the
progeny seed to produce a progeny plant and crossing the progeny plant with
itself or a
second plant to produce further progeny seed; and, optionally c) repeating
step (b) one or
more times to produce the hemp plant derived from the variety 'AF14b15-21'.
[0017] In some embodiments, the present disclosure teaches a method of
producing a hemp
plant derived from the variety 'AF14b15-21', further comprising crossing the
hemp plant
derived from the variety 'AF14b15-21', with a plant of a different genotype to
produce
seed of a hybrid plant derived from the hemp variety 'AF14b15-21'.
[0018] In some embodiments, the present disclosure teaches a method for
producing
nucleic acids, comprising isolating nucleic acids from the seed, plant, plant
part, or plant
cell of the present disclosure.
[0019] In some embodiments, the present disclosure teaches a hemp plant, plant
part, or
plant cell comprising a single locus conversion and otherwise essentially all
the
morphological and physiological characteristics of the hemp plant 'AF14b15-21'
when
grown in the same environmental conditions. In some embodiments, the present
disclosure
teaches that the single locus conversion confers said plant with herbicide
resistance. In
some embodiments, the present disclosure teaches that the single locus
conversion is an
artificially mutated gene or nucleotide sequence. In some embodiments, the
present
disclosure teaches that the single locus conversion is a gene that has been
modified through
the use of breeding techniques taught in the present disclosure.
[0020] In some embodiments, the present disclosure teaches a cultivar of hemp
designated
'AF14b15-21' as described and detailed in the present disclosure.
[0021] In some embodiments, the present disclosure teaches a method of
producing a
cannabinoid extract, said method comprising the steps (a) contacting the plant
of the
present disclosure with a solvent or heat, thereby producing a cannabinoid
extract.
4
Date Recue/Date Received 2022-04-12
[0022] In some embodiments, the present disclosure teaches a dry, non-viable
plant, or dry
non-viable part thereof, of hemp variety 'AF14b15-21', wherein representative
seed of the
variety has been deposited under NCMA No. 202203083.
[0023] In some embodiments, the present disclosure teaches an assemblage of
dry, non-
viable female inflorescences from a hemp plant variety designated 'AF14b15-
21', wherein
representative seed the variety has been deposited under NCMA No. 202203083.
In some
embodiments, the present disclosure teaches that a dry, non-viable plant part
is an
inflorescence and/or a flower.
[0024] In some embodiments, the present disclosure teaches a hemp plant of the
present
disclosure is asexually reproduced. In some embodiments, the present
disclosure teaches
a hemp plant of the present disclosure is capable of producing an asexual
clone of said
hemp plant. In some embodiments, the present disclosure teaches that the
asexual clone is
capable of producing said hemp plant taught in the present disclosure.
[0025] A further embodiment relates to a method for developing a hemp plant in
a hemp
plant breeding program, comprising applying plant breeding techniques
comprising
crossing, recurrent selection, mutation breeding, wherein said mutation
breeding selects
for a mutation that is spontaneous or artificially induced, backcrossing,
pedigree breeding,
marker enhanced selection, haploid/double haploid production, or
transformation to the
hemp plant of 'AF14b15-21', or its parts, wherein application of said
techniques results in
development of a hemp plant.
[0026] A further embodiment relates to a method of introducing a mutation into
the
genome of hemp plant 'AF14b15-21', said method comprising mutagenesis of the
plant,
or plant part thereof, of 'AF14b15-21', wherein said mutagenesis is selected
from the group
consisting of temperature, long-term seed storage, tissue culture conditions,
ionizing
radiation, chemical mutagens, and targeting induced local lesions in genomes,
and wherein
the resulting plant comprises at least one genome mutation and producing
plants therefrom.
[0027] A further embodiment relates to a method of editing the genome of hemp
plant
'AF14b15-21', wherein said method is selected from the group comprising zinc
finger
nucleases, transcription activator-like effector nucleases (TALENs),
engineered homing
Date Recue/Date Received 2022-04-12
endonucleases/meganucleases, and the clustered regularly interspaced short
palindromic
repeat (CRISPR)-associated protein9 (Cas9) system, and plants produced
therefrom.
[0028] A further embodiment relates to a plant cell of a hemp plant, which is
a descendant
of `AF14b15-21', the descendant expressing the physiological and morphological
characteristics of variety `AF14b15-21' listed in Tables 1-5 as determined at
the 5%
significance level when grown under substantially similar environmental
conditions,
wherein representative seed of 'AF14b15-21' has been deposited under NCMA No.
202203083.
[0029] A further embodiment relates to use of a hemp plant variety designated
`AF14b15-
21', wherein representative seed of the variety has been deposited under NCMA
No.
202203083, for isolating nucleic acids from the variety.
[0030] A further embodiment relates to use of a hemp plant variety designated
`AF14b15-
21', wherein representative seed of the variety has been deposited under NCMA
No.
202203083, to produce a commodity plant product.
[0031] A further embodiment relates to use of a hemp plant variety designated
`AF14b15-
21', wherein representative seed of the variety has been deposited under NCMA
No.
202203083, to produce a cannabinoid and/or terpene extract.
[0032] A further embodiment relates to use of a hemp plant variety designated
`AF14b15-
21', wherein representative seed of the variety has been deposited under NCMA
No.
202203083, to grow subsequent generations.
[0033] A further embodiment relates to use of a hemp plant variety designated
`AF14b15-
21', wherein representative seed of the variety has been deposited under NCMA
No.
202203083, for vegetative propagation.
[0034] A further embodiment relates to use of a hemp plant variety designated
`AF14b15-
21', wherein representative seed of the variety has been deposited under NCMA
No.
202203083, as a recipient of a conversion locus.
6
Date Recue/Date Received 2022-04-12
[0035] A further embodiment relates to use of a hemp plant variety designated
'AF14b15-
21', wherein representative seed of the variety has been deposited under NCMA
No.
202203083, to produce a genetic marker profile.
[0036] A further embodiment relates to use of a hemp plant variety designated
'AF14b15-
21', wherein representative seed of the variety has been deposited under NCMA
No.
202203083, as a source of seed.
[0037] A further embodiment relates to use of a hemp plant variety designated
'AF14b15-
21', wherein representative seed of the variety has been deposited under NCMA
No.
202203083, as a crop.
[0038] A further embodiment relates to use of a hemp plant variety designated
'AF14b15-
21', wherein representative seed of the variety has been deposited under NCMA
No.
202203083, as a recipient of a transgene.
[0039] A further embodiment relates to use of a hemp plant variety designated
'AF14b15-
21', wherein representative seed of the variety has been deposited under NCMA
No.
202203083, as a source of breeding material.
[0040] A further embodiment relates to use of a hemp plant variety designated
'AF14b15-
21', wherein representative seed of the variety has been deposited under NCMA
No.
202203083, for producing a plant derived from the variety.
DESCRIPTION OF THE DRAWINGS
[0041] The accompanying photographs depict characteristics of the new 'AF14b15-
21'
plants as nearly true as possible reproductions.
[0042] FIG. 1 shows an overall view of the 'AF14b15-21' plant with main axis
dominance
at the early flower stage.
[0043] FIG. 2 shows an overall view of the 'AF14b15-21' plant with main axis
dominance
at the mid-flower stage.
[0044] FIG. 3 shows a field of 'AF14b15-21' at the mid-flower stage.
[0045] FIG. 4 shows an overall view of the 'AF14b15-21' plant in full flower.
7
Date Recue/Date Received 2022-04-12
100461 FIG. 5 shows a close view of upper part (including flowers) of the
'AF14b15-21'
plant with main axis dominance at floral maturity.
100471 FIG. 6 shows a close view of upper part (including flowers) of the
'AF14b15-21'
plant with main axis dominance at floral maturity.
100481 FIG. 7 is a stacked bar graph of the 'AF14b15-21' Terpene Profile at
maturity.
DETAILED DESCRIPTION OF THE INVENTION
100491
100501 The following description includes information that may be useful in
understanding
the present disclosure. It is not an admission that any of the information
provided herein
is prior art or relevant to the presently claimed disclosures, or that any
publication
specifically or implicitly referenced is prior art.
Definitions
100511 As used herein, the term "about" refers to plus or minus 10% of the
referenced
number, unless otherwise stated or otherwise evident by the context (such as
when a range
would exceed 100% of a possible value or fall below 0% of a possible value).
For example,
reference to an absolute content of a particular cannabinoid of "about 1%"
means that that
cannabinoid can be present at any amount ranging from 0.9% to 1.1% content by
weight.
The term "about," when applied to a range modifies each of the end points, as
discussed
above. For example, about 1-2% includes the complete range of 0.9%- to 2.2%.
Unless
otherwise stated, ranges in this document include end points, such that 1-2%
includes
values of 1% and 2%.
100521 The disclosure provides cannabis hemp plants. As used herein, the term
"plant"
refers to plants in the genus of Cannabis and plants derived thereof. Such as
cannabis plants
produced via asexual reproduction, tissue culture, and via seed production.
8
Date Recue/Date Received 2022-09-13
[0053] The disclosure provides plant parts. As used herein, the term "plant
part" refers to
any part of a plant including but not limited to the embryo, shoot, root,
stem, seed, stipule,
leaf, petal, flower, inflorescence, bud, ovule, bract, trichome, branch,
petiole, internode,
bark, pubescence, tiller, rhizome, frond, blade, ovule, pollen, stamen, and
the like. The two
main parts of plants grown in some sort of media, such as soil or vermiculite,
are often
referred to as the "above-ground" part, also often referred to as the
"shoots", and the
"below-ground" part, also often referred to as the "roots". Plant parts may
also include
certain extracts such as kief or hash, which include cannabis plant trichomes
or glands. In
some embodiments, plant part should also be interpreted as referring to
individual cells
from the plant.
[0054] As used herein, the term "plant cell" refers to any plant cell from a
cannabis plant.
Plant cells of the present disclosure include cells from a cannabis plant
shoot, root, stem,
seed, stipule, leaf, petal, inflorescence, bud, ovule, bract, trichome,
petiole, internode. In
some embodiments, the disclosed plant cell is from a cannabis trichome.
[0055] The term "a" or "an" refers to one or more of that entity; for example,
"a gene"
refers to one or more genes or at least one gene. As such, the terms "a" (or
"an"), "one or
more" and "at least one" are used interchangeably herein. In addition,
reference to "an
element" by the indefinite article "a" or "an" does not exclude the
possibility that more than
one of the elements is present, unless the context clearly requires that there
is one and only
one of the elements.
[0056] The International Code of Zoological Nomenclature defines rank, in the
nomenclatural sense, as the level, for nomenclatural purposes, of a taxon in a
taxonomic
hierarchy (e.g., all families are for nomenclatural purposes at the same rank,
which lies
between superfamily and subfamily). While somewhat arbitrary, there are seven
main
ranks defined by the international nomenclature codes: kingdom,
phylum/division, class,
order, family, genus, and species. Further taxonomic hierarchies used in this
disclosure are
described below.
[0057] The term "variety" as used herein has identical meaning to the
corresponding
definition in the International Convention for the Protection of New Varieties
of Plants
(UPOV treaty), of Dec. 2, 1961, as Revised at Geneva on Nov. 10, 1972, on Oct.
23, 1978,
9
Date Recue/Date Received 2022-04-12
and on Mar. 19, 1991. Thus, "variety" means a plant grouping within a single
botanical
taxon of the lowest known rank, which grouping, irrespective of whether the
conditions for
the grant of a breeder's right are fully met, can be i) defined by the
expression of the
characteristics resulting from a given genotype or combination of genotypes,
ii)
distinguished from any other plant grouping by the expression of at least one
of the said
characteristics and iii) considered as a unit with regard to its suitability
for being
propagated unchanged. As used herein, the term "variety" also refers to a
"cultivar"
(cultivar being a cultivated variety) and thus these terms are synonymous.
[0058] As used herein, the term "inbreeding" refers to the production of
offspring via the
mating between relatives. The plants resulting from the inbreeding process are
referred to
herein as "inbred plants" or "inbreds."
[0059] The term "single locus conversion" or "single locus converted plant" as
used herein
refers to those plants that are developed by backcrossing or transformation
wherein
essentially all of the desired morphological and physiological characteristics
are recovered
in addition to the single locus transferred.
[0060] The disclosure provides samples. As used herein, the term "sample"
includes a
sample from a plant, a plant part, a plant cell, or from a transmission
vector, or a soil, water
or air sample.
[0061] The disclosure provides offspring. As used herein, the term "offspring"
refers to
any plant resulting as progeny from a vegetative or sexual reproduction from
one or more
parent plants or descendants thereof.
[0062] The disclosure provides methods for crossing a first plant with a
second plant. As
used herein, the term "cross", "crossing", "cross pollination" or "cross-
breeding" refer to
the process by which the pollen of one flower on one plant is applied
(artificially or
naturally) to the ovule (stigma) of a flower on another plant. Backcrossing is
a process in
which a breeder repeatedly crosses hybrid progeny, for example a first
generation hybrid
(F1), back to one of the parents of the hybrid progeny. Backcrossing can be
used to
introduce one or more single locus conversions from one genetic background
into another.
Date Recue/Date Received 2022-04-12
[0063] In some embodiments, the present disclosure provides methods for
obtaining plant
genotypes. As used herein, the term "genotype" refers to the genetic makeup of
an
individual cell, cell culture, tissue, organism (e.g., a plant), or group of
organisms.
[0064] The disclosure provides self-pollination populations. As used herein,
the term "self-
crossing", "self-pollinated" or "self-pollination", "self-fertilized" or "self-
fertilization"
means the pollen of one flower on one plant is applied (artificially or
naturally) to the ovule
(stigma) of the same or a different flower on the same plant. In some
embodiments, plants
of the present disclosure are genetically stable, such that pollination
between plants of the
same cultivar produces offspring are still considered part of the same
cultivar.
[0065] In some embodiments, the present disclosure teaches cannabis plants,
which are an
annual, dioecious, flowering herb. Its leaves are typically palmately compound
or digitate,
with serrated leaflets. Cannabis normally has imperfect flowers, with
staminate "male" and
pistillate "female" flowers occurring on separate plants. It is not unusual,
however, for
individual plants of some cannabis varieties to separately bear both male and
female
flowers (i.e., have monoecious plants). Although monoecious plants are often
referred to
as "hermaphrodites," true hermaphrodites (which are less common in cannabis)
bear
staminate and pistillate structures on individual flowers, whereas monoecious
plants bear
male and female flowers at different locations on the same plant. In some
embodiments,
plants of the 'AF14b15-21' variety have been feminized, and only produce
female
inflorescences. In some embodiments, seeds of the 'AF14b15-21' variety produce
plants
that are greater than 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90%
female.
[0066] Persons having skill in the art will be familiar with ways of inducing
male flowers
in otherwise female plants, including rodelization or colloidal silver
treatments. Briefly,
rodelization is the process of stressing female plants to induce pollen sac
formations. This
can be done by allowing unfertilized female flowers to go beyond harvest
maturity in
flowering conditions, which will trigger the formation of pollen sacs in the
plant's last
effort to self-fertilize before the end of the life cycle. Another way of
triggering the
formation pollen in otherwise feminized plants is to spray the feminized
plants at the
flowering stage with colloidal silver solutions (e.g. > 30ppm). After several
sprays, the
plants will start forming pollen sacks. Other forms of silver, such as silver
nitrate and silver
11
Date Recue/Date Received 2022-04-12
thiosulfate are also effective. Also, hormones such as gibberellins can be
used to induce
male flowers on female cannabis plants. Additional methods of inducing male
flowers have
been known to one of ordinary skill in the art, e.g., methods discussed in Ram
and Sett
(Theoretical and Applied Genetics, 1982, 62(4):369-375) and methods discussed
in Ram
and Jaiswal (Plant, 1972, 105(3):263-266).
[0067] As used herein, a "dioecious" plant refers to a plant having either
only male flowers
(androecious) or female flowers (gynoecious).
[0068] As used herein, a "monoecious" plant is a plant having both male and
female or
bisexual flowers, or both female and male or bisexual flowers. Plants bearing
separate
flowers of both sexes at the same time are called simultaneously or
synchronously monoecious. Plants bearing flowers of one sex at one time are
called
consecutively monoecious.
[0069] The disclosure provides ovules and pollens of plants. As used herein
when
discussing plants, the term "ovule" refers to the female gametophyte, whereas
the term
"pollen" means the male gametophyte.
100701 The disclosure provides methods for obtaining plants comprising
recombinant
genes through transformation. As used herein, the term "transformation" refers
to the
transfer of nucleic acid (i.e., a nucleotide polymer) into a cell. As used
herein, the term
"genetic transformation" refers to the transfer and incorporation of DNA,
especially
recombinant DNA, into a cell.
100711 The disclosure provides transformants comprising recombinant genes. As
used
herein, the term "transformant" refers to a cell, tissue or organism that has
undergone
transformation. The original transformant is designated as "TO." Selfing the
TO produces
a first transformed generation designated as "Fl" or "Tl."
100721 In some embodiments, the present disclosure refers to inflorescences
from a
cannabis plant comprising particular cannabinoid and terpene contents (i.e.,
inflorescences
comprising no more than 0.3% THC). In some embodiments, inflorescences, such
as dried
inflorescences, described as having cannabinoid content are female
inflorescences. In some
12
Date Recue/Date Received 2022-09-13
embodiments, the inflorescences are grown "sinsemilla," in the absence of male
plants to
avoid pollination. Thus in some embodiments, the female inflorescences of the
present
disclosure are seedless, and in many cases, un-pollinated. The term
"inflorescence" and
"flower" are used interchangeably throughout this document.
[0073] As used herein, the term "autoflower" refers to a plant that is
insensitive to
photoperiods and is triggered to flower by environmental signals.
[0074] As used herein, the term "essential characteristics" has identical
meaning to the
corresponding definition in the "Explanatory Notes on Essentially Derived
Varieties Under
the 1991 Act of the UPOV Convention (dated April 6, 2017). Thus, essential
characteristics means i) heritable traits that are determined by the
expression of one or more
genes, or other heritable determinants, that contribute to the principal
features, performance
or value of the variety; (ii) characteristics that are important from the
perspective of the
producer, seller, supplier, buyer, recipient, or user; (iii) characteristics
that are essential for
the variety as a whole, including, for example, morphological, physiological,
agronomic,
industrial and biochemical characteristics; (iv) essential characteristics may
or may not be
phenotypic characteristics used for the examination of distinctness,
uniformity, and
stability; (v) essential characteristics are not restricted to those
characteristics that relate
only to high performance or value (for instance, disease resistance may be
considered as
an essential characteristic when the variety has susceptibility to disease);
and (vi) essential
characteristics may be different in different crops/species.
[0075] As used herein, "essentially derived from" has identical meaning to the
corresponding definition in the "Explanatory Notes on Essentially Derived
Varieties Under
the 1991 Act of the UPOV Convention (dated April 6, 2017). Thus, a variety is
essentially
derived from another variety when (i) it is predominantly derived from the
initial variety,
or from a variety that is itself predominantly derived from the initial
variety, while retaining
the expression of the essential characteristics that result from the genotype
or combination
of genotypes of the initial variety; (ii) it is clearly distinguishable from
the initial variety
and (iii) except for the differences which result from the act of derivation,
it conforms to
the initial variety in the expression of the essential characteristics that
result from the
genotype or combination of genotypes of the initial variety. Essentially
derived varieties
13
Date Recue/Date Received 2022-04-12
may be obtained, for example, by the selection of a natural or induced mutant,
or of a
somaclonal variant, the selection of a variant individual from plants of the
initial variety,
backcrossing, or transformation by genetic engineering. A single locus
conversion plant is
an example of an essentially derived variety.
[0076] Unless otherwise noted, references to cannabinoids in a plant, plant
part, extract, or
composition of the present disclosure should be understood as references to
both the acidic
and decarboxylated versions of the compound (e.g., potential THC as determined
by the
conversion guidelines described in this document, and understood by those
skilled in the
art). For example, unless otherwise stated or clear from the context,
references to high CBD
contents of a cannabis plant in this disclosure should be understood as
references to the
combined CBD and CBDA content (accounting for weight loss during
decarboxylation).
DETAILED BOTANICAL DESCRIPTION
[0077] The present disclosure relates to a new and distinct hemp (Cannabis
saliva L.)
cultivar designated as 'AF14b15-21'. Whole-plant hemp extracts from 'AF14b15-
21'
contain an assortment of phytocannabinoids (e.g., CBD), terpenes, flavonoids
and other
minor but valuable hemp compounds that work synergistically to heighten
effects of
products produced from 'AF14b15-21'. This synergistic effect is sometimes
referred to as
the "entourage effect." 'AF14b15-21' extracts can be used to produce a variety
of products,
including liquid and capsule forms for oral administration, topical products,
cosmetic
products, infused beverages, sport products and hemp-infused pet treats.
[0078] Despite cannabis being consumed since at least the third millennium BC,
complete
scientific corroboration for uses of CBD are still in their infancy. Industry
reports suggest
CBD is used for a variety of health and wellness purposes, including as a
sleep aid, coping
with daily stress, fighting anxiety, relieving pain, assisting with cognitive
function and
boosting immune health. Significant research is currently being conducted at a
variety of
laboratories on the use of CBD as it relates to epilepsy, Post-Traumatic
Stress Disorder
(PTSD), cancer, autism, neuroprotection, anti-inflammatory effects, anti-tumor
effects and
anti-psychotic effects.
14
Date Recue/Date Received 2022-04-12
[0079] The primary goal of the breeding program was to develop a new hemp
variety with
early maturity for Northern climates, as well as high cannabidiolic acid
(CBDA)
concentrations and low tetrahydrocannabinolic acid (THCA) concentrations in
its mature
female flowers. `AF14b15-21' is an Fl hybrid maintained by crossing two
parental lines
maintained at applicant's facilities. Each parental line was the result of
controlled crossings
and recurrent selections. The female parental line has exhibited uniformity
over five
generations. The male parental line has exhibited uniformity over three
generations.
[0080] `AF14b15-21' has not been observed under all possible environmental
conditions,
and the phenotype may vary significantly with variations in environment. The
following
observations, measurements, and comparisons describe this plant as grown in
the field in
Colorado.
[0081] Plants for the botanical measurements in the present application are
annual plants.
In the following description, the color determination is in accordance with
The Royal
Horticultural Society Colour Chart, Sixth Edition (2015), except where general
color terms
of ordinary dictionary significance are used.
[0082] Tables 1-5, below, provide the morphological and physiological
characteristics of
the `AF14b15-21' variety as measured on August 20, 2021 in Greeley, Colorado,
United
States. Morphological and physiological characteristics of male parental line
`AF15'
gathered in the same location and on the same date are provided for
comparative purposes.
Note that `AF15' is maintained as a feminized inbred line, such that the
phenotypic
comparison below conducted on a female plant of `AF15'. Rodelization was
utilized to
produce pollen for the cross that produced AF14b15-21' .
Table 1. General Characteristics
Characteristic New Variety ('AF14b15-21') Check Variety ('AF15')
Plant life forms An herbaceous annual plant (herb). An herbaceous annual
plant
(herb).
Plant growth Upright, dwarf structure with a Upright, dwarf structure
with
habit conical shape. Branches emerging .. a candelabra shape when
from a dominant apical stalk. flowering. Branches
emerging from a dominant
apical stalk.
Date Recue/Date Received 2022-04-12
Characteristic New Variety ('AF14b15-21') Check Variety ('AF15')
Plant origin First generation cross (F1 hybrid) Inbred line maintained
at
between two parent lines maintained Applicant's facilities.
at Applicant's facilities.
Plant Seed. Parental lines maintained Maintained as a feminized
propagation through targeted seed increases, inbred seed line.
Parental seed crossed to generate Fl
hybrid seed.
Propagation Easy, using rodelization. Easy, using rodelization.
ease
Plant Height 0.55 m - 1.06 m 0.48 m -0.79 m
Plant Width 45.72 cm -92.71 cm 38.1 cm - 60.96 cm
Plant vigor During early vegetative stages Less vigorous than
AF14b15-21 ' exhibits rapid, `AF14b15-21'.
vigorous growth. By week 3 of
vegetative growth it has
accumulated approximately 50-60%
more biomass than `AF15'.
Time to Harvest 50-60 days after sowing 60-70 days after sowing
AF14b15-21 ' is a photoperiod `AF15' is a photoperiod
insensitive plant; "autoflowers" are insensitive plant;
triggered by environmental signals "autoflowers" are triggered
other than photoperiod. Time to by environmental signals
harvest will depend on plant health, other than photoperiod. Time
stress levels, heat units (growing to harvest will depend on
degree days), and other factors not plant health, stress levels,
listed here. heat units (growing degree
days), and other factors not
listed here.
Is this plant a NO NO
Genetically
Modified
Organism?
16
Date Recue/Date Received 2022-04-12
Table 2. Leaf/Foliage
Characteristic New Variety ('AF14b15-21') Check Variety ('AF15')
Leaf structure Leaflets gladiate to somewhat linear Leaflets gladiate to
with the tip tapering to an acute to somewhat linear with the tip
acuminate apex, with the widest tapering to an acute to
point above the leaflet midpoint, acuminate apex, with the
Serrated margins, widest point above the
leaflet
midpoint. Serrated margins.
Leaf shape Palmately compound. Palmately compound.
Leaf During vegetative growth, branch During vegetative
growth,
arrangement points and leaf arrangement exhibit branch points and leaf
(Phyllotaxy) opposite phyllotaxy with a 180- arrangement exhibit
opposite
degree rotation at each node. When phyllotaxy with a 180-degree
the reproductive stage is initiated rotation at each node. When
phyllotaxy changes from opposite to the reproductive stage is
alternate, initiated phyllotaxy changes
from opposite to alternate.
Leaf margin Serrated, with the outer sides of the Serrated, with the
outer sides
serrations slightly convex and the of the serrations slightly
inner sides slightly concave. Teeth convex and the inner sides
are pointed toward the leaflet apex. slightly concave. Teeth are
Sometimes teeth tips raise up above pointed toward the leaflet
the leaf plane. apex. Sometimes teeth tips
raise up above the leaf plane.
Leaf hairs Present. Present.
(Presence or
absence)
Location of leaf Leaf hairs present on the abaxial Leaf hairs present on
the
hairs if present surface of the leaf, most prevalent abaxial surface of
the leaf,
on the veinal structures. Leaf hair most prevalent on the veinal
structure is setose and silky. Few structures. Leaf hair
structure
leaf hairs present on the adaxial is setose and silky. Few leaf
surface, with hairs present in higher hairs present on the adaxi al
density on the leaf margins and surface, with hairs present
in
lower density in the center of the higher density on the leaf
leaflets. margins and lower density in
the center of the leaflets.
17
Date Recue/Date Received 2022-04-12
Characteristic New Variety ('AF14b15-21') Check Variety ('AF15')
Leaf length 15 cm-24 cm 12.5 cm-18 cm
with petiole at
maturity
No. of leaflets 5-8; 7 being most common 5-7 on all
Middle largest 8.5 cm-13 cm 8.0 cm-11.5 cm
(longest) leaflet
length
Middle largest 0.7 cm-1.9 cm 1.5 cm-2.9 cm
(longest) leaflet
width
Middle largest 6:1-16:1 3:1-6:1
(longest) leaflet
length/width
ratio
No. teeth of 21-29 19-26
middle leaflet
Leaf (upper side 147B for green phenotype, N186A 137C green phenotype only.
- adaxial) color for mature purple phenotype.
(RHS No.)
Leaf (lower side 147C 146C
- abaxial) color
(RHS No.)
Leaf glossiness Absent. Absent.
Vein/midrib Midvein is straight down the middle Midvein is straight down
the
shape (general of each leaflet (percurrent). middle of each leaflet
description) Secondary venation branches off the (percurrent). Secondary
main vein in a slightly alternate venation branches off the
pattern and goes all the way to the main vein in a slightly
tooth edge without connecting to alternate pattern and goes
all
other veins. On the top side of the the way to the tooth edge
leaf, veins look slightly recessed, but without connecting to other
on the bottom veins protrude off the veins. On the top side of the
surface. leaf, veins look slightly
recessed, but on the bottom
side veins protrude off the
surface.
18
Date Recue/Date Received 2022-04-12
Characteristic New Variety ('AF14b15-21') Check Variety ('AF15')
Vein/midrib N148C 147C
color
(RHS No.)
Petiole length 6 cm-12 cm 4 cm-7 cm
Petiole color N148C 147C
(RHS No.)
Petiole Absent. Absent.
anthocyanin
coloration
(presence or
absence)
Stipule shape A non-rigid, spine-like leaf A non-rigid, spine-like leaf
(general structure. With white, paper-like structure. With white,
paper-
description) edges. Wider at the base, tapering to like edges. Wider at
the base,
the apex. Often absent on mature tapering to the apex. Often
leaves due to senescence. absent on mature leaves due
to senescence.
Stipule length 3 mm-8 mm 4 mm-7 mm
Stipule color 147D or 142B 144B
(RHS No.)
Foliage Reminiscent of fresh cut grass when Reminiscent of tomato
with a
fragrance crushed, with musky undertone. hint of celery.
Table 3. Stem
Characteristic New Variety ('AF14b15-21') Check Variety ('AF15')
Stem shape Generally round, slightly fluted with Generally round to
slightly
(general vertical concave furrows running ovate, moderately fluted
with
description) parallel on entire stem length. vertical concave furrows
Furrows are moderately deep, but running parallel on entire
narrow in diameter. stem length. Furrows are
more pronounced than
`AF14b15-21' and in some
cases wider and deeper.
Stem diameter 0.8 cm-2.5 cm 1 cm-2 cm
at base
Stem color 146C 146B
19
Date Recue/Date Received 2022-04-12
Characteristic New Variety ('AF14b15-21') Check Variety ('AF15')
(RHS No.)
Stem pith type Moderate. Thin.
(absent, thin,
moderate, or
thick)
Depth of main Shallow to medium in the main Medium.
stem stalk, shallow in younger stem tissue
ribs/grooves such as side branches.
(shallow,
medium or
deep)
Internode 7.9 cm-11.9 cm 5.5 cm-11.3 cm
length
Table 4. Inflorescence (Female/Pistillate Flowers)
Characteristic New Variety ('AF141315-21') Check Variety ('AF15')
Flowering Plant begins flowering at 20-30 days Plant begins flowering
at 30-
(blooming) post sowing, i.e. hybrid is day- .. 40 days post sowing,
i.e.
habit (general neutral; photoperiod insensitive, hybrid is day-neutral;
description) photoperiod insensitive.
Inflorescence Inflorescences are generally above .. Inflorescences are
generally
position relative the foliage. However, reduced leaves above the foliage.
However,
to foliage are interspersed throughout the reduced leaves are
compound flowers, interspersed throughout the
compound flowers.
Flower Compound Raceme. Compound Raceme.
arrangement
Type of flowers Incomplete; imperfect. Incomplete; imperfect.
Flower shape Cylindrical shape that tapers to a Cylindrical shape that
tapers
(general rounded point, to a rounded point.
description)
Flower 130 mm-315 mm 150 mm-340 mm
(compound
cyme) length
Date Recue/Date Received 2022-04-12
Characteristic New Variety ('AF14b15-21') Check Variety ('AF15')
Flower 5.6 cm-8.4 cm 5.4 cm-8.2 cm
(compound
cyme) diameter
Bract shape Tear drop shaped. Tear drop shaped.
Bract length 3 mm-7 mm 3 mm-7 mm
Bract color 147B at tip fading to 144B at base. 147B
(RHS No.)
Stigma shape Filiform structure, widened at the Filiform structure,
widened
(general base and narrows to a tip, protruding at the base and
narrows to a
description) from bracts. tip, protruding from bracts.
Stigma length 2 mm-5 mm 2 mm-6 mm
Stigma color A mixture of moderate yellow 160A Most stigmas exhibit a
shade
(RHS No.) and light pink 186A when flowers of pink ranging from 58D
to
are forming. Brownish orange 63A with a smaller
N170A when senesced. proportion exhibiting 160A
when flowers are forming.
Stigmas moderate reddish
brown 166B when senesced.
Trichome shape Stalked glandular capitate, bulbous, Stalked glandular
capitate,
capitate sessile, cystolithic hairs. bulbous, capitate sessile,
cystolithic hairs.
Trichome color NN155 A-D translucent-clear-milky; NN155 A-D translucent-
(RHS No.) can also turn yellow-brown-amber clear-milky; can also
turn
when over mature. yellow-brown-amber when
over mature.
Terminal bud Cylindrical shape that is elongated Cylindrical shape
that is
shape and tapers to a rounded point, elongated and tapers to a
rounded point.
Terminal bud 148B for green phenotype, Ni 86A- 147B green phenotype only.
color (RHS Ni 86B for mature purple phenotype.
No.)
Pedicel Absent. Absent.
Sepal Absent. Absent.
Petal Absent. Absent.
Staminate N/A N/A
flower if
present
21
Date Recue/Date Received 2022-04-12
Characteristic New Variety ('AF14b15-21') Check Variety ('AF15')
Pollen if present N/A N/A
Seed shape Nearly lens-shaped, with a round Nearly lens-shaped, with
a
base and a tapered rounded opposite round base and a tapered
point, rounded opposite point.
Seed length 3.7 mm-5 mm 3.1 mm-4.0 mm
Seed color 197A-B 197A-D
(RHS No.)
Marbling of Weak. Weak, some absent of
seed marbling.
(weak, medium
or stong)
Total CBD 5-6% (wt/wt) total CBD on average 3.5-4.5% (wt/wt) total CBD
content after decarboxylation of sample on average if sampled at
the
under measured conditions, though same timepoint as
significantly higher contents have 'AF14b15-21'.
been measured under optimal
growing conditions. On average,
'AF14b15-21' has 40% more total
CBD content on a dry weight basis
than 'AF15'. See Tables 6-7 for
additional information.
Total THC 0.25-0.29% (wt/wt) total THC on 0.10-0.20 (wt/wt) total
THC
content average after decarboxylation of on average after
sample. See Tables 6-7 for decarboxylation of sample.
additional information.
Table 5. Other Characteristics
Characteristic New Variety ('AF14b15-21') Check Variety ('AF15')
Aroma Citrus, musky, cut lumber. Sweet citrus leading into
minty pine.
Proportion of Less than 1%. Less than 1%.
Hermaphrodite
Time period Flower initiation typically begins Flower initiation
typically
and condition of 20-30 days after sowing. Plants are begins 30-40 days
after
flowering/ day-neutral. sowing. Plants are day-
blooming neutral.
22
Date Recue/Date Received 2022-04-12
Characteristic New Variety ('AF14b15-21') Check Variety ('AF15')
Plant Hardiness Hardy once stand is established. Hardy once stand is
Difficult to transplant; best if direct established. Difficult to
seeded. transplant; best if direct
seeded.
Breaking action Does not lodge nor break easily. Does not lodge nor break
easily.
Rooting rate Clones can produce roots in 10-14 Clones can produce
roots in
after days under ideal lighting and above 10-14 days under
ideal
cutting/cloning 80% RH, however cuttings do not lighting and above 80% RH,
revert back to a vegetative state if however cuttings do not
taken during flowering, revert back to a vegetative
state if taken during
flowering.
Types of Stem. Stem.
Cutting for
Cloning
Market use of Cannabinoid dietary supplements Not Available.
flower if and other consumable and cosmetic
available products.
Productivity of An average of 188 g dried weight An average of 109 g dried
flower if per plant with stem, fiber, and leaf weight per plant
with stem,
available removed was achieved from a small fiber, and leaf removed
was
(weight per sample size of 20 plants, under achieved from a small
plant) tested conditions. Weight will vary sample size of 20
plants.
depending on moisture content of Weight will vary depending
dried sample. Productivity upwards on moisture content of dried
of 300 g have been measured in sample.
optimal climate conditions.
[0083] Tables 6-8 include detailed information of the hemp plant named
'AF14b15-21' for
profiles of cannabinoids and terpenes as tested by third-party service
providers.
[0084] As used herein, the term "maturity," "harvest maturity," or "floral
maturity" refers
to the developmental stage at which the 'AF14b15-21' plant is harvested.
Persons having
skill in the art will recognize maturity based on the plant's morphologies. It
is also good
practice to conduct periodic cannabinoid content (i.e., potency) tests
throughout the
development of the plant to ensure that harvest occurs at maturity.
23
Date Recue/Date Received 2022-04-12
[0085] In some embodiments, stigma color can also be an indication of
maturity. For
example, in some embodiments, if all stigmas are browning it could indicate
AF14b15-
21' is past maturity.
[0086] Growing conditions throughout the plant's life cycle, nutrient
variations, and
environmental factors can all influence the amount of time for 'AF14b15-21'
plants to
reach harvest maturity. The present disclosure uses the terms "maturity,"
"harvest
maturity," and "floral maturity" interchangeably. In some embodiments, harvest
maturity
can encompass any period after the emergence of inflorescences, but before the
THC
content of any inflorescence surpasses 0.3%.
[0087] Total Potential THC/CBD contents presented in this document reflect the
total
potential (i.e., decarboxylated) THC and CBD content after decarboxylation of
the THCA
and CBDA contents of the sample. The formula used for this calculation is
reproduced
below for the Office's convenience. Total THC = THC + (THCA * (0.877)). Total
CBD =
CBD + (CBDA * (0.877)).
[0088] 'AF14b15-21' was bred primarily for high a CBD, low THC chemotypic
profile.
When compared to the check variety 'AF15', 'AF14b15-21' has faster maturity
and higher
yield than 'AF 15'.
[0089] AF14b15-21' shows good vigor, health, and uniformity. 'AF14b15-21'
inflorescences will turn purple at maturity if exposed to cold temperatures,
whereas 'AF15'
only produces green inflorescences. The aroma of 'AF14b15-21' is more musky
and like
cut lumber, while 'AF15' has an aroma that is sweeter and more like minty
pine.
Cannabis hemp breeding methods
[0090] In some embodiments, the plants of the present disclosure can be used
to produce
new plant varieties. In some embodiments, the plants are used to develop new,
unique and
superior varieties or hybrids with desired phenotypes. As used herein, the
term "plant
breeding techniques" comprises all of the plant breeding techniques disclosed
in this
section of the application, and well known to persons having skill in the art.
Thus, in some
embodiments, plant breeding methods encompass the application of recurrent
selection,
24
Date Recue/Date Received 2022-04-12
mass selection, hybridization, open-pollination, backcrossing, pedigree
breeding, marker
assisted selection breeding, mutation breeding, gene editing, and combinations
thereof.
100911 In some embodiments, selection methods, e.g., molecular marker assisted
selection,
can be combined with breeding methods to accelerate the process. Additional
breeding
methods have been known to one of ordinary skill in the art, e.g., methods
discussed in
Chahal and Gosal (Principles and procedures of plant breeding:
biotechnological and
conventional approaches, CRC Press, 2002, ISBN 084931321X, 9780849313219),
Taji et
al. (In vitro plant breeding, Routledge, 2002, ISBN 156022908X,
9781560229087),
Richards (Plant breeding systems, Taylor & Francis US, 1997, ISBN 0412574500,
9780412574504), Hayes (Methods of Plant Breeding, Publisher: READ BOOKS, 2007,
ISBN1406737062, 9781406737066).
The Cannabis genome has been sequenced recently (van Bakel et
al., The draft genome and transcriptome of Cannabis saliva, Genome Biology,
12(10):R102, 2011). Molecular markers for cannabis plants are described in
Datwyler et
al. (Genetic variation in hemp and marijuana (Cannabis saliva L.) according to
amplified
fragment length polymorphisms, J Forensic Sci 2006 Mar;51(2):371-5.),
Pinarkara et al.,
(RAPD analysis of seized marijuana (Cannabis saliva L.) in Turkey, Electronic
Journal of
Biotechnology, 12(1), 2009), Hakki et al., (Inter simple sequence repeats
separate
efficiently hemp from marijuana (Cannabis saliva L.), Electronic Journal of
Biotechnology, 10(4), 2007), Datwyler et al., (Genetic Variation in Hemp and
Marijuana
(Cannabis saliva L.) According to Amplified Fragment Length Polymorphisms, J
Forensic
Sci, March 2006, 51(2):371-375), Gilmore et al. (Isolation of microsatellite
markers in
Cannabis saliva L. (marijuana), Molecular Ecology Notes, 3(1):105-107, March
2003),
Pacifico et al., (Genetics and marker-assisted selection of chemotype in
Cannabis saliva
L.), Molecular Breeding (2006) 17:257-268), and Mendoza et al., (Genetic
individualization of Cannabis saliva by a short tandem repeat multiplex
system, Anal
Bioanal Chem (2009) 393:719-726).
100921 In some embodiments, molecular markers are designed and made, based on
the
genome of the plants of the present application. In some embodiments, the
molecular
Date Recue/Date Received 2022-09-13
markers are selected from Isozyme Electrophoresis, Restriction Fragment Length
Polymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),
Arbitrarily
Primed Polymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting
(DAF),
Sequence Characterized Amplified Regions (SCARs). Amplified Fragment Length
Polymorphisms (AFLPs), and Simple Sequence Repeats (SSRs) which are also
referred to
as Microsatellites, etc. Methods of developing molecular markers and their
applications are
described by Avise (Molecular markers, natural history, and evolution,
Publisher: Sinauer
Associates, 2004, ISBN
0878930418, 9780878930418), Srivastava et al. (Plant
biotechnology and molecular markers, Publisher: Springer, 2004,
ISBN1402019114,
9781402019111), and Vienne (Molecular markers in plant genetics and
biotechnology,
Publisher: Science Publishers, 2003).
100931 The molecular markers can be used in molecular marker assisted
breeding. For
example, the molecular markers can be utilized to monitor the transfer of the
genetic
material. In some embodiments, the transferred genetic material is a gene of
interest, such
as genes that contribute to one or more favorable agronomic phenotypes when
expressed
in a plant cell, a plant part, or a plant.
100941 Details of existing cannabis plants varieties and breeding methods are
described in
Potter et al. (2011, World Wide Weed: Global Trends in Cannabis Cultivation
and Its
Control), Holland (2010, The Pot Book: A Complete Guide to Cannabis, Inner
Traditions
/ Bear & Co, ISBN1594778981, 9781594778988), Green I (2009, The Cannabis Grow
Bible: The Definitive Guide to Growing Marijuana for Recreational and Medical
Use,
Green Candy Press, 2009, ISBN 1931160589, 9781931160582), Green 11 (2005, The
Cannabis Breeder's Bible: The Definitive Guide to Marijuana Genetics, Cannabis
Botany
and Creating Strains for the Seed Market, Green Candy Press, 1931160279,
9781931160278), Starks (1990, Marijuana Chemistry: Genetics, Processing &
Potency,
ISBN 0914171399, 9780914171393), Clarke (1981, Marijuana Botany, an Advanced
Study: The Propagation and Breeding of Distinctive Cannabis, Ronin Publishing,
ISBN
091417178X, 9780914171782), Short (2004, Cultivating Exceptional Cannabis: An
Expert
Breeder Shares His Secrets, ISBN 1936807122, 9781936807123), Cervantes (2004,
26
Date Recue/Date Received 2022-09-13
Marijuana Horticulture: The Indoor/Outdoor Medical Grower's Bible, Van Patten
Publishing, ISBN 187882323X, 9781878823236), Franck et al. (1990, Marijuana
Grower's
Guide, Red Eye Press, ISBN 0929349016, 9780929349015), Grotenhermen and Russo
(2002, Cannabis and Cannabinoids: Pharmacology, Toxicology, and Therapeutic
Potential,
Psychology Press, ISBN 0789015080, 9780789015082), Rosenthal (2007, The Big
Book
of Buds: More Marijuana Varieties from the World's Great Seed Breeders, ISBN
1936807068, 9781936807062), Clarke, RC (Cannabis: Evolution and Ethnobotany
2013
(In press)), King, J (Cannabible Vols 1-3, 2001-2006), and four volumes of
Rosenthal's
Big Book of Buds series (2001, 2004, 2007, and 2011).
[0095] Classical breeding methods can be included in the present disclosure to
introduce
one or more recombinant expression cassettes of the present disclosure into
other plant
varieties, or other close-related species that are compatible to be crossed
with the transgenic
plant. In some embodiments, the recombinant expression cassette can encode for
a
desirable phenotype, including herbicide resistance, disease or pest
resistance, insect
resistance, resistance to antibiotics, or additional traits, as disclosed in
this application.
[0096] In some embodiments, said method comprises (i) crossing any one of the
plants of
the present disclosure comprising the expression cassette as a donor to a
recipient plant line
to create a Fl population; (ii) selecting offspring that have expression
cassette. Optionally,
the offspring can be further selected by testing the expression of the gene of
interest. Thus
in some embodiments, the present disclosure teaches crossing a transgenic
plant with the
presently disclosed AF14b 15-21 ' plant.
[0097] In some embodiments, complete chromosomes of the donor plant are
transferred.
For example, the transgenic plant with the expression cassette can serve as a
male or female
parent in a cross pollination to produce offspring plants, wherein by
receiving the transgene
from the donor plant, the offspring plants have the expression cassette.
100981 In a method for producing plants having the expression cassette,
protoplast fusion
can also be used for the transfer of the transgene from a donor plant to a
recipient plant
Protoplast fusion is an induced or spontaneous union, such as a somatic
hybridization,
between two or more protoplasts (cells in which the cell walls are removed by
enzymatic
27
Date Recue/Date Received 2022-09-13
treatment) to produce a single bi- or multi-nucleate cell. The fused cell that
may even be
obtained with plant species that cannot be interbred in nature is tissue
cultured into a hybrid
plant exhibiting the desirable combination of traits. More specifically, a
first protoplast can
be obtained from a plant having the expression cassette. A second protoplast
can be
obtained from a second plant line, optionally from another plant species or
variety,
preferably from the same plant species or variety, that comprises commercially
desirable
characteristics, such as, but not limited to disease resistance, insect
resistance, valuable
grain characteristics (e.g., increased seed weight and/or seed size) etc. The
protoplasts are
then fused using traditional protoplast fusion procedures, which are known in
the art to
produce the cross.
100991 Alternatively, embryo rescue may be employed in the transfer of the
expression
cassette from a donor plant to a recipient plant. Embryo rescue can be used as
a procedure
to isolate embryos from crosses wherein plants fail to produce viable seed. In
this process,
the fertilized ovary or immature seed of a plant is tissue cultured to create
new plants (see
Pierik, 1999, In vitro culture of higher plants, Springer, ISBN 079235267x,
9780792352679).
1001001 In some embodiments, the recipient plant is an elite line having one
or more certain
desired traits. Examples of desired traits include but are not limited to
those that result in
increased biomass production, production of specific chemicals, increased seed
production,
improved plant material quality, increased seed oil content, etc. Additional
examples of
desired traits include pest resistance, vigor, development time (time to
harvest), enhanced
nutrient content, novel growth patterns, aromas or colors, salt, heat, drought
and cold
tolerance, and the like. Desired traits also include selectable marker genes
(e.g., genes
encoding herbicide or antibiotic resistance used only to facilitate detection
or selection of
transformed cells), hormone biosynthesis genes leading to the production of a
plant
hormone (e.g., auxins, gibberellins, cytokinins, abscisic acid and ethylene
that are used
only for selection), or reporter genes (e.g. luciferase, 13-glucuronidase,
chloramphenicol
acetyl transferase (CAT, etc.). The recipient plant can also be a plant with
preferred
chemical compositions, e.g., compositions preferred for medical use or
industrial
applications.
28
Date Recue/Date Received 2022-09-13
1001011 Classical breeding methods can be used to produce new varieties of
cannabis
according to the present disclosure. Newly developed Fl hybrids can be
reproduced via
asexual reproduction.
1001021 Open-Pollinated Populations. The improvement of open-pollinated
populations of
such crops as cannabis, rye, many maizes and sugar beets, herbage grasses,
legumes such
as alfalfa and clover, and tropical tree crops such as cacao, coconuts, oil
palm and some
rubber, depends essentially upon changing gene-frequencies towards fixation of
favorable
alleles while maintaining a high (but far from maximal) degree of
heterozygosity.
Uniformity in such populations is impossible and trueness-to-type in an open-
pollinated
variety is a statistical feature of the population as a whole, not a
characteristic of individual
plants. Thus, the heterogeneity of open-pollinated populations contrasts with
the
homogeneity (or virtually so) of inbred lines, clones and hybrids.
1001031 Population improvement methods fall naturally into two groups, those
based on
purely phenotypic selection, normally called mass selection, and those based
on selection
with progeny testing. Interpopulation improvement utilizes the concept of open
breeding
populations; allowing genes to flow from one population to another. Plants in
one
population (cultivar, strain, ecotype, or any germplasm source) are crossed
either naturally
(e.g., by wind) or by hand or by bees (commonly ilpis mellifera L. or
Megachile rotundata
F.) with plants from other populations. Selection is applied to improve one
(or sometimes
both) population(s) by isolating plants with desirable traits from both
sources.
1001041 There are basically two primary methods of open-pollinated population
improvement First, there is the situation in which a population is changed en
masse by a
chosen selection procedure. The outcome is an improved population that is
indefinitely
propagatable by random-mating within itself in isolation. Second, the
synthetic variety
attains the same end result as population improvement but is not itself
propagatable as such;
it has to be reconstructed from parental lines or clones. These plant breeding
procedures
for improving open-pollinated populations are well known to those skilled in
the art and
comprehensive reviews of breeding procedures routinely used for improving
cross-
pollinated plants are provided in numerous texts and articles, including:
Allard, Principles
of Plant Breeding, John Wiley & Sons, Inc. (1960); Simmonds, Principles of
Crop
29
Date Recue/Date Received 2022-04-12
Improvement, Longman Group Limited (1979); Hallauer and Miranda, Quantitative
Genetics in Maize Breeding, Iowa State University Press (1981); and, Jensen,
Plant
Breeding Methodology, John Wiley & Sons, Inc. (1988).
[00105] Mass Selection. In mass selection, desirable individual plants are
chosen,
harvested, and the seed composited without progeny testing to produce the
following
generation. Since selection is based on the maternal parent only, and there is
no control
over pollination, mass selection amounts to a form of random mating with
selection. As
stated herein, the purpose of mass selection is to increase the proportion of
superior
genotypes in the population.
[00106] Mutation breeding is another method of introducing new traits into the
hemp
plants of the present disclosure. Mutations that occur spontaneously or are
artificially
induced can be useful sources of variability for a plant breeder. The goal of
artificial
mutagenesis is to increase the rate of mutation for a desired characteristic.
Mutation rates
can be increased by many different means including temperature, long-term seed
storage,
tissue culture conditions, radiation; such as X-rays, Gamma rays (e.g., cobalt
60 or cesium
137), neutrons, (product of nuclear fission by uranium 235 in an atomic
reactor), Beta
radiation (emitted from radioisotopes such as phosphorus 32 or carbon 14), or
ultraviolet
radiation (preferably from 2500 to 2900 nm), or chemical mutagens (such as
base
analogues (5-bromo-uracil)), related compounds (8-ethoxy caffeine),
antibiotics
(streptonigrin), alkylating agents (sulfur mustards, nitrogen mustards,
epoxides,
ethyleneamines, sulfates, sulfonates, sulfones, lactones), azide,
hydroxylamine, nitrous
acid, or acridines. Once a desired trait is observed through mutagenesis the
trait may then
be incorporated into existing germplasm by traditional breeding techniques.
Details of
mutation breeding can be found in Allard, Principles of Plant Breeding, John
Wiley &
Sons, Inc. (1960). In addition, mutations created in other hemp plants may be
used to
produce a backcross conversion of hemp plants having all phenotypes of the
AF14b15-
21' line while comprising the mutation obtained from the other hemp plants.
[00107] Additional methods include, but are not limited to, expression vectors
introduced
into plant tissues using a direct gene transfer method, such as
microprojectile-mediated
delivery, DNA injection, electroporation, and the like. Additionally,
expression vectors
Date Recue/Date Received 2022-04-12
are introduced into plant tissues by using either microprojectile-mediated
delivery with a
biolistic device or by using Agrobacterium-mediated transformation.
Transformant plants
obtained with the protoplasm of the subject hemp plants are intended to be
within the scope
of the embodiments of the application.
[00108] Synthetics. A synthetic variety is produced by crossing inter se a
number of
genotypes selected for good combining ability in all possible hybrid
combinations, with
subsequent maintenance of the variety by open pollination. Whether parents are
(more or
less inbred) seed-propagated lines, makes no difference in principle. Parents
are selected
on general combining ability, sometimes by test crosses or toperosses, more
generally by
polycrosses. Parental seed lines may be deliberately inbred (e.g. by selfing
or sib crossing).
However, even if the parents are not deliberately inbred, selection within
lines during line
maintenance will ensure that some inbreeding occurs. Clonal parents will, of
course,
remain unchanged and highly heterozygous.
[00109] Whether a synthetic can go straight from the parental seed production
plot to the
farmer or must first undergo one or two cycles of multiplication depends on
seed
production and the scale of demand for seed.
[00110] While mass selection is sometimes used, progeny testing is generally
preferred for
polycrosses, because of their operational simplicity and obvious relevance to
the objective,
namely exploitation of general combining ability in a synthetic.
[00111] The numbers of parental lines or clones that enter a synthetic vary
widely. In
practice, numbers of parental lines range from 10 to several hundred, with 100-
200 being
the average. Broad based synthetics formed from 100 or more clones would be
expected
to be more stable during seed multiplication than narrow based synthetics.
[00112] Pedigreed varieties. A pedigreed variety is a superior genotype
developed from
selection of individual plants out of a segregating population followed by
propagation and
seed increase of self-pollinated offspring and careful testing of the genotype
over several
generations. This is an open pollinated method that works well with naturally
self-
pollinating species. This method can be used in combination with mass
selection in variety
31
Date Recue/Date Received 2022-04-12
development. Variations in pedigree and mass selection in combination are the
most
common methods for generating varieties in self-pollinated crops.
101001 Hybrids. A hybrid is an individual plant resulting from a cross between
parents of
differing genotypes. Commercial hybrids are now used extensively in many
crops,
including corn (maize), sorghum, sugar beet, sunflower and broccoli. Hybrids
can be
formed in a number of different ways, including by crossing two parents
directly (single
cross hybrids), by crossing a single cross hybrid with another parent (three-
way or triple
cross hybrids), or by crossing two different hybrids (four-way or double cross
hybrids).
[0101] Strictly speaking, most individuals in an out breeding (i.e., open-
pollinated)
population are hybrids, but the term is usually reserved for cases in which
the parents are
individuals whose genomes are sufficiently distinct for them to be recognized
as different
species or subspecies. Hybrids may be fertile or sterile depending on
qualitative and/or
quantitative differences in the genomes of the two parents. Heterosis, or
hybrid vigor, is
usually associated with increased heterozygosity that results in increased
vigor of growth,
survival, and fertility of hybrids as compared with the parental lines that
were used to form
the hybrid. Maximum heterosis is usually achieved by crossing two genetically
different,
highly inbred lines.
101021 Targeting Induced Local Lesions in Genomes (TILLING). Breeding schemes
of
the present disclosure can include crosses with TILLING plant lines. TILLING
is a
method in molecular biology that allows directed identification of mutations
in a specific
gene. TILLING was introduced in 2000, using the model plant Arabidopsis
thaliana.
TILLING has since been used as a reverse genetics method in other organisms
such as
zebrafish, corn, wheat, rice, soybean, tomato and lettuce. The method combines
a standard
and efficient technique of mutagenesis with a chemical mutagen (e.g., Ethyl
methanesulfonate (EMS)) with a sensitive DNA screening-technique that
identifies single
base mutations (also called point mutations) in a target gene. EcoTILLING is a
method
that uses TILLING techniques to look for natural mutations in individuals,
usually for
population genetics analysis (see Comai, et al., 2003 The Plant Journal 37,
778-786;
Gilchrist et a/.2006 Mol. Ecol. 15, 1367-1378; Mejlhede el a/.2006 Plant
Breeding 125,
461-467; Nieto et aL 2007 BMC Plant Biology 7, 34-42).
32
Date Recue/Date Received 2022-09-13
DEcoTILUNG is a modification of TILLING and
EcoTILLING which uses an inexpensive method to identify fragments (Garvin et
aL, 2007,
DEco-TILLING: An inexpensive method for SNP discovery that reduces
ascertainment
bias. Molecular Ecology Notes 7, 735-746). More detailed description on
methods and
compositions on l'ILIING can be found in US 5994075, US 2004/0053236 Al, WO
2005/055704, and WO 2005/048692..
101031 In some embodiments, TILLING can also be utilized for plants of the
cannabis
genus including hemp plants. Thus, in some embodiments, the breeding methods
of the
present disclosure include breeding with one or more TILLING plant lines with
one or
more identified mutations.
101041 Gene editing technologies. Breeding and selection schemes of the
present
disclosure can include crosses with plant lines that have undergone genome
editing. In
some embodiments, the breeding and selection methods of the present disclosure
are
compatible with plants that have been modified using any gene and/or genome
editing tool,
including, but not limited to: ZFNs, TALENS, CRISPR, and Mega nuclease
technologies.
In some embodiments, persons having skill in the art will recognize that the
breeding
methods of the present disclosure are compatible with many other gene editing
technologies. In some embodiments, the present disclosure teaches gene-editing
technologies can be applied for a single locus conversion, for example,
conferring hemp
plant with herbicide resistance. In some embodiments, the present disclosure
teaches that
the single locus conversion is an artificially mutated gene or nucleotide
sequence that has
been modified through the use of breeding techniques taught herein.
[0105] In some embodiments, the breeding and selection methods of the present
disclosure
are compatible with plants that have been modified through Zinc Finger
Nucleases. Three
variants of the ZFN technology are recognized in plant breeding (with
applications ranging
from producing single mutations or short deletions/insertions in the case of
ZFN-1 and -2
techniques up to targeted introduction of new genes in the case of the ZFN-3
technique);
1) ZFN-1: Genes encoding ZFNs are delivered to plant cells without a repair
template. The
ZFNs bind to the plant DNA and generate site specific double-strand breaks
(DSBs). The
33
Date Recue/Date Received 2022-09-13
natural DNA-repair process (which occurs through nonhomologous end-joining,
NHEJ)
leads to site specific mutations, in one or only a few base pairs, or to short
deletions or
insertions; 2) ZFN-2: Genes encoding ZFNs are delivered to plant cells along
with a repair
template homologous to the targeted area, spanning a few kilo base pairs. The
ZFNs bind
to the plant DNA and generate site-specific DSBs. Natural gene repair
mechanisms
generate site-specific point mutations e.g. changes to one or a few base pairs
through
homologous recombination and the copying of the repair template; and 3) ZFN-3:
Genes
encoding ZFNs are delivered to plant cells along with a stretch of DNA which
can be
several kilo base pairs long and the ends of which are homologous to the DNA
sequences
flanking the cleavage site. As a result, the DNA stretch is inserted into the
plant genome in
a site-specific manner.
[0106] In some embodiments, the breeding and selection methods of the present
disclosure
are compatible with plants that have been modified through Transcription
activator-like
(TAL) effector nucleases (TALENs). TALENS are polypeptides with repeat
polypeptide
arms capable of recognizing and binding to specific nucleic acid regions. By
engineering
the polypeptide arms to recognize selected target sequences, the TAL nucleases
can be
used to direct double stranded DNA breaks to specific genomic regions. These
breaks can
then be repaired via recombination to edit, delete, insert, or otherwise
modify the DNA of
a host organism. In some embodiments, TALENSs are used alone for gene editing
(e.g.,
for the deletion or disruption of a gene). In other embodiments, TALs are used
in
conjunction with donor sequences and/or other recombination factor proteins
that will
assist in the Non-homologous end joining (NHEJ) process to replace the
targeted DNA
region. For more information on the TAL-mediated gene editing compositions and
methods of the present disclosure, see US Patent Nos. 8,440,432; US 8,450,471;
US
8,586,526; US 8,586,363; US 8,592,645; US 8,697,853; 8,704,041; 8,921,112; and
8,912,138.
[0107] In some embodiments, the breeding and selection methods of the present
disclosure
are compatible with plants that have been modified through Clustered Regularly
Interspaced Short Palindromic Repeats (CRISPR) or CRISPR-associated (Cas) gene
editing tools. CRISPR proteins were originally discovered as bacterial
adaptive immunity
34
Date Recue/Date Received 2022-09-13
systems which protected bacteria against viral and plasmid invasion. There are
at least three
main CRISPR system types (Type 1,11, and III) and at least 10 distinct
subtypes (Makarova,
KS., et.al., Nat Rev Microbiol. 2011 May 9; 9(6):467-477). Type I and Ill
systems use Cas
protein complexes and short guide polynucleotide sequences to target selected
DNA
regions. Type II systems rely on a single protein (e.g. Cas9) and the
targeting guide
polynucleotide, where a portion of the 5' end of a guide sequence is
complementary to a
target nucleic acid. For more information on the CRISPR gene editing
compositions and
methods of the present disclosure, see US Patent Nos. 8,697,359; 8,889,418;
8,771,945;
and 8,871,445.
101081 In some embodiments, the breeding and selection methods of the present
disclosure
are compatible with plants that have been modified through meganucleases. In
some
embodiments, meganucleases are engineered endonucleases capable of targeting
selected
DNA sequences and inducing DNA breaks. In some embodiments, new meganucleases
targeting specific regions are developed through recombinant techniques which
combine
the DNA binding motifs from various other identified nucleases. In other
embodiments,
new meganucleases are created through semi-rational mutational analysis, which
attempts
to modify the structure of existing binding domains to obtain specificity for
additional
sequences. For more information on the use of meganucleases for genome
editing, see Silva
et al., 2011 Current Gene Therapy 11 pg 11-27; and Stoddard et al., 2014
Mobile DNA 5
pg 7.
Plant Transformation
101091 Hemp plants of the present disclosure, such as `AF14b15-21' can be
further
modified by introducing one or more transgenes which when expressed lead to
desired
phenotypes. The most common method for the introduction of new genetic
material into a
plant genome involves the use of living cells of the bacterial pathogen
Agrobacterium
tumefaciens to literally inject a piece of DNA, called transfer or T-DNA, into
individual
plant cells (usually following wounding of the tissue) where it is targeted to
the plant
nucleus for chromosomal integration. There
are numerous patents governing
Agrobacterium mediated transformation and particular DNA delivery plasmids
designed
specifically for use with Agrobacterium, for example, US4536475, EP0265556,
Date Recue/Date Received 2022-09-13
EP0270822, W08504899, W08603516, US5591616, EP0604662, EP0672752,
W08603776, W09209696, W09419930, W09967357, US4399216, W08303259,
US5731179, EP068730, W09516031, US5693512, US6051757 and EP904362A1.
Agrobacterium-mediated plant transformation involves as a first step the
placement of
DNA fragments cloned on plasmids into living Agrobacterium cells, which are
then
subsequently used for transformation into individual plant cells.
Agrobacterium-mediated
plant transfoimation is thus an indirect plant transformation method. Methods
of
Agrobacterium-mediated plant transformation that involve using vectors with no
T-DNA
are also well known to those skilled in the art and can have applicability in
the present
disclosure. See, for example, U.S. Patent No. 7,250,554, which utilizes P-DNA
instead of
T-DNA in the transformation vector.
[0110] Direct plant transformation methods using DNA have also been reported.
The first
of these to be reported historically is electroporation, which utilizes an
electrical current
applied to a solution containing plant cells (M. E. Fromm et al., Nature, 319,
791 (1986);
H. Jones et al., Plant Mol. Biol., 13, 501 (1989) and H. Yang et al., Plant
Cell Reports, 7,
421 (1988).
Another direct method, called "biolistic bombardment", uses ultrafine
particles, usually tungsten or gold, that are coated with DNA and then sprayed
onto the
surface of a plant tissue with sufficient force to cause the particles to
penetrate plant cells,
including the thick cell wall, membrane and nuclear envelope, but without
killing at least
some of them (US 5,204,253, US 5,015,580). A third direct method uses fibrous
forms of
metal or ceramic consisting of sharp, porous or hollow needle-like projections
that literally
impale the cells, and also the nuclear envelope of cells. Both silicon carbide
and aluminum
borate whiskers have been used for plant transformation (Mizuno et al., 2004;
Petolino et
al., 2000; US5302523 US Application 20040197909) and also for bacterial and
animal
transformation (Kaepler et al., 1992; Raloff, 1990; Wang, 1995). There are
other methods
reported, and undoubtedly, additional methods will be developed. However, the
efficiencies of each of these indirect or direct methods in introducing
foreign DNA into
plant cells are invariably extremely low, making it necessary to use some
method for
selection of only those cells that have been transformed, and further,
allowing growth and
regeneration into plants of only those cells that have been transformed.
36
Date Recue/Date Received 2022-04-12
101111 For efficient plant transformation, a selection method must be employed
such that
whole plants are regenerated from a single transformed cell and every cell of
the
transformed plant carries the DNA of interest. These methods can employ
positive
selection, whereby a foreign gene is supplied to a plant cell that allows it
to utilize a
substrate present in the medium that it otherwise could not use, such as
mannose or xylose
(for example, refer US 5,767,378; US 5994629). More typically, however,
negative
selection is used because it is more efficient, utilizing selective agents
such as herbicides
or antibiotics that either kill or inhibit the growth of nontransformed plant
cells and
reducing the possibility of chimeras. Resistance genes that are effective
against negative
selective agents are provided on the introduced foreign DNA used for the plant
transformation. For example, one of the most popular selective agents used is
the antibiotic
kanamycin, together with the resistance gene neomycin phosphotransferase
(nptII), which
confers resistance to kanamycin and related antibiotics (see, for example,
Messing &
Vierra, Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187 (1983)).
However,
many different antibiotics and antibiotic resistance genes can be used for
transformation
purposes (refer US 5034322, US 6174724 and US 6255560). In addition, several
herbicides and herbicide resistance genes have been used for transformation
purposes,
including the bar gene, which confers resistance to the herbicide
phosphinothricin (White
et al., Nucl Acids Res 18: 1062 (1990), Spencer et al., Theor Appl Genet 79:
625-631(1990),
US 4795855, US 5378824 and US 6107549). In addition, the dhfr gene, which
confers
resistance to the anticancer agent methotrexate, has been used for selection
(Bourouis et
al., EMBO J. 2(7): 1099-1104 (1983).
[0112] Genes can be introduced in a site directed fashion using homologous
recombination. Homologous recombination permits site specific modifications in
endogenous genes and thus inherited or acquired mutations may be corrected,
and/or novel
alterations may be engineered into the genome. Homologous recombination and
site-
directed integration in plants are discussed in, for example, U.S. Patent Nos.
5,451,513,
5,501,967 and 5,527,695.
[0113] Methods of producing transgenic plants are well known to those of
ordinary skill
in the art. Transgenic plants can now be produced by a variety of different
transformation
37
Date Recue/Date Received 2022-04-12
methods including, but not limited to, electroporation; microinjection;
microprojectile
bombardment, also known as particle acceleration or biolistic bombardment;
viral-
mediated transformation; and Agrobacterium-mediated transformation. See, for
example,
U.S. Patent Nos. 5,405,765; 5,472,869; 5,538,877; 5,538,880; 5,550,318;
5,641,664; and
5,736,369; and International Patent Application Publication Nos.
WO/2002/038779 and
WO/2009/117555; Lu et al., (Plant Cell Reports, 2008, 27:273-278); Watson et
al.,
Recombinant DNA, Scientific American Books (1992); Hinchee et al., Bio/Tech.
6:915-
922 (1988); McCabe et al., Bio/Tech. 6:923-926 (1988); Toriyama et al.,
Bio/Tech.
6:1072-1074 (1988); Fromm et al., Bio/Tech. 8:833-839 (1990); Mullins et at,
Bio/Tech.
8:833-839 (1990); Hiei et al., Plant Molecular Biology 35:205-218 (1997);
Ishida et at,
Nature Biotechnology 14:745-750 (1996); Zhang et al., Molecular Biotechnology
8:223-
231(1997); Ku et at, Nature Biotechnology 17:76-80(1999); and, Raineri et at,
Bio/Tech.
8:33-38 (1990)).
Other references teaching the transformation of cannabis plants and the
production
of callus tissue include Raharjo et al 2006, "Callus Induction and
Phytochemical
Characterization of Cannabis sativa Cell Suspension Cultures", Ind . J. Chem 6
(1) 70-
74; and "The biotechnology of Cannabis sativa" by Sam R. Zwenger,
electronically
published April, 2009.
101141 Microprojecfile bombardment is also known as particle acceleration,
biolistic
bombardment, and the gene gun (Biolistic Gene Gun). The gene gun is used to
shoot
pellets that are coated with genes (e.g., for desired traits) into plant seeds
or plant tissues
in order to get the plant cells to then express the new genes. The gene gun
uses an actual
explosive (.22 caliber blank) to propel the material. Compressed air or steam
may also be
used as the propellant. The Biolistic Gene Gun was invented in 1983-1984 at
Cornell
University by John Sanford, Edward Wolf, and Nelson Allen. It and its
registered
trademark are now owned by E. I. du Pont de Nemours and Company. Most species
of
plants have been transformed using this method.
101151 Agrobacterium tumefaciens is a naturally occurring bacterium that is
capable of
inserting its DNA (genetic information) into plants, resulting in a type of
injury to the plant
known as crown gall. Most species of plants can now be transformed using this
method,
38
Date Recue/Date Received 2022-09-13
including cucurbitaceous species. A transgenic plant formed using
Agrobacterium
transformation methods typically contains a single gene on one chromosome,
although
multiple copies are possible. Such transgenic plants can be referred to as
being hemizygous
for the added gene. A more accurate name for such a plant is an independent
segregant,
because each transformed plant represents a unique T-DNA integration event
(U.S. Patent
No. 6,156,953). A transgene locus is generally characterized by the presence
and/or
absence of the transgene. A heterozygous genotype in which one allele
corresponds to the
absence of the transgene is also designated hemizygous (U.S. Patent No.
6,008,437).
[0116] General transformation methods, and specific methods for transforming
certain
plant species (e.g., maize) are described in U.S. Patent Nos. 4940838,
5464763, 5149645,
5501967, 6265638, 4693976, 5635381, 5731179, 5693512, 6162965, 5693512,
5981840,
6420630, 6919494, 6329571, 6215051, 6369298, 5169770, 5376543, 5416011,
5569834,
5824877, 5959179, 5563055, and 5968830.
[0117] Non-limiting examples of methods for transforming cannabis plants and
cannabis
tissue culture methods are described in Zweger (The Biotechnology of Cannabis
sativa,
April 2009); MacKinnon (Genetic transformation of Cannabis saliva Linn: a
multipurpose
fiber crop, doctoral thesis, University of Dundee, Scotland, 2003), MacKinnon
et al.
(Progress towards transformation of fiber hemp, Scottish Crop Research, 2000),
and US
20120311744.
The transformation can be physical, chemical and/or biological.
Herbicide Resistance
[0118] Numerous herbicide resistance genes are known and may be employed with
the
invention. A non-limiting example is a gene conferring resistance to a
herbicide that
inhibits the growing point or meristem such as imidazolinone or sulfonylurea
herbicides.
As imidazolinone and sulfonylurea herbicides are acetolactate synthase (ALS)-
inhibiting
herbicides that prevent the formation of branched chain amino acids, exemplary
genes in
this category code for ALS and AHAS enzymes as described, for example, by Lee
et
39
Date Recue/Date Received 2022-09-13
al., EMBO J., 7:1241, 1988; Gleen et al., Plant Molec. Biology, 18:1185, 1992;
and Miki
et al., Theor. Appl. Genet., 80:449, 1990. As a non-limiting example, a gene
may be
employed to confer resistance to the exemplary sulfonylurea herbicide
nicosulfuron.
[0119] Resistance genes for glyphosate (resistance conferred by mutant 5-
enolpyruvylshikimate-3-phosphate synthase (EPSPS) and aroA genes,
respectively) and
other phosphono compounds such as glufosinate (phosphinothricin
acetyltransferase
(PAT) and Streptomyces hygroscopicus phosphinothricin acetyltransferase (bar)
genes)
may also be used. See, for example, U.S. Pat. No. 4,940,835 to Shah et al.,
which discloses
the nucleotide sequence of a form of EPSPS that can confer glyphosate
resistance. Non-
limiting examples of EPSPS transformation events conferring glyphosate
resistance are
provided by U.S. Pat. Nos. 6,040,497 and 7,632,985. The M0N89788 event
disclosed in
U.S. Pat. No. 7,632,985 in particular is beneficial in conferring glyphosate
tolerance in
combination with an increase in average yield relative to prior events.
[0120] A DNA molecule encoding a mutant aroA gene can be obtained under ATCC
Accession No. 39256, and the nucleotide sequence of the mutant gene is
disclosed in U.S.
Pat. No. 4,769,061 to Comai. A hygromycin B phosphotransferase gene from E.
coli that
confers resistance to glyphosate in tobacco callus and plants is described in
Penaloza-
Vazquez et al., Plant Cell Reports, 14:482, 1995. European Patent Application
Publication
No. EP0333033 to Kumada et al., and U.S. Pat. No. 4,975,374 to Goodman et al.,
disclose
nucleotide sequences of glutamine synthetase genes that confer resistance to
herbicides
such as L-phosphinothricin. The nucleotide sequence of a phosphinothricin
acetyltransferase gene is provided in European Patent Application Publication
No.
EP0242246 to Leemans et al. DeGreef et al. (Biotechnology, 7:61, 1989)
describe the
production of transgenic plants that express chimeric bar genes coding for
phosphinothricin
acetyl transferase activity. Exemplary genes conferring resistance to a
phenoxy class
herbicide haloxyfop and a cyclohexanedione class herbicide sethoxydim are the
Acct-S1,
Acct-S2 and Acct-53 genes described by Marshall et al., (Theor. App!. Genet.,
83:435,
1992). As a non-limiting example, a gene may confer resistance to other
exemplary
phenoxy class herbicides that include, but are not limited to, quizalofop-p-
ethyl and 2,4-
dichlorophenoxy acetic acid (2,4-D).
Date Recue/Date Received 2022-04-12
[0121] Genes are also known that confer resistance to herbicides that inhibit
photosynthesis such as, for example, triazine herbicides (psbA and gs+ genes)
and
benzonitrile herbicides (nitrilase gene). As a non-limiting example, a gene
may confer
resistance to the exemplary benzonitrile herbicide bromoxynil. Przibila et al.
(Plant
Cell, 3:169, 1991) describe the transformation of Chlamydomonas with plasmids
encoding
mutant psbA genes. Nucleotide sequences for nitrilase genes are disclosed in
U.S. Pat. No.
4,810,648 to Stalker, and DNA molecules containing these genes are available
under
ATCC Accession Nos. 53435, 67441, and 67442. Cloning and expression of DNA
coding
for a glutathione S-transferase is described by Hayes et at. (Biochem. 1,
285:173, 1992).
4-hych-oxyphenylpyruvate dioxygenase (HPPD) is a target of the HPPD-inhibiting
herbicides, which deplete plant plastoquinone and vitamin E pools. Rippert et
al. (Plant
PhysioL, 134:92, 2004) describes an HPPD-inhibitor resistant tobacco plant
that was
transformed with a yeast-derived prephenate dehydrogenase (PDH) gene.
Protoporphyrinogen oxidase (PPO) is the target of the PPO-inhibitor class of
herbicides; a
PPO-inhibitor resistant PPO gene was recently identified in Amaranthus
tuberculatus (Patzoldt et al., PNAS, 103(33):12329, 2006). The herbicide
methyl viologen
inhibits CO,assimilation. Foyer et al. (Plant PhysioL, 109:1047, 1995)
describe a plant
overexpressing glutathione reductase (GR) that is resistant to methyl viologen
treatment.
[0122] Siminszky (Phytochemistry Reviews, 5:445, 2006) describes plant
cytochrome
P450-mediated detoxification of multiple, chemically unrelated classes of
herbicides.
Modified bacterial genes have been successfully demonstrated to confer
resistance to
atrazine, a herbicide that binds to the plastoquinone-binding membrane protein
QBin
photosystem II to inhibit electron transport. See, for example, studies by
Cheung et al.
(PNAS, 85:391, 1988), describing tobacco plants expressing the chloroplast
psbA gene
from an atrazine-resistant biotype of Amaranthus hybridus fused to the
regulatory
sequences of a nuclear gene, and Wang et al. (Plant Biotech. J, 3:475, 2005),
describing
transgenic alfalfa, Arabidopsis, and tobacco plants expressing the atzA gene
from Pseudomonas sp. that were able to detoxify atrazine.
[0123] Bayley et al. (Theor. AppL Genet., 83:645, 1992) describe the creation
of 2,4-D-
resistant transgenic tobacco and cotton plants using the 2,4-D monooxygenase
gene tfdA
41
Date Recue/Date Received 2022-04-12
from Akaligenes eutrophus plasmid pJP5. U.S. Patent Application Publication
No.
20030135879 describes the isolation of a gene for dicamba monooxygenase (DMO)
from Psueodmonas maltophilia that is involved in the conversion of dicamba to
a non-
toxic 3,6-dichlorosalicylic acid and thus may be used for producing plants
tolerant to this
herbicide.
[0124] Other examples of herbicide resistance have been described, for
instance, in U.S.
Pat. Nos. 6,803,501; 6,448,476; 6,248,876; 6,225,114; 6,107,549; 5,866,775;
5,804,425;
5,633,435; 5,463,175.
Disease and Pest Resistance
[0125] Plant defenses are often activated by specific interaction between the
product of a
disease resistance gene (R) in the plant and the product of a corresponding
avirulence (Avr)
gene in the pathogen. A plant line can be transformed with a cloned resistance
gene to
engineer plants that are resistant to specific pathogen strains. See, for
example Jones et al.
(Science, 266:789-793, 1994) (cloning of the tomato Cf-9 gene for resistance
to Cladosporium flavum); Martin et al. (Science, 262:1432-1436, 1993) (tomato
Pto gene
for
resistance to Pseudomonas syringae pv. tomato); and Mindrinos et al.
(Cell, 78(6):1089-1099, 1994) (Arabidopsis RPS2 gene for resistance to
Pseudomonas
syringae).
[0126] A viral-invasive protein or a complex toxin derived therefrom may also
be used for
viral disease resistance. For example, the accumulation of viral coat proteins
in transformed
plant cells imparts resistance to viral infection and/or disease development
effected by the
virus from which the coat protein gene is derived and related viruses. See
Beachy et al.
(Ann. Rev. Phytopathol., 28:451, 1990). Coat protein-mediated resistance has
been
conferred upon transformed plants against alfalfa mosaic virus, cucumber
mosaic virus,
tobacco streak virus, potato virus X, potato virus Y, tobacco etch virus,
tobacco rattle virus,
and tobacco mosaic virus.
[0127] A virus-specific antibody may also be used. See, for example,
Tavladoraki et al.
(Nature, 366:469-472, 1993), who show that transgenic plants expressing
recombinant
antibody genes are protected from virus attack. Virus resistance has also been
described in,
42
Date Recue/Date Received 2022-04-12
for example, U.S. Pat. Nos. 6,617,496; 6,608,241; 6,015,940; 6,013,864;
5,850,023 and
5,304,730. Additional means of inducing whole-plant resistance to a pathogen
include
modulation of the systemic acquired resistance (SAR) or pathogenesis related
(PR) genes,
for example genes homologous to the Arabidopsis thaliana NIM1/NPR1/SAI1,
and/or by
increasing salicylic acid production (Ryals etal., Plant Cell, 8:1809-1819,
1996).
101281 Logemann et a1. (Biotechnology, 10:305-308, 1992), for example,
disclose
transgenic plants expressing a barley ribosome-inactivating gene that have an
increased
resistance to fungal disease. Plant defensins may be used to provide
resistance to fungal
pathogens (Thomma et al., Planta, 216:193-202, 2002). Other examples of fungal
disease
resistance are provided in U.S. Pat. Nos. 6,653,280; 6,573,361; 6,506,962;
6,316,407;
6,215,048; 5,516,671; 5,773,696; 6,121,436; and 6,316,407.
101291 Nematode resistance has been described in, for example, U.S. Pat. No.
6,228,992,
and bacterial disease resistance has been described in, for example, U.S. Pat.
No.
5,516,671.
101301 The use of the herbicide glyphosate for disease control in hemp plants
containing
event M0N89788, which confers glyphosate tolerance, has also been described in
U.S.
Pat. No. 7,608,761.
Insect Resistance
101311 One example of an insect resistance gene includes a Bacillus
thuringiensis protein,
a derivative thereof, or a synthetic polypeptide modeled thereon. See, for
example, Geiser
et al. (Gene, 48(1):109-118, 1986), who disclose the cloning and nucleotide
sequence of
a Bacillus thuringiensis 8-endotoxin gene. Moreover, DNA molecules encoding 8-
endotoxin genes can be purchased from the American Type Culture Collection,
Manassas,
Va., for example, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.
Another
example is a lectin. See, for example, Van Damme et al., (Plant Molec. Biol.,
24:825-830,
1994), who disclose the nucleotide sequences of several Clivia miniata mannose-
binding
lectin genes. A vitamin-binding protein may also be used, such as, for
example, avidin. See
PCT Application No. US93/06487.
43
Date Recue/Date Received 2022-09-13
This application teaches the use of avidin and avidin homologues as larvicides
against insect pests.
101321 Yet another insect resistance gene is an enzyme inhibitor, for example,
protease,
proteinase, or amylase inhibitors. See, for example, Abe et al. (J. Biol.
Chem., 262:16793-
16797, 1987) describing the nucleotide sequence of a rice cysteine proteinase
inhibitor;
Linthorst et al. (Plant Molec. BioL, 21:985-992, 1993) describing the
nucleotide sequence
of a cDNA encoding tobacco proteinase inhibitor I; and Sumitani et al.
(Biosci. Biotech.
Biochem., 57:1243-1248, 1993) describing the nucleotide sequence of a
Streptomyces
nitrosporeus a-amylase inhibitor.
101331 An insect-specific hormone or pheromone may also be used. See, for
example, the
disclosure by Hammock et al. (Nature, 344:458-461, 1990) of baculovirus
expression of
cloned juvenile hormone esterase, an inactivator of juvenile hormone; Gade and
Goldsworthy (Eds. Physiological System in Insects, Elsevier Academic Press,
Burlington,
Mass., 2007), describing allostatins and their potential use in pest control;
and Patti et at.
(Vitam. Horm., 73:59-100, 2005), disclosing use of ecdysteroid and ecdysteroid
receptor
in agriculture. The diuretic hormone receptor (DHR) was identified in Price et
al. (Insect
MoL Biol., 13:469-480, 2004) as another potential candidate target of
insecticides.
101341 Still other examples include an insect-specific antibody or an
immunotoxin derived
therefrom and a developmental-arrestive protein. See Taylor et at. (Seventh
Intl
Symposium on Molecular Plant-Microbe Interactions, Edinburgh, Scotland,
Abstract W97,
1994), who described enzymatic inactivation in transgenic tobacco via
production of
single-chain antibody fragments. Numerous other examples of insect resistance
have been
described. See, for example, U.S. Pat. Nos. 6,809,078; 6,713,063; 6,686,452;
6,657,046;
6,645,497; 6,642,030; 6,639,054; 6,620,988; 6,593,293; 6,555,655; 6,538,109;
6,537,756;
6,521,442; 6,501,009; 6,468,523; 6,326,351; 6,313,378; 6,284,949; 6,281,016;
6,248,536;
6,242,241; 6,221,649; 6,177,615; 6,156,573; 6,153,814; 6,110,464; 6,093,695;
6,063,756;
6,063,597; 6,023,013; 5,959,091; 5,942,664; 5,942,658, 5,880,275; 5,763,245
and
5,763,241.
44
Date Recue/Date Received 2022-09-13
Resistance to Abiotic Stress
[0135] Abiotic stress includes dehydration or other osmotic stress, salinity,
high or low
light intensity, high or low temperatures, submergence, exposure to heavy
metals, and
oxidative stress. Delta-pyrroline-5-carboxylate synthetase (P5CS) from
mothbean has been
used to provide protection against general osmotic stress. Mannitol- 1 -
phosphate
dehydrogenase (mt1D) from E. coil has been used to provide protection against
drought
and salinity. Choline oxidase (codA from Arthrobactor globiformis) can protect
against
cold and salt. E. coil choline dehydrogenase (betA) provides protection
against salt.
Additional protection from cold can be provided by omega-3-fatty acid
desaturase (fad7)
from Arabidopsis thaliana. Trehalose-6-phosphate synthase and levan sucrase
(SacB) from
yeast and Bacillus subtilis, respectively, can provide protection against
drought
(summarized from Annex II Genetic Engineering for Abiotic Stress Tolerance in
Plants,
Consultative Group On International Agricultural Research Technical Advisory
Committee). Overexpression of superoxide dismutase can be used to protect
against
superoxides, see U.S. Pat. No. 5,538,878.
Additional Traits
[0136] Additional traits can be introduced into the hemp variety of the
present invention.
A non-limiting example of such a trait is a coding sequence which decreases
RNA and/or
protein levels. The decreased RNA and/or protein levels may be achieved
through RNAi
methods, such as those described in U.S. Pat. No. 6,506,559.
[0137] Another trait that may find use with the hemp variety of the invention
is a sequence
which allows for site-specific recombination. Examples of such sequences
include the FRT
sequence used with the FLP recombinase (Zhu and Sadowski, J. Biol. Chem.,
270:23044-
23054, 1995) and the LOX sequence used with CRE recombinase (Sauer, MoL
Biol., 7:2087-2096, 1987). The recombinase genes can be encoded at any
location within
the genome of the hemp plant and are active in the hemizygous state.
[0138] In certain embodiments hemp plants may be made more tolerant to or more
easily
transformed with Agrobacterium tumefaciens. For example, expression of p53 and
iap, two
baculovirus cell-death suppressor genes, inhibited tissue necrosis and DNA
cleavage.
Date Recue/Date Received 2022-04-12
Additional targets may include plant-encoded proteins that interact with
the Agrobacterium Vir genes; enzymes involved in plant cell wall formation;
and histones,
histone acetyltransferases and histone deacetylases
(reviewed in
Gelvin, Microbiology &MoL Biol. Reviews, 67:16-37, 2003).
[0139] In addition to the modification of oil, fatty acid, or phytate content
described above,
certain embodiments may modify the amounts or levels of other compounds. For
example,
the amount or composition of antioxidants can be altered. See, for example,
U.S. Pat. Nos.
6,787,618 and 7,154,029 and International Patent Application Publication No.
WO
00/68393, which disclose the manipulation of antioxidant levels, and
International Patent
Application Publication No. WO 03/082899, which discloses the manipulation of
an
antioxidant biosynthetic pathway.
[0140] Additionally, seed amino acid content may be manipulated. U.S. Pat. No.
5,850,016
and International Patent Application Publication No. WO 99/40209 disclose the
alteration
of the amino acid compositions of seeds. U.S. Pat. Nos. 6,080,913 and
6,127,600 disclose
methods of increasing accumulation of essential amino acids in seeds.
[0141] U.S. Pat. No. 5,559,223 describes synthetic storage proteins of which
the levels of
essential amino acids can be manipulated. International Patent Application
Publication No.
WO 99/29882 discloses methods for altering amino acid content of proteins.
International
Patent Application Publication No. WO 98/20133 describes proteins with
enhanced levels
of essential amino acids. International Patent Application Publication No. WO
98/56935
and U.S. Pat. Nos. 6,346,403; 6,441,274; and 6,664,445 disclose plant amino
acid
biosynthetic enzymes. International Patent Application Publication No. WO
98/45458
describes synthetic seed proteins having a higher percentage of essential
amino acids than
wild-type.
[0142] U.S. Pat. No. 5,633,436 discloses plants comprising a higher content of
sulfur-
containing amino acids; U.S. Pat. No. 5,885,801 discloses plants comprising a
high
threonine content; U.S. Pat. Nos. 5,885,802 and 5,912,414 disclose plants
comprising a
high methionine content; U.S. Pat. No. 5,990,389 discloses plants comprising a
high lysine
content; U.S. Pat. No. 6,459,019 discloses plants comprising an increased
lysine and
threonine content; International Patent Application Publication No. WO
98/42831
46
Date Recue/Date Received 2022-04-12
discloses plants comprising a high lysine content; International Patent
Application
Publication No. WO 96/01905 discloses plants comprising a high threonine
content; and
International Patent Application Publication No. WO 95/15392 discloses plants
comprising
a high lysine content.
Cannabis Hemp Extracts and Compositions
[0143] In some embodiments, the present disclosure provides for extracts and
compositions from the hemp plants of the present disclosure. Cannabis extracts
or products
or the present disclosure include:
[0144] Solvent reduced oils- also sometimes known as oil, BHO, CO2 extract,
among other
names. This type of extract is made by soaking plant material in a chemical
solvent capable
of solubilizing one or more chemical constituents of the plant (e.g.,
cannabinoids and/or
terpenes). After separating the solvent from plant material, the solvent can
be boiled or
evaporated off, leaving the extract "oil" behind. Butane Hash Oil is produced
by passing
butane over cannabis and then letting the butane evaporate. Rick Simpson Oil
is produced
through isopropyl, or ethanol extraction of cannabis. The resulting substance
is a wax like
golden brown paste. Another common extraction solvent for creating cannabis
oil is CO2.
Persons having skill in the art will be familiar with CO2 extraction
techniques and devices,
including those disclosed in US 20160279183, US 2015/01505455, US 9,730,911,
and US
2018/0000857.
[0145] Heat extractions- The present disclosure also teaches extracts produced
via heat-
based extraction methods, such as those disclosed in US Patent Application
Nos. US
2018/0078874, US 2019/0151771, US 2019/0076753, and US 10,159,908.
In some embodiments, the plants of
the present disclosure can be extracted by exposing tissue to a hot air gas
stream that
volatizes cannabinoids and/or other secondary metabolites of the plant, which
are then
condensed and recovered in tanks.
[0146] In some embodiments, the present disclosure teaches exposing plants,
plant parts
or plant cells to vaporizing heat. As used herein, the term "vaporizing heat"
refers to heat
sufficient to volatize one or more terpene on camiabinoid components of said
plant, plant
47
Date Recue/Date Received 2022-09-13
part or plant cell. The boiling points for each of the cannabinoid and terpene
constituents
of a hemp plant are well known or readily ascertainable. In some embodiments,
vaporizing
heat comprises 1500 F, 155 F, 160 F, 165 F, 170 F, 175 F, 180 F, 185 F,
1900
F, 195 F, 200 F, 205 F, 210 F, 215 F, 220 F, 225 F, 230 F, 235
F, 240 F,
245 F, 250 F, 255 F, 260 F, 265 F, 270 F, 275 F, 280 F, 285
F, 290 F, 295
0F, 3000 F, 3050 F, 3100 F, 3150 F, 3200 F, 3250 F, 3300 F, 3350 F, 3400 F,
3450 F,
or 350 F, and all ranges and subranges therebetween.
[0147] Tinctures- are alcoholic extracts of cannabis. These are usually made
by mixing
cannabis material with high proof ethanol and separating out plant material.
Within the
dietary supplement industry "tincture" may also describe an oil dilution of
hemp extract.
[0148] In some embodiments, the specialty cannabis of the present disclosure
is extracted
via methods that preserve the cannabinoid and terpenes. In other embodiments,
said
methods can be used with any cannabis plants. The extracts of the present
disclosure are
designed to produce products for human or animal consumption via inhalation
(via
combustion, vaporization and nebulization), buccal absorption within the
mouth, oral
administration (e.g., eating/drinking), and topical application delivery
methods.
[0149] The chemical extraction of specialty cannabis can be accomplished
employing
polar and non-polar solvents in various phases at varying pressures and
temperatures to
selectively or comprehensively extract terpenes, cannabinoids and other
compounds of
flavor, fragrance or pharmacological value for use individually or combination
in the
formulation of our products. The solvents employed for selective extraction of
our cultivars
may include water, carbon dioxide, 1,1,1,2-tetrafluoroethane, butane, propane,
ethanol,
isopropyl alcohol, hexane, and limonene, in combination or series. It is also
possible to
extract compounds of interest mechanically by sieving the plant parts that
produce those
compounds. Measuring the plant part, i.e. trichome gland head, to be sieved
via optical or
electron microscopy can aid the selection of the optimal sieve pore size,
ranging from 30
to 130 microns, to capture the plant part of interest. The chemical and
mechanical
extraction methods of the present disclosure can be used to produce products
that combine
chemical extractions with plant parts containing compounds of interest.
48
Date Recue/Date Received 2022-04-12
[0150] The extracts of the present disclosure may also be combined with pure
compounds
of interest to the extractions, e.g. cannabinoids or terpenes to further
enhance or modify the
resulting foiinulation' s fragrance, flavor or pharmacology. Thus, in some
embodiments,
the present disclosure teaches compositions comprising at least one ingredient
extracted
from the `AF14b15-21' plant. In some embodiments, extracts from the hemp lines
of the
present disclosure are combined with one or more additional compounds. In some
embodiments, extracts of the present disclosure, such as whole hemp extracts,
or a purified
cannabinoid from said hemp plant, can be combined with another cannabinoid or
terpene
to produce a composition.
[0151] The compositions of the present disclosure encompass many forms. In
some
embodiments, the present disclosure provides CBD oils and tinctures. In some
embodiments, the present disclosure provides CBD capsules. In some
embodiments, the
present disclosure provides CBD infused edibles, such as gummies, gum,
lollipops, taffy,
cookies, brownies, ice cream, chocolate, jerky, animal dry and wet foods,
animal treats,
etc. In some embodiments, the present disclosure provides for cosmetics
comprising CBD,
such as lip stick, balms, creams, shampoo, conditioners, lotions, rubbing
oils, lubricants,
Etc. In some embodiments, the CBD oils comprise extracts from `AF14b15-21',
such as
solvent extracted oils, heat extracted oils. In some embodiments, the capsules
comprise
extracts from AF14b15-21' .
[0152] In some embodiments, the present disclosure teaches hemp commodity
products,
including processed hemp inflorescences, fiber, hemp extract, catmabinoids,
and terpenes.
As used herein, the term "processed hemp inflorescences" means inflorescences
from a
hemp plant that have been harvested and dried to a moisture content of less
than 20% wt/wt.
In some embodiments, the processed hemp inflorescences are ground or broken up
in
smaller pieces.
Infused Inflorescences
[0153] In some embodiments, the hemp strains of the present disclosure can be
processed
to produce infused inflorescences or infused inflorescence material. Briefly,
inflorescences
49
Date Recue/Date Received 2022-04-12
of the present disclosure can be contacted with one or more cannabinoid
fortifiers prior to
use/sale. In some embodiments, the cannabinoid fortifier(s) are added to dried
inflorescence material. In some embodiments the cannabinoid fortifier(s) are
added to
processed/ground plant material. In some embodiments, the cannabinoid
fortifiers
comprise THC, THCA, CBD, CBDa, delta 8 THC, delta 10 THC, delta 11 THC, THC-0-
acetate (THC-0), hexahydrocannabinol (HCC), tetrahydrocannabiphorol (THCP),
THCVA, THCV, CBDV, CBDVA, and derivatives thereof.
DEPOSIT INFORMATION
[0154] A deposit of the AF14b15-21' hemp cultivar is maintained by Charlotte's
Web,
Inc., 700 Tech Court, Louisville, CO 80027, USA. In addition, a sample of 625
seeds of
the `AF14b15-21' variety of this disclosure has been deposited with an
International
Depositary Authority as established under the Budapest Treaty according to 37
CFR
1.803(a)(1). Applicant has deposited seeds at the Provasoli-Guillard National
Center for
Marine Algae and Microbiota (NCMA), located at the Bigelow Laboratory for
Ocean
Science at 60 Bigelow Drive East Boothbay, ME 04544.
[0155] The `AF14b15-21' seeds (also known as Ambassador line of hemp) have
been
deposited and accepted under the Budapest Treaty as NCMA No. 202203083 on
March 22,
2022.
[0156] To satisfy the enablement requirements of 35 U.S.C. 112, and to certify
that the
deposit of the isolated strain (i.e., hemp plant) of the present disclosure
meets the criteria
set forth in 37 C.F.R. 1.801-1.809 and Manual of Patent Examining Procedure
(MPEP)
2402-2411.05, Applicants hereby make the following statements regarding the
deposited
`AF14b15-21' hemp cultivar (deposited as NCMA No. 202203083):
1. During the pendency of this application, access to the disclosure will
be
afforded to the Commissioner upon request;
2. All restrictions on availability to the public will be irrevocably
removed
upon granting of the patent under conditions specified in 37 CFR 1.808;
Date Recue/Date Received 2022-04-12
3. The deposit will be maintained in a public repository for a period of 30
years or 5 years after the last request or for the effective life of the
patent,
whichever is longer;
4. A test of the viability of the biological material at the time of
deposit will
be conducted by the public depository under 37 C.F.R. 1.807; and
5. The deposit will be replaced if it should ever become unavailable.
[0157] Access to this deposit will be available during the pendency of this
application to
persons determined by the Commissioner of Patents and Trademarks to be
entitled thereto
under 37 C.F.R. 1.14 and 35 U.S.C. 122. Upon allowance of any claims in
this
application, all restrictions on the availability to the public of the variety
will be irrevocably
removed by affording access to a deposit of at least 625 seeds of the same
variety with the
NCMA.
[0158] Unless defined otherwise, all technical and scientific terms herein
have the same
meaning as commonly understood by one of ordinary skill in the art to which
this invention
belongs. Although any methods and materials, similar or equivalent to those
described
herein, can be used in the practice or testing of the present invention, the
non-limiting
exemplary methods and materials are described herein.
[0159] All publications and patent applications mentioned in the specification
are
indicative of the level of those skilled in the art to which this invention
pertains.
Nothing herein is to be construed as an admission
that the present disclosure is not entitled to antedate such publication by
virtue of prior
disclosure.
[0160] Many modifications and other embodiments of the disclosures set forth
herein will
come to mind to one skilled in the art to which these disclosures pertain
having the benefit
of the teachings presented in the foregoing descriptions and the associated
drawings.
Therefore, it is to be understood that the disclosures are not to be limited
to the specific
embodiments disclosed and that modifications and other embodiments are
intended to be
51
Date Recue/Date Received 2022-09-13
included within the scope of the appended claims. Although specific terms are
employed
herein, they are used in a generic and descriptive sense only and not for
purposes of
limitation.
[0161] While the disclosure has been described in connection with specific
embodiments
thereof, it will be understood that it is capable of further modifications and
this application
is intended to cover any variations, uses, or adaptations of the disclosure
following, in
general, the principles of the disclosure and including such departures from
the present
disclosure as come within known or customary practice within the art to which
the
disclosure pertains and as may be applied to the essential features
hereinbefore set forth
and as follows in the scope of the appended claims.
Example 1. Cannabinoid Potency Tests.
[0162] Samples of inflorescences from the 'AF14b15-21' hemp line harvested at
maturity
were provided to a third-party vendor for analysis. using high performance
liquid
chromatography. The results of these analyses are provided below in Table 6.
Values
shown below in Table 6 are % (w/w) = (weight of analyte/weight of product),
wherein the
"weight of product" is dry weight of the inflorescence.
Table 6. Cannabinoid Potency Analysis Results for 'AF14b15-21'
Sample Location Total THC Total CBD CBD:THC
Potential Potential Ratio
1 Greeley, CO, U.S. 0.29 5.74 20.1
2 Greeley, CO, U.S. 0.29 5.90 20.7
3 Greeley, CO, U.S. 0.26 5.45 21.1
4 Greeley, CO, U.S. 0.26 5.38 20.8
Greeley, CO, U.S. 0.29 6.02 21.1
6 Greeley, CO, U.S. 0.27 5.84 21.9
7 Greeley, CO, U.S. 0.27 5.79 21.7
8 Greeley, CO, U.S. 0.27 5.83 21.8
9 Greeley, CO, U.S. 0.27 5.79 21.7
Greeley, CO, U.S. 0.27 5.80 21.7
11 Coaldale, Canada 0.14 4.60 32.6
12 Coaldale, Canada 0.17 6.22 35.9
13 La Salle, Canada 0.26 7.04 27.0
14 Russell, Canada 0.26 7.81 30.0
Russell, Canada 0.25 7.27 29.1
52
Date Recue/Date Received 2022-04-12
Sample Location Total THC Total CBD CBD:THC
Potential Potential Ratio
16 Coaldale, Canada 0.14 4.66 33.2
[0163] Additional sampling of cannabinoids was done according to Health
Canada's
"Policy for the Management of Industrial Hemp Varieties on the List of
Approved
Cultivars, that came into effect on March 11, 2020." Sampling was done on a
minimum
of 60 randomly selected plants per plot, or 180 per experiment (640 plant on 4
locations).
The samples represented the entire fruit bearing part of the plant (female
inflorescence),
taken from the top third of the plant. Samples in the experiments with
cultivars that are
intended to be harvested with no seeds present were taken when the plants were
anticipated
to achieve a maximum level of total THC+THCA. Samples in the experiment with
cultivars
that were intended to be harvested with seed present were taken when the first
seeds of
50% of the plants were resistant to compression. 30 specimens were used for
analysis and
30 specimens were set-aside. Samples were dried to 8-13% and stored in
darkness at
temperature not exceeding 20 degrees Celsius. Each sample representing a plot
were
analyzed separately, allowing a relatively robust analysis and calculation of
the confidence
interval.
[0164] Canadian samples were analyzed by Intertek Testing Services (ITS)
Canada Ltd.,
Saskatoon, SK, a Health Canada accredited lab. The method is based on the
standard
reference method AOAC 2018.11 (First Action 2018) "Quantitation of
Cannabinoids in
Cannabis Dried Plant Materials, Concentrates, and Oils Liquid
Chromatography¨Diode
Array Detection Technique with Optional Mass Spectrometric Detection", and
Agilent
Application Note 5991-9285-en as the reference method. Deviations include: 1.
The
official method uses only Ethanol for extraction, the in-house method was also
validated
using Methanol. 2. The official referred methods use different columns and LC
system.
The in-house method was validated using Waters Acquity ARC UHPLC system with
Waters ARC 2489 UV/VIS Detector and XBridge BEH XP C18 column (130A, 2.5
um,150 x 3 mm). 3. The official referred methods use different sample
injection volume
runtime. In-house method was validated with 6 1_, sample injection volume and
20 min
run time.
53
Date Recue/Date Received 2022-04-12
[0165] The amount of Cannabinoid in sample extract was determined by standard
curve
followed by the following calculation: Cannabinoid in Sample (%, w/w) =CxVx DF
x
(1/W) x (1/10,000) Where C = analyte conc from std curve (g/m1) V = Volume of
extraction solvent (m1) DF= Dilution.
Table 7. Canadian LOAC trial of cannabinoid Potency for `AF14b15-21'
Location Plot No. Total THC% Total CBD % CBD:THC Ratio
La Salle 117 0.26 7.04 27.08
La Salle 212 0.218 5.84 26.79
La Salle 313 0.196 5.34 27.24
RM Dufferin 109 0.176 5.05 28.69
RM Dufferin 204 0.213 6.03 28.31
RM Dufferin 306 0.192 5.43 28.28
Russell 104 0.26 7.81 30.04
Russell 206 0.256 7.27 28.40
Russell 306 0.24 7.27 30.29
Coaldale 117 0.141 4.608 32.68
Coaldale 213 0.144 4.66 32.36
Coaldale 310 0.173 6.22 35.95
Example 2. Terpene Profile Tests.
[0166] Samples of inflorescences from the 'AF14b15-21' hemp line harvested at
maturity
and grown in Greeley, Colorado were provided to a third-party vendor for
analysis.
Samples of the ` AF14b15-21' inflorescences were analyzed using gas
chromatography.
The results of these analyses are provided below in Table 8 and shown in Fig.
7. Values
shown below in Table 8 are % (w/w) = (weight of analyte/weight of product),
wherein the
"weight of product" is dry weight of the inflorescence.
54
Date Recue/Date Received 2022-04-12
Table 8. Terpene Profile Analysis Results for 'AF14b15-21'
Beta-Myrcene Caryophyllene D-Limonene Alpha- Linalool Alpha-
Trans- Caryophyllene
Humulene
Bisabolol Nerolidol oxide
1 0.087 0.243 0.019 0.075 0.018
0.174 0.026 0.011
2 0.101 0.217 0.020 0.067 0.019
0.161 0.021 0.007
3 0.087 0.201 0.027 0.079 0.038
0.108 0.016 0.009
4 0.097 0.177 0.018 0.054 0.025
0.132 0.016 0.007
0.075 0.201 0.014 0.060 0.018 0.128 0.014
0.007
6 0.090 0.169 0.018 0.051 0.019
0.151 0.018 0.000
7 0.138 0.124 0.023 0.048 0.024
0.131 0.015 0.000
8 0.132 0.132 0.023 0.042 0.032
0.085 0.015 0.000
9 0.109 0.134 0.019 0.042 0.024
0.094 0.018 0.000
Date Recue/Date Received 2022-04-12
[0167]
Mention of any reference, article, publication, patent, patent publication,
and
patent application cited herein is not, and should not be taken as, an
acknowledgment or
any form of suggestion that they constitute valid prior art or form part of
the common
general knowledge in any country in the world.
NUMBERED EMBODIMENTS
[0168] Further embodiments contemplated by the disclosure are listed below.
[0169] 1. A seed, plant, plant part, or plant cell of hemp plant variety
designated
'AF14b15-21', wherein representative seed of the variety has been deposited
under NCMA
No. 202203083.
[0170] 2. The hemp plant part of embodiment 1, wherein the plant part is an
inflorescence.
[0171] 3. A hemp plant or a plant part or a plant cell thereof, having all of
the
characteristics of the hemp plant variety designated 'AF14b15-21' listed in
Tables 1-5.
10172] 4. A hemp plant, or a plant part or a plant cell thereof, having all of
the essential
physiological and morphological characteristics of the hemp plant of any one
of
embodiments 1-3.
[0173] 5. A hemp plant, or a part or a plant cell thereof, having all of the
essential
physiological and morphological characteristics of the hemp plant variety
designated
'AF14b15-21', wherein a representative sample of seed of said variety was
deposited under
NCMA No. 202203083.
[0174] 6. A tissue culture of regenerable cells produced from the plant, plant
part or plant
cell of any one of embodiments 1-5, wherein a new plant regenerated from the
tissue culture
has all of the characteristics of the hemp plant variety designated 'AF14b15-
21' listed in
Tables 1-5 when grown under the same environmental conditions.
[0175] 7. A hemp plant regenerated from the tissue culture of embodiment 6,
said plant
having all the characteristics of the hemp plant variety designated 'AF14b15-
21' listed in
Tables 1-5 when grown under the same environmental conditions.
56
Date Recue/Date Received 2022-09-13
[0176] 8. A hemp plant regenerated from the tissue culture of embodiment 6,
wherein the
regenerated plant has all of the characteristics of the hemp plant variety
designated
'AF14b15-21', wherein a representative sample of seed of said variety was
deposited under
NCMA No. 202203083.
[0177] 9. A method for producing a hemp seed, comprising a) selfing the hemp
plant of
any one of embodiments 1-5 and 7-8, and b) harvesting the resultant hemp seed.
[0178] 10. A hemp seed produced by the method of embodiment 9.
[0179] 11. A method for producing a hemp seed comprising crossing the hemp
plant of
any one of embodiments 1-5 and 7-8 with a second, distinct plant.
[0180] 12. An Fl hemp seed produced by the method of embodiment 11.
[0181] 13. An Fl hemp plant, or a part or a plant cell thereof, produced by
growing the
seed of embodiment 12.
[0182] 14. A method of producing a hemp plant derived from the variety
'AF14b15-21',
comprising: a) crossing the plant of any one of embodiments 1-5 and 7-8, with
itself or a
second plant to produce progeny seed; b) growing the progeny seed to produce a
progeny
plant and crossing the progeny plant with itself or a second plant to produce
further progeny
seed; and c) repeating steps (a) and (b) with sufficient inbreeding until a
seed of an hemp
plant derived from the variety 'AF14b15-21' is produced.
[0183] 15. The method of embodiment 14, further comprising crossing the hemp
plant
derived from the variety 'AF14b15-21', with a plant of a different genotype to
produce
seed of a hybrid plant derived from the hemp variety 'AF14b15-21'.
[0184] 16. A method for producing nucleic acids, the method comprising
isolating nucleic
acids from the seed, plant, plant part, or plant cell of any one of
embodiments 1-15.
[0185] 17. The hemp plant of any one of embodiments 1-5 and 7-8, comprising a
single
locus conversion and otherwise essentially all of the characteristics of the
hemp plant of
any one of embodiments 1-5 and 7-8 when grown in the same environmental
conditions.
[0186] 18. The hemp plant of embodiment 17, wherein the single locus
conversion confers
said plant with herbicide resistance.
[0187] 19. The hemp plant of embodiment 17, wherein the single locus
conversion is an
artificially mutated gene or nucleotide sequence.
57
Date Recue/Date Received 2022-04-12
[0188] 20. The hemp plant of embodiment 17, wherein the single locus
conversion is a
gene that has been modified through the use of breeding techniques.
[0189] 21. A cultivar of hemp designated 'AF14b15-21' as described and
detailed herein.
[0190] 22. A method of producing a cannabinoid extract, said method comprising
the steps
a) contacting the plant of any one of embodiments 1-5 and 7-8 with a solvent,
thereby
producing a cannabinoid extract.
[0191] 23. A dry, sinsemilla non-viable plant or part thereof, wherein seed of
hemp plants
producing said dry plant and part thereof has been deposited under NCMA No.
202203083.
[0192] 24. An assemblage of dry, non-viable sinsemilla female inflorescences
from a hemp
plant variety designated 'AF14b15-21' wherein representative seed of the
variety has been
deposited under NCMA No. 202203083.
[0193] 25. The dry, non-viable plant part of embodiment 23 or 24, wherein the
plant part
is an inflorescence.
[0194] 26. The dry, non-viable plant part of embodiment 23 or 24, wherein the
plant part
is a trichome.
[0195] 27. Dry, non-viable kief powder comprising cannabidiol (CBD), wherein
seed of
hemp plants producing said kief has been deposited under NCMA No. 202203083.
[0196] 28. A method of producing a hemp plant with cannabidiol (CBD), said
method
comprising propagating a vegetative cutting from a hemp plant variety
designated
'AF14b15-21' wherein representative seed of the variety has been deposited
under NCMA
No. 202203083.
[0197] 29. The hemp plant with CBD, produced according to the methods of
embodiment
28.
[0198] 30. The hemp plant of embodiment 5, wherein the plant is asexually
reproduced.
[0199] 31. A method for producing a hemp plant with inflorescences that
produce
cannabidiol (CBD), said method comprising:
propagating a vegetative cutting from a stock hemp plant, thereby producing
the
hemp plant having CBD;
wherein the stock hemp plant is a product of applying a plant breeding
technique
to a variety designated 'AF14b15-21', wherein representative seed of the
variety has been
deposited under NCMA No. 202203083.
58
Date Recue/Date Received 2022-04-12
[0200] 32. The method of embodiment 31, wherein said plant breeding technique
is
recurrent selection.
[0201] 33. The method of embodiment 31, wherein said plant breeding technique
is mass
selection.
[0202] 34. The method of embodiment 31, wherein said plant breeding technique
is
hybridization.
[0203] 35. The method of embodiment 31, wherein said plant breeding technique
is open-
pollination.
[0204] 36. The method of embodiment 31, wherein said plant breeding technique
is
backcrossing.
[0205] 37. The method of embodiment 31, wherein said plant breeding technique
is pedigree
breeding.
[0206] 38. The method of embodiment 31, wherein said plant breeding technique
is mutation
breeding, and wherein said mutation selected is spontaneous or artificially
induced.
[0207] 39. A method for producing a Cannabis plant derived from a hemp variety
designated 'AF14b15-21', said method comprising crossing 'AF14b15-21' with
another
Cannabis plant, thereby producing a progeny Cannabis plant; wherein
representative seed
of the 'AF14b15-21' variety has been deposited under NCMA No. 202203083.
[0208] 40. The method of embodiment 39, further comprising the steps of:
a) crossing the progeny Cannabis plant from a previous step with itself or
another Cannabis
plant to produce a progeny Cannabis plant of a subsequent generation;
b) repeating step (a) for one or more additional generations to produce a
Cannabis plant
further derived from the hemp variety designated 'AF14b15-21'.
[0209] 41. The method of embodiment 39 or 40, further comprising the step of
contacting
the Cannabis plant further derived from the hemp variety designated 'AF14b15-
21' or a
plant part derived therefrom with a solvent, or exposing said Cannabis plant
or plant part
to vaporizing heat, thereby producing a cannabinoid extract.
[0210] 42. A method for producing a Cannabis plant derived from a hemp variety
designated 'AF14b15-21', said method comprising:
propagating a vegetative cutting from a stock Cannabis plant, thereby
producing the
Cannabis plant derived from the hemp variety designated 'AF14b15-21';
59
Date Recue/Date Received 2022-04-12
wherein the stock Cannabis plant is a product of applying a plant breeding
technique to
AF14b15-21' , wherein representative seed of the `AF14b15-21' variety has been
deposited under NCMA No. 202203083.
[0211] 43. The method of embodiment 42, further comprising the step of
contacting the
Cannabis plant derived from the hemp variety designated AF14b15-21' or a plant
part
derived therefrom with a solvent, or exposing said Cannabis plant or plant
part to
vaporizing heat, thereby producing a cannabinoid extract.
[0212] 44. The method of embodiment 42, wherein said plant breeding technique
is
recurrent selection.
[0213] 45. The method of embodiment 42, wherein said plant breeding technique
is mass
selection.
[0214] 46. The method of embodiment 42, wherein said plant breeding technique
is
hybridization.
[0215] 47. The method of embodiment 42, wherein said plant breeding technique
is open-
[0216] 48. The method of embodiment 42, wherein said plant breeding technique
is
backcrossing.
[0217] 49. The method of embodiment 42, wherein said plant breeding technique
is
pedigree breeding.
[0218] 50. The method of embodiment 42, wherein said plant breeding technique
is
mutation breeding, and wherein said mutation selected is spontaneous or
artificially
induced.
[0219] 51. The method of embodiment 42, wherein said plant breeding technique
is marker
enhanced selection.
[0220] 52. A method for producing a Cannabis plant derived from a hemp variety
designated AF14b15-21', said method comprising:
crossing a stock Cannabis plant with itself or another Cannabis plant, thereby
producing
the Cannabis plant derived from the hemp variety designated AF14b15-21';
wherein the stock Cannabis plant is a product of applying a plant breeding
technique to
AF14b15-21' , wherein representative seed of the `AF14b15-21' variety has been
deposited under NCMA No. 202203083.
Date Recue/Date Received 2022-04-12
[0221] 53. The method of embodiment 52, further comprising the step of
contacting the
Cannabis plant derived from the hemp variety designated AF14b15-21' or a plant
part
derived therefrom with a solvent, or exposing said Cannabis plant or plant
part to
vaporizing heat, thereby producing a cannabinoid extract.
[0222] 54. The method of embodiment 52, wherein said plant breeding technique
is
recurrent selection.
[0223] 55. The method of embodiment 52, wherein said plant breeding technique
is mass
selection.
[0224] 56. The method of embodiment 52, wherein said plant breeding technique
is
hybridization.
[0225] 57. The method of embodiment 52, wherein said plant breeding technique
is open-
pollination.
[0226] 58. The method of embodiment 52, wherein said plant breeding technique
is
backcrossing.
[0227] 59. The method of embodiment 52, wherein said plant breeding technique
is
pedigree breeding.
[0228] 60. The method of embodiment 52, wherein said plant breeding technique
is
mutation breeding, and wherein said mutation selected is spontaneous or
artificially
induced.
[0229] 61. The method of embodiment 52, wherein said plant breeding technique
is marker
enhanced selection.
[0230] 62. The hemp plant of embodiment 5, wherein the plant is capable of
producing an
asexual clone of said hemp plant.
[0231] 63. The hemp plant of embodiment 62, wherein the asexual clone is
capable of
producing said hemp plant of embodiment 1.
[0232] 64. A method of producing a commodity plant product, the method
comprising
producing the commodity plant product from the plant of embodiment 1.
[0233] 65. A method of producing a commodity plant product comprising
collecting the
commodity plant product from a seed, plant, plant part, or plant cell of hemp
plant variety
designated AF14b15-21' , wherein representative seed of the variety has been
deposited
under NCMA No. 202203083.
61
Date Recue/Date Received 2022-04-12
[0234] 66. The method of embodiments 64 or 65, wherein the commodity plant
product is
selected from a group consisting of processed hemp inflorescence, hemp fiber,
hemp oil
extract, terpenes, and cannabinoids.
[0235] 67. A method of producing an infused inflorescence comprising
collecting an
inflorescence of hemp plant variety designated 'AF14b15-21', wherein
representative seed
of the variety has been deposited under NCMA No. 202203083, and contacting the
inflorescence with at least one cannabinoid fortifiers.
[0236] 68. The method of embodiment 67, wherein the inflorescence is dried.
[0237] 69. The method of embodiment 67, wherein the inflorescence is processed
and/or
ground plant material.
[0238] 70. The method of any one of embodiments 67-69, wherein the cannabinoid
fortifier is selected from the group consisting of THC, THCA, CBD, CBDa, delta
8 THC,
delta 10 THC, delta 11 THC, THC-0-acetate (THC-0), hexahydrocannabinol (HCC),
tetrahydrocannabiphorol (THCP), THCVA, THCV, CBDV, CBDVA, and derivatives
thereof.
[0239] 71. The method of embodiment 70, wherein the cannabinoid fortifier is
delta 8
THC.
[0240] 72. A plant cell from a hemp plant variety designated 'AF14b15-21',
wherein
representative seed of the variety has been deposited under NCMA No.
202203083.
[0241] 73. The plant cell of embodiment 72, wherein the plant cell is from a
plant part
selected from the group consisting of a seed, a leaf, a stem, an
inflorescence, and a
trichome.
[0242] 74. The plant cell of embodiment 73, wherein the plant cell is from an
inflorescence.
[0243] 75. A plant cell of a hemp plant, which is a descendant of 'AF14b15-
21', the
descendant expressing the physiological and morphological characteristics of
variety
'AF14b15-21' as determined at the 5% significance level when grown under
substantially
similar environmental conditions, wherein representative seed of 'AF14b15-21'
has been
deposited under NCMA No. 202203083.
[0244] 76. A plant cell of a hemp plant, which is a descendant of 'AF14b15-
21', the
descendant expressing the physiological and morphological characteristics of
variety
'AF14b15-21' listed in Tables 1-5 as determined at the 5% significance level
when grown
62
Date Recue/Date Received 2022-04-12
under substantially similar environmental conditions, wherein representative
seed of
'AF14b15-21' has been deposited under NCMA No. 202203083.
[0245] 77. A plant cell from a tissue culture produced from the plant cell of
any one of
embodiments 72-76.
[0246] 78. A plant cell from a hemp plant regenerated from the tissue culture
defined in
embodiment 77, said plant expressing the physiological and morphological
characteristics
of variety 'AF14b15-21' listed in Tables 1-5, as determined at the 5%
significance level
when grown under substantially similar environmental conditions, wherein
representative
seed of 'AF14b15-21' has been deposited under NCMA No. 202203083.
[0247] 79. A locus converted plant cell from a locus converted plant obtained
by
introducing a locus conversion into hemp plant variety designated 'AF14b15-
21', wherein
the locus converted plant cell is the same as a plant cell from 'AF14b15-21'
except for the
locus conversion, and the plant expresses the physiological and morphological
characteristics of variety 'AF14b15-21' listed in Tables 1-5 as determined at
the 5%
significance level except for a trait conferred by the locus conversion, when
grown under
substantially similar environmental conditions, and wherein representative
seed of the
variety has been deposited under NCMA No. 202203083.
[0248] 80. The locus converted plant cell of embodiment 79, wherein the locus
conversion
comprises a transgene.
[0249] 81. The locus converted plant cell of any one of embodiments77-80,
wherein the
locus conversion confers a trait selected from the group consisting of male
sterility,
herbicide tolerance, insect resistance, pest resistance, disease resistance,
and abiotic stress
resistance.
[0250] 82. The locus converted plant cell of embodiment 81, wherein the locus
that confers
herbicide tolerance confers tolerance to benzonitrile herbicides,
cyclohexanedione
herbicides, imidazolinone herbicides, phenoxy herbicides, sulfonylurea
herbicides, triazine
herbicides, 1-aminocyclopropane-1 -carboxylic acid synthase-inhibiting
herbicides, 4-
hydroxyphenylpyruvate dioxygenase-inhibiting herbicides, acetolactate synthase-
inhibiting herbicides, protoporphyrinogen oxidase-inhibiting herbicides, 2,4-
dichlorophenoxyacetic acid (2,4-D), bromoxynil, dicamba, glufosinate,
glyphosate,
nicosulfuron, or quiza1ofop-p-ethyl.
63
Date Recue/Date Received 2022-04-12
[0251] 83. Use of a hemp plant variety designated 'AF14b15-21', wherein
representative
seed of the variety has been deposited under NCMA No. 202203083, for isolating
nucleic
acids from the variety.
[0252] 84. Use of a hemp plant variety designated 'AF14b15-21', wherein
representative
seed of the variety has been deposited under NCMA No. 202203083, to produce a
commodity plant product.
[0253] 85. The use of embodiment 84, wherein the commodity plant product is
selected
from a group consisting of processed hemp inflorescence, infused hemp
inflorescence,
infused ground hemp plant material, hemp fiber, hemp oil extract, terpenes,
and
cannabinoids.
[0254] 86. Use of a hemp plant variety designated 'AF14b15-21', wherein
representative
seed of the variety has been deposited under NCMA No. 202203083, to produce a
cannabinoid and/or terpene extract.
[0255] 87. Use of a hemp plant variety designated 'AF14b15-21', wherein
representative
seed of the variety has been deposited under NCMA No. 202203083, to grow
subsequent
generations.
[0256] 88. Use of a hemp plant variety designated 'AF14b15-21', wherein
representative
seed of the variety has been deposited under NCMA No. 202203083, for
vegetative
propagation.
[0257] 89. The use of embodiment 88, further comprising a use to produce a
hemp plant
comprising cannabidiol (CBD).
[0258] 90. Use of a hemp plant variety designated 'AF14b15-21', wherein
representative
seed of the variety has been deposited under NCMA No. 202203083, as a
recipient of a
conversion locus.
[0259] 91.Use of a hemp plant variety designated 'AF14b15-21', wherein
representative
seed of the variety has been deposited under NCMA No. 202203083, to produce a
genetic
marker profile.
[0260] 92. Use of a hemp plant variety designated 'AF14b15-21', wherein
representative
seed of the variety has been deposited under NCMA No. 202203083, as a source
of seed.
[0261] 93. Use of a hemp plant variety designated 'AF14b15-21', wherein
representative
seed of the variety has been deposited under NCMA No. 202203083, as a crop.
64
Date Recue/Date Received 2022-04-12
[0262] 94. Use of a hemp plant variety designated 'AF14b15-21', wherein
representative
seed of the variety has been deposited under NCMA No. 202203083, as a
recipient of a
transgene.
[0263] 95. Use of a hemp plant variety designated 'AF14b15-21', wherein
representative
seed of the variety has been deposited under NCMA No. 202203083, as a source
of
breeding material.
[0264] 96. Use of a hemp plant variety designated 'AF14b15-21', wherein
representative
seed of the variety has been deposited under NCMA No. 202203083, for producing
a plant
derived from the variety.
[0265] 97. The use of embodiment 96, wherein the plant derived from the
variety is a first,
second, third or fourth generation progeny plant.
[0266] 98. The use of any one of embodiments 96-97, further comprising a use
for
producing a cannabinoid extract from the plant derived from the variety.
[0267] 99. A cannabis extract comprising the cell of any one of embodiments 72-
82.
[0268] 100. A non-viable edible product comprising the cell of any one of
embodiments
72-82.
[0269] 101. A non-viable edible product comprising the cannabis extract of
embodiment
99.
[0270] 102. A dry, non-viable plant, or thy non-viable plant part thereof, of
hemp plant
variety designated 'AF14b15-21', wherein representative seed of the variety
has been
deposited under NCMA No. 202203083.
[0271] 103. A cannabis extract comprising the dry, non-viable plant, or dry
non-viable
plant part thereof, of embodiment 102.
[0272] 104. A non-viable edible product comprising the dry, non-viable plant,
or dry non-
viable plant part thereof, of embodiment 102.
[0273] 105. A non-viable edible product comprising the cannabis extract of
embodiment
103.
[0274] 106. An assemblage of dry, non-viable female inflorescences from hemp
plant
variety designated 'AF14b15-21', wherein representative seed of the variety
has been
deposited under NCMA No. 202203083.
Date Recue/Date Received 2022-04-12
[0275] 107. A cannabis extract comprising the non-viable female inflorescences
of
embodiment 106.
[0276] 108. A non-viable edible product comprising the non-viable female
inflorescences
of embodiment 106.
[0277] 109. A non-viable edible product comprising the cannabis extract of
embodiment
107.
[0278] 110. Use of the cell of any one of embodiments 72-82, the dry non-
viable plant, or
part thereof of embodiment 102 or the assemblage of dry non-viable female
inflorescences
of embodiment 106, as a medicament.
[0279] 111. Use of the extract of any one of embodiments 99, 103 or 107, as a
medicament.
[0280] 112. Use of the non-viable edible product of any one of embodiments
100, 101,
104, 105, 108 or 109, as a medicament.
[0281] 113. A method of producing a cannabinoid extract, comprising:
contacting a (i) plant, or plant part thereof, of hemp plant variety
designated 'AF14b15-21'
or (ii) plant cell of a descendant of hemp plant variety designated 'AF14b15-
21' as defined
in embodiment 75 or 76, with a solvent, or exposing said plant, plant part, or
plant cell to
vaporizing heat, thereby producing a cannabinoid extract, wherein
representative seed of
the variety has been deposited under NCMA No. 202203083.
[0282] 114. The method of embodiment 113, wherein the plant, plant part
thereof or plant
cell of a descendant comprises the cell of any one of embodiments 72-82, the
dry non-
viable plant, or part thereof of embodiment 102 or the assemblage of dry non-
viable female
inflorescences of embodiment 106.
[0283] 115. The method of any one of embodiments 113-114, wherein the
cannabinoid
extract is further diluted in one or more edible oils.
[0284] 116. Hemp fiber comprising the cell of any one of embodiments 72-82.
[0285] 117. Use of a hemp plant variety designated 'AF14b15-21', wherein
representative
seed of the variety has been deposited under NCMA No. 202203083, as a source
of seed,
wherein the seed is clean seed.
[0286] 118. Use of a hemp plant variety designated 'AF14b15-21', wherein
representative
seed of the variety has been deposited under NCMA No. 202203083, as a source
of seed,
wherein the seed is treated seed.
66
Date Recue/Date Received 2022-04-12
[0287] 119. The use of embodiment 118, wherein the treated seed is treated
with a
fungicide or pesticide.
[0288] 120. The plant cell of any one of embodiments 72, 73, 75, 76, or 79-82,
wherein
the plant cell is a seed cell and the seed is clean seed.
[0289] 121. The plant cell of any one of embodiments 72, 73, 75, 76, or 79-82,
wherein
the plant cell is a seed cell and the seed is treated seed.
67
Date Recue/Date Received 2022-04-12
